Advancing Precision Medicine

March 2020 | Volume 68 | Issues 1–3 BioTechniques.com

Tech News: Advancing precision medicine

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2019-24573-RS_Stericup-E_8-25x10-81_MSIG.indd 1 9/11/19 9:46 AM Advert template.indd 164 28/02/2020 10:14:52 Contents Raw SCN HiCNorm ICE 0 m GeNorm le GeNorm GSC le ence gene RT-qPCR NormFinder r fe GSC

50 m ence gene nal re RT-qPCR NormFinder r fe er Obtain the most stab Coefficient of int

variation nal re er Obtain the most stab Coefficient of int variation p4 100 m p50 p59 Monolayer

Features Monolayer KR Opinion chromoR multiHiCcompare 3 From the editor 34 What are the off-target effects of CRISPR Welcome to BioTechniques 2020! and why are they concerning? Gaetan Burgio 4 Tech News 0 m 36 On high-throughput sample processing and 4 Advancing precision medicine laboratory techniques: concerns, pitfalls and more 7 Taming the T cell Mariana Salas Garcia & Jack Gilbert 11 Developing new ways to diagnose cancer 38 A hammer doesn’t need a nail 3 Joachim Goedhart 15 News in Brief 40 Top Comments 18 Top 5 The 5 most read 50items m in BioTechniques in 2019 Reports in Brief 2 45 Expert Opinion Step emulsification: high-throughput production of 48 An efficient and novel technology for the monodisperse droplets extraction of parasite genomic DNA from whole Linbo Liu, Nan Xiang, Zhonghua Ni, Xing Huang, Juanjuan Zheng, blood or culture Yunhua Wang & Xingcai Zhang 1 DJ Clark, CM Moore, M Flanagan, K Van Bocxlaer, E-T Piperaki, V Yardley, SL Croft, J Tyson, SP Whitehouse, J O’Halloran, S Krishna & HM Staines

Viewpoints 100 m 49 Evaluation of DNA degradation and establishment of a degradation analysis model for Lepidoptera 0 60 Seconds specimens 21 Leslie Biesecker discusses the issue of genetic Y Xu, XY Ren, HB Wang, M Wang & GH Li exceptionalism and the importance of making FULL ARTICLE genetics open to all 50 Comparison of normalization methods for Hi-C data 23 Robert Philibert on his new digital PCR test H Lyu, E Liu & Z Wu for addiction 59 Selection of reference genes suitable for 25 Anne Carpenter discusses her work in cell imaging normalization of RT-qPCR data in glioma stem cells software and how AI is revolutionizing the field W Dang, X Zhang, Q Ma, L Chen, M Cao, J Miao, Y Cui & X Zhang Interviews 60 ELIMU-MDx: a web-based, open-source platform 27 A peek behind the paper: Jeremy Clark on the new for storage, management and analysis of method for the at-home collection of urine samples diagnostic qPCR data for prostate cancer detection S Krähenbühl, F Studer, E Guirou, A Deal, P Mächler, S Hosch, M Mpina, S Mswata, C Daubenberger & T Schindler 28 Ge Wang and Margarida Barroso on the HOTGEM imaging technology for breast cancer 61 A rapid approach to profiling diverse fungal communities using the MinION™ nanopore Inscopix’s Jonathan Nassi and Kunal Ghosh on 31 sequencer their cutting-edge neuroscience technology KK Mafune, BJ Godfrey, DJ Vogt & KA Vogt samples for prostate cancer detection

62 A simplified method to calculate telomere length Benchmarks in Brief from Southern blot images of terminal restriction fragment lengths 79 Isolation of DNA-free RNA from human bone LF Lincz, FE Scorgie, MB Garg, J Gilbert & JA Sakoff marrow mononuclear cells: comparison of FULL ARTICLE laboratory methods T Alabi, SB Patel, S Bhatia, JA Wolfson & P Singh 63 Erythrosin B: a versatile colorimetric and fluorescent vital dye for bacteria 80 Ultra-fast conductive media for RNA JD Franke, AL Braverman, AM Cunningham, EE Eberhard electrophoretic mobility shift assays & GA Perry SZ Brown, LC Agostini, HL Thomsett & JR Brody 70 Noninvasive cell counting of adherent, suspended 81 Improved next-generation sequencing pre-capture and encapsulated mammalian cells using optical library yields and sequencing parameters using density on-bead PCR A Aijaz, D Trawinski, S McKirgan & B Parekkadan CR McEvoy, T Semple, B Yellapu, DY Choong, H Xu, GM Arnau, AP Fellowes & SB Fox 71 Methodology for the at-home collection of urine samples for prostate cancer detection 82 An improved shotgun antisense method for M Webb, K Manley, M Olivan, I Guldvik, M Palczynska, R Hurst, mutagenesis and gene identification SP Connell, IG Mills, DS Brewer, R Mills, CS Cooper & J Clark Y Tang & RH Gomer 72 A proof-of-concept analysis of carbohydrate- 83 Simple workflow for genome and methylation deficient transferrin by imaged capillary isoelectric analyses of ejaculated bovine spermatozoa with focusing and in-capillary immunodetection low sperm input J Wu, CH Haitjema, CD Heger & A Boge BW Daigneault, SK Rajput & GW Smith 73 Noninvasive prenatal diagnosis of hemophilia A 84 An improved Xer-cise technology for the by a haplotype-based approach using cell-free generation of multiple unmarked mutants in fetal DNA mycobacteria C Chen, J Sun, Y Yang, L Jiang, F Guo, Y Zhu, D Li, R Wu, R Lu, Y-M Boudehen, M Wallat, P Rousseau, O Neyrolles & C Gutierrez M Zhao, F Chen, P Ni, Z He & Z Peng 85 A simple method for non-denaturing purification of 74 A cautionary tale of cross-contamination among biotin-tagged proteins through competitive elution plasmids from commercial suppliers with free biotin Jinli Sun, Yaping Tian, Yingying Du, Zhenzhen Wang, K Lin, Q Yan, A Mitchell, N Funk, C Lu & H Xiao Guodong Zhao, Yong Ma & Minxue Zheng 86 A customized program for the identification of 75 An improved molecular tool for screening bacterial conserved protein sequence motifs colonies using GFP expression enhanced by a M Mian, J Talada, A Klobas, S Torres, Y Rasheed, H Javed, Dictyostelium sequence Perfusion Z Lughmani & R Forough Direct immersion T Kondo & S Yumura 76 Detecting G protein-coupled receptor complexes in postmortem human brain with proximity ligation Trending assay and a Bayesian classifier Y Zhu, J Mészáros, R Walle, R Fan, Z Sun, AJ Dwork, P Trifilieff 41 Events Roundup & JA Javitch 42 Webinar Roundup 77 Parallel sample processing using dispersive INtip Original Ladder Normalized micro-purification on programmable multichannel 43 Upcoming Events pipettes PA Kates, JJ Tomashek, DA Miles & LA Lee 44 Cover Competition 78 An improved method for the rescue of recombinant 87 Application Note Untreated + EB Heat shockedNewcastle + EB Ethanol disease treated + EB virusCaCl treated + EB Mixture + EB 2 P-S Cheow, TK Tan, AA-L Song, K Yusoff & SL Chia 90 New Products 92 In the Next Issue E. coli 93 Sponsors 3000 bp

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Welcome to BioTechniques Staff Editorial, Production & Circulation BioTechniques 2020! Chairman: James Drake Managing Director: Phil Garner Editor in Chief: Francesca Lake Publisher: Sarah Mayes his issue marks the first of 2020 and thus a close to 2019. 2019 Managing Editor: Joseph Martin was (as promised in my introduction to that year’s volume!) an Digital Editor: Tristan Free T exciting year, which saw us continue to push towards Head of Production: Zara Robinson reproducibility and launch a new website. Sales & Business Offices 2020 promises more of the same – our print edition has been updated to Advertising: JT Hronich • [email protected] provide you more content and yet use fewer trees (not using magic, Subscriptions: Dominik March • [email protected] I promise you!), and our online content is Reprints: Sam Cavana • [email protected] List Rental: Leela Ripton • [email protected] expanding to bring you more of the methods and This issue “ Permissions: Adriana Gonzalez • [email protected] protocols you love, as well as more news and contains a expert discussion of matters important to the life science laboratory researcher. look back at Editorial Board 2019 from Richard J. Ablin, University of Arizona Bill Brizzard, Brizzard Consulting LLC This issue contains a look back at 2019 from the the Editorial Bruce Budowle, UNT Health Science Center Editorial team, as well as a variety of content, team… Piotr Chomczynski, Molecular Research Center including a new open source platform for qPCR ” Rita R. Colwell, University of Maryland-College Park and Johns data analysis and a look at the latest technological advances in basic Hopkins University research that are pushing us toward the goal of precision medicine. Joshua J. Coon, University of Wisconsin-Madison David Cronk, Charles River Laboratories I hope you enjoy this new, revamped print issue, as well as all the issues Manel Esteller, Spanish National Cancer Centre (CNIO) from the rest of the 68th volume of BioTechniques! Jeffrey Felton, Western University of Health Sciences Andrew French, University of Nottingham Erica A. Golemis, Fox Chase Cancer Center Peter M. Gresshoff, The University of Queensland Yoshihide Hayashizaki, RIKEN Jörg Hoheisel, German Cancer Research Center Shenglin Huang, Fudon University Pui-Yan Kwok, University of California, San Francisco Rachael L. Neve, Massachusetts Institute of Technology Peter J. Oefner, University of Regensburg Stephen W. Paddock, University of Wisconsin-Madison Scott D. Patterson, Gilead Sciences, Inc. Leonard F. Peruski, Jr., Centers for Disease Control George Poste, Arizona State University John Quackenbush, Harvard School of Public Health Joshua Rappoport, Northwestern University School of Medicine John Rossi, City of Hope Michel Goedert, MRC Francesca Lake Herbert P. Schweizer, Colorado State University Editor in Chief, Future Science Group, Unitec Barton Slatko, New England Biolabs House, 2 Albert Place, London, UK. Steve S. Sommer, MEDomics, LLC [email protected] Igor Stagljar, University of Toronto Mathias Uhlén, The Royal Institute of Technology Timothy Veenstra, SAIC-Frederick, Inc. Kent E. Vrana, Penn State College of Medicine BioTechniques is a peer-reviewed journal dedicated to the publication of original laboratory methods, related technical tools, and methods-oriented review articles Michael Weiner, AxioMx that are of broad interest to scientists engaged in basic applied life science research. Complete Instructions for Authors are available at: https://mc04.manuscriptcentral. com/fs-btn, BioTechniques’ website for online manuscript submission. All manuscripts should be submitted at this site.

ADVANCING PRECISION MEDICINE Francesca Lake explores the latest goings on in techniques advancing precision medicine.

recision medicine is arguably ‘the next big thing’ in modern that in order for participants to really want to stay engaged in this study medicine. Various initiatives have been launched and have for as many years as we’re asking them to, there needs to be some sort P gathered huge steam and funding in a bid to allow precise of give back to participants.” medical treatments that will in turn increase treatment success and Another interesting aspect discussed was the difficulty in decrease money lost owing to failures. To name a few, there is the ensuring the data are as widely available as possible in order to ‘Precision Medicine Initiative’ (renamed All of Us), announced by maximize innovation whilst also protecting the participants. Barack Obama in his 2015 State of the Union Address [1], and the 100,000 Genomes Project, announced by then-UK Prime Minister HIDDEN IN THE GENOME David Cameron in 2013, which focused on sequencing the genomes Our genomics session was begun by Elaine Mardis (The Ohio State of patients with rare diseases and their families, and those with cancer, University College of Medicine, OH, USA) and focused primarily on in a bid to improve scientific understanding and thus bring about new oncology. Since Janet Rowley first proved that cancer is a genetic medical insights [2]. Funding has, of course, followed and a huge disease(s) in the 1970s and the human genome reference sequence amount of research has been conducted, or is currently underway. was created, strides have been made in understanding the The BioTechniques Advancing Precision Medicine virtual relationship between genetic variants and cancer, and what that symposium was a free-to-attend online event held over 13 and means for healthcare (Figure 1). 14 November 2019 that saw a variety of experts discuss the latest What’s more, in the last 15 years or so we’ve moved from capillary in laboratory research, which is helping to drive the development of technology to next-generation sequencing and, as a result, the cost of precision medicine approaches and their adoption into the clinic [3]. sequencing has plummeted from millions to just $1000 per genome The meeting covered a variety of aspects including genomics, big and the time taken has reduced from years to hours. “Of course, all of data, microbiomics, wearables and more. this high throughput is a great thing, but we really need computational pipelines to be able to tease apart the information that we have and to DIVING INTO THE ALL OF US RESEARCH PROGRAM make sense of it in the context of the individual patient,” Mardis noted. Stephanie Devaney, Deputy Director of the All of Us Research Program, Mardis went on to discuss the particularly tricky example of gave the plenary talk discussing the work accomplished by the pediatric cancers. These are often difficult to study owing to their program thus far and what is yet to be achieved. Of the participants, rarity, the increased ethical complications and difficulties in trans- 80% are from groups under-represented in biomedical research, a lating adult-approved drugs to children. Essentially, this leads to result of All of Us focusing on these groups. an ‘N of one’. Some particularly interesting aspects of the All of Us Research However, now that some of the differences and similarities Program include the emphasis on ‘participants as partners’, ensuring between pediatric and adult cancer are better understood, Mardis’ those providing the data know what data are being collected, what team is building a system for cancer next-generation sequencing analysis and research will be performed, and how data will be returned diagnostics in pediatrics. “We really have benefited incredibly in to them. oncology from the use and development of large-scale resources coming “Traditionally in research studies, going back decades, we have talked out of cancer discovery,” she concluded. “In pediatric cancer we have about research participants as human subjects,” Devaney noted. “We a little bit more of a difficult path… because of the different nature of have decided that it really needs to be a bi-directional relationship, in genomic drivers in this disease, we’re really benefitting from a combi-

Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 4 www.BioTechniques.com deplete, however, while we’re also seeing a rise in some diseases. Sequencing the genome: then and now And that’s not just a coincidence. Knight highlighted how with human DNA you can only classify someone as lean or obese with 57% accuracy, but with the genes of your microbiome that rises to 90%. His team is now studying environmental bacteria, which are key for both climate and health. One striking example of this is the correlation of Kawasaki disease incidence with wind direction [4]. “The

Capillary technology conjecture here is that dust from a particular region of China is carrying “Next-generation sequencing” Applied Biosystems 3730xl (2004) Illumina NovaSeq 6000 (2018) with it the causative pathogen of Kawasaki disease,” he noted. He is now $15,000,000/genome $1,000/genome working with Jane Burns’ group at UC San Diego to match up ~5 years, international project 24 whole human genomes, 44 hours microbes in clouds with those on an afflicted child’s fingertips and airways to see if they can identify the particular causative agent. He’s also now working on water. “Almost nothing is known about Figure 1. A look at the advances in genome sequencing, as presented how the microbes in the oceans might be getting into the atmosphere by Elaine Mardis. and affecting climate,” he explained. “Are those good or bad microbes preferentially? And do they lead to any health impacts?” nation of both DNA and RNA sequencing data and multiple analytical The sheer size of the outputs of this research then led to further approaches.” discussion of big data, which was also a key theme of our next John Quackenbush (Harvard TH Chan School of Public Health, speaker, Michael Snyder (Stanford University, CA, USA). Snyder’s MA, USA) spoke next, looking at the interesting approach of exploring talk highlighted how the current way we do medicine is somewhat cancer using network medicine. His research centers around the flawed, being reactive and based on population-based measure- core ideas that biological systems are driven by complex networks, ments. However, most ‘healthy’ measurements are really an average and that network structure is important to understanding system of a range, and what is healthy for some might not be for others. biology. Differentially expressed genes are controlled by regulatory His group has been employing various wearables and technologies networks, and his group works to develop methods to analyze and to perform ‘personal omics profiling’, following a cohort of around compare network topology. “By comparing network topology between 109 people for approximately 6.5 years. “In its most deluxe form healthy and diseased populations what we can start to get a handle on we’ll measure 13 or 14 different -omes in people. The genome is is not just what genes are differentially expressed, but what processes just one; we’ll measure all these other -omes as well.” This includes may regulate that differential expression”, he noted. This leads to an the epigenome, transcriptome, proteome, cytokines, metabolome, understanding of what genes are differentially expressed as well as lipidome, autoantibody-ome and microbiome (Figure 2). the how and the why. “We’re literally taking billions of measurements from people,” he This has led to the development of ‘The Methodological Zoo’, a continued. “The goal is to try and understand, what does it mean to be group of models and methods useful for different sets of data that healthy? What does the health state look like? How does it differ between can be gleaned from various experiments and which are named after different people? How does it change over time?” But where will this lead? animals, such as PANDA and CONDOR. He then went on to discuss “I see a world where people will be getting sequenced before they’re born,” CONDOR and eQTL analysis in more detail. he concluded. “And then together with other deep data profiling, omics Finalizing the genomics session, Jun J Yang (St. Jude Children’s of various sorts, as well as wearables, you’ll be able to better predict Research Hospital, TN, USA) discussed pharmacogenetics for drug people’s disease risk, catch disease earlier and treat it before it becomes response; specifically how bioinformatics are generally poor at symptomatic.” predicting response while high-throughput functional genomics can be extremely powerful. He illustrated this with the case study CELLS AS MODELS of NUDT15 and Thiopurine, where modern techniques allowed a David Tuveson (Cold Spring Harbor Laboratory, NY, USA) discussed tenfold speed up of discovery to implementation, taking 2 years and how organoids are being harnessed for the study of pancreatic 6 months for NUDT15 compared with TPMT, which took 21 years. “This cancer. Affecting around 300,000 patients a year in the Western is a particularly exciting time for pharmacogenetics because you world, it has a low survival rate, with 9% living 5 years [5]. Early can make genetic discovery and transfer that into clinical practice diagnosis is problematic, treatment options are limited and often at a really amazing pace,” he noted. patients are too sick to try a second treatment, so a further understanding of the disease is critical to choose the right first treatment UNDERSTANDING HOST–PATHOGEN and improve survival. His team has created organoids from & HOST–ENVIRONMENT INTERACTIONS pancreatic cancers, metastases and healthy tissues, determining Rob Knight (UC San Diego, CA, USA) discussed the 3D mapping of protein and gene signatures, using whole-genome sequencing to microbes and molecules. The human body is, in some ways, more compare the models to the primary tumor and performing ‘pharma- microbe than human – we have around 30 trillion human cells, but cotyping’. The different samples have demonstrated a huge level of 39 trillion microbial cells. This is even starker at the DNA level – for disparity in drug sensitivities. “This was quite surprising to me, that our 20,000, we have an estimated 2–20 million microbial genes. humans would be this different,” he noted. “We don’t actually measure Knight discussed how we are seeing our microbiome diversity the amount of chemotherapy that’s in the body of a person when we

give it to them. We just give it to them based upon parameters that are set up to limit toxicity, and don’t really study efficacy in the cancer.” His Longitudinal Personal Omics Profiling team is now looking to stratify patients based on organoid signatures. Omics Genome Measurements Epigenome The organoid theme was continued by Sylvia Boj from Transcriptome Hubrecht Organoid Technology (Utrecht, The Netherlands), who Proteome spoke about a new organoid model for cystic fibrosis.“We believe Cytokines Billions of Metabolome Measurements that with this in vitro model, we are able to bring patients back to the bench, Lipidomics Autoantibody-ome Clinical Tests into preclinical drug development, in order to improve and change the way Microbiome (gut, urine, nasal, Questionnaires tongue, skin) Biosensors that until now drugs have been developed,” she highlighted. Her team Stress Echos Glucose Control can now generate organoids from almost any patient, character- Year 1 Viral infection Year 2… izing and stratifying them in a bid to improve treatment. 6 109 Individuals 6.5 years James Clinton (ATCC, VA, USA) completed the session, discussing next-generation cancer models as part of the Human Cancer Models Initiative [6]. “Cancer cell lines for many specific tissues – pancreas and Figure 2. Longitudinal personal omics profiling, as presented by liver, for example – are really underrepresented,” he highlighted. “I think Michael Snyder. there’s also increasing realization that many lines have been mis-identified or have been in culture for so long… there’s really no telling how they analysis, as well as development of outcome measures and design and compare to the original tumor.” He also noted how heterogeneity is performance of trials that will really be representative.” missing from existing cell lines, as well as patient history and the Finally, George Patrinos (University of Patras, Greece) discussed lack or relevancy caused by 2D models. The Human Cancer Models how pharmacogenomics are “paving the path to genomic medicine”. Initiative is seeking to change this, and Clinton took us through “More than 50% of the general population and patients carry DNA variants their goals. that affect their response to the majority of the most commonly prescribed drugs,” he noted. This can lead to adverse drug reactions which, in the TRANSLATIONAL RESEARCH USA, are one of the leading causes of death [9]. He took us through For our final session, Heidi Rehm (The Broad Institute, MA, USA) began a number of examples of successful translation of genomic under- by discussing a variety of projects that are looking to translate basic standing to reducing adverse events (and cost) in the clinic, finishing research into the clinic, including MedSeq, BabySeq and ClinGen. She by discussing the U-PGx project, which is implementing pre-emptive highlighted that something underlying all these projects is the need pharmacogenomics testing in real-world clinical settings in Europe [10]. for good standards and data-sharing platforms, for example the He discussed the various technologies and infrastructure required for ClinVar database, Matchmaker Exchange for rare disease data and the project to be successful in achieving its goal of making pharma- newer platforms such as AnVIL and Terra, which are intended to cogenomics available to every European citizen. support large-scale genomics. “I would argue that for the best outcomes Overall, the meeting was a fascinating and in-depth showcase for all patients, we need to share data on a global scale, and critically of the benchside technology that is enabling precision medicine at evaluate evidence as a collective community,” she noted. the bedside, and this tech feature provides only a superficial insight She then focused in on ClinGen, a consortium of people sharing into what was discussed. We encourage readers to visit the event data, developing standards and curating knowledge who work with page to watch the full talks [3] and we will look forward to more the ClinVar database, which allows ingestion and sharing of variant- advances to come! level knowledge [7]. This platform goes on to enable the creation of robust gene-testing panels. Written by Francesca Lake Next, Marni Falk (The Children’s Hospital of Philadelphia, PA, USA) zoomed in on precision mitochondrial medicine, a field where REFERENCES there are no proven effective therapies. Again, big data is critical 1. What is the Precision Medicine Initiative? https://ghr.nlm.nih.gov/primer/precisionmed- icine/initiative here, and Falk discussed the best way to collect, integrate, interpret 2. The 100,000 Genomes Project. https://www.genomicsengland.co.uk/about-genom- and share relevant data, be it from the genome, transcriptome, ics-england/the-100000-genomes-project/ 3. BioTechniques Online Event: Advancing Precision Medicine 2019. https://www.biotech- physiome and so on, and then report that in a way that is under- niques.com/events/biotechniques-online-event-advancing-precision-medicine-2019/ 4. Rodo X, Ballester J, Cayan D et al. Association of Kawasaki disease with tropospheric standable to the family and physician. Falk took us through how wind patterns. Sci. Rep. 1, 152 (2011). her team overcomes these issues, using various systems including 5. Cancer Facts & Figures 2019. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2019/cancer-facts- some that are actually designed for business. She went on to and-figures-2019.pdf discuss various initiatives aiming to help develop therapeutics 6. HCMI: Human Cancer Models Initiative. https://ocg.cancer.gov/programs/HCMI for mitochondrial diseases, including the mitochondrial medicine 7. Explore the clinical relevance of genes & variants. https://clinicalgenome.org/ 8. Preventable Adverse Drug Reactions: A Focus on Drug Interactions. https://www.chop. frontier program [8]. “The goals of this are really to have a continual edu/centers-programs/mitochondrial-disease-clinical-center pipeline from early gene discovery, modelling, therapeutic de-risking and 9. US FDA. Preventable Adverse Drug Reactions: A Focus on Drug Interactions. https:// www.fda.gov/drugs/drug-interactions-labeling/preventable-adverse-drug-reactions-fo- developing of companion diagnostics and translational research to enable cus-drug-interactions us to bring this to patients on a regular basis, specific to each patient,”she 10. U-PGx. https://upgx.eu/ explained. “[This] requires us to harness our integrated data systems, a clear registry of patient genetics, phenotypes and their samples for

Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 6 www.BioTechniques.com Tech News TAMING THE T CELL Joseph Martin and Tristan Free explore the latest developments in the T-cell-based therapy CAR-T and the challenges that need to be overcome for them to become common clinical practice.

s genetic engineering techniques have become more The initial evolution included the addition of a costimulatory sophisticated over the last decade, our ability to manip- domain to the CAR complex, such as CD28 or CD137 [3]. These A ulate cells for a specific purpose has advanced. Nowhere second-generation CARs display an increase in T-cell proliferation, has this been more clearly demonstrated in the last few years than anti-apoptotic protein and cytokine release [4]. The third generation by the T cell. simply introduced a further costimulatory signal, while the fourth The rise of the much-lauded chimeric antigen receptor (CAR) involves additional stimulation of transcription factors to induce T-cell therapy has been ably assisted by gene-editing techniques the production and secretion of specific cytokines and interleukins such as CRISPR and zinc finger nucleases [1]. These techniques have in order to promote the potency of T-cell action [5]. The scope of allowed researchers to integrate ever more intricate structures and these additional functional transgenic proteins in the fourth gener- adjustments into T cells in order to fine tune the therapy. ation does not end with the simple amplification of T-cell potency. Despite the encouraging rate of development in these therapies, These proteins could be selected to provide cells with an on/off many challenges remain in their commercialization and implemen- switch or a suicide gene to enable greater, more granular control tation while characterization remains an evolving area. of the therapy [1]. To further promote CAR-T cells as a viable therapeutic CAR-T CELLS: ADDRESSING THE CHALLENGES option capable of surviving in the TME, researchers have developed CAR-T therapy relies on the incorporation of CARs into a patient’s dual receptor CAR-T cells that contain the traditional TAA targeting extracted T cells. The key components of these CAR molecules are CARs and an additional receptor for cytokine-mediated growth the single-chain variable antibody domain (scFv), which can be stimulation helping to offset the effects of the immune checkpoint designed to target specific tumor-associated antigens (TAAs), and signals in the TME [5]. the intracellular signaling domain, primarily consisting of the CD3ζ The next generation of CAR-T cells is hypothesized to include chain of a T-cell receptor (TCR). On binding to the TAA, the CAR initiates the knockout of the T cell’s human leukocyte antigen alongside proliferation of the CAR-T cells and the death of tumor cells through TCR genes, allowing T cells taken from healthy donors to be the release of cytokines, bypassing the need to bind to antigens used and avoiding their rejection by the cancer-patient host, presented by the tumor’s multi-histocompatibility complex (MHC) [2]. therefore enabling their application in multiple patients [1].

SURVIVING IN HOSTILE TERRITORY TACKLING TOXICITY From the inception of CAR-T, the hostile tumor microenvironment A further challenge in CAR-T is dealing with the toxicity of the (TME) has been established as a challenge to the therapy, reducing therapy. The typical toxicities observed with CAR-T are cytokine the survival and proliferation of CAR-T cells. Over the past few years, release syndrome (CRS), neurotoxicity and brain edema. As these the basic formula of the therapy has been updated to include the cells target tumor surface proteins, unrestricted by the MHC and current fourth generation of CARs. Each evolution has been designed are engineered to be vehemently potent, the off-target effects of to address barriers such as the TME and to increase the potency the treatment have the potential to be devastating. As the ligands of T cells. found on the surface of tumors can often be found in healthy,

Single antigen Simultaneous Sequential antigen targeting antigen targeting targeting

Universal Universal CAR immune immune receptor receptor Targeting Targeting ligand ligand TAAs TAAs TAAs

Cell killing Resistance/relapse Cell killing Cell killing Resistance/relapse • TAA heterogeneity • Splice variants • TAA loss

Cell killing

Figure 1. Demonstrating the different binding modalities of (A) classical CAR-T cells, (B) simultaneous and (C) sequential UIRs [5].

functional cells in different regions of the body, the selection evolution, the engineered T cells can be retargeted to TAAs on the of an appropriate target is vital. This is perhaps most painfully newly evolved tumor cells [7]. exemplified by the first in-human use of third-generation CARs that had been designed to target HER2 [6]. Minutes after the CAR-T CHARACTERIZATION administration of 1011 HER2-targeted CAR cells, the patient experi- Due to the complexity of live products, variability in starting material enced extreme pain and entered a comatose state from which and difficult characterization, CAR-T cell manufacturing is considered they did not recover, dying 5 days later. It is likely that the natural to be much more challenging than that of traditional biologics. CAR-T presence of HER2 in pulmonary epithelial cells was a significant cell therapy manufacturing entails several carefully performed steps factor in the observed accumulation of CAR-T cells in the patient’s from lymphocyte collection to genetic modification with the CAR lungs [5]. construct. These complex steps, alongside the requirement of To address this pressing concern, researchers are designing reagents and source materials with differing complexity, are all key universal immune receptors (UIRs) for use in CAR-T cells that can elements that must be completely understood to ensure that the target multiple TAAs. Unlike standard CARs, these UIRs contain product has the correct identity, potency and purity. Therefore, an additional extracellular adaptor domain that connects intracel- product characterization is key for all successful biological drug lular T-cell signaling domains to a soluble tumor antigen targeting development. ligand (TL) (Figure 1). In this instance, T-cell activation is induced Breaking down the manufacturing framework, it is important by cell binding to the ‘tag’ or binding domain on the TAA-bound TL. to identify chemical, biological and physical attributes, known as This enables post-translational alteration of the T cell’s target speci- critical quality attributes, that can be measured continually. ficity by alteration of the soluble TL [7]. Ramello et al. recently addressed the uncertainty involved in This development means that if the T cells begin to elicit a CAR-T cells’ mechanism of action, specifically how CARs interact cytotoxic response in an undesired region of the body, they can with endogenous T-cell molecules. This characterization is essential be retargeted by changing the TL. Additionally, if the tumor evades to provide tools to develop novel receptors with more tightly initial administration due to tumor heterogeneity or subsequent controlled functions [8].

Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 8 www.BioTechniques.com CAR-T COMMERCIALIZATION with ever more sophisticated solutions, new unforeseen The early stage of this field, when combined with the complex challenges are sure to happen. However, with each manufacturing that gene-edited cell therapies require, means evolution these challenges should grow less dramatic and inhib- that their price is particularly expensive. Two recently approved itory to the application of CAR-T cells. The current exciting rate CAR-T therapies – Yescarta and Kimriah – are currently priced of development bodes well for the theoretical application of at US$373,000 and US$475,000, respectively [10]. these cells in a wide variety of settings. The manufacturing process is obviously a key driver behind Challenges for the development of a more controlled and cost- the price tag. However, this is not so straightforward to fix. First, effective manufacturing process have however yet to be overcome. the manufacturing process must result in a safe and clinically Despite there only being a few therapies approved and on the effective cell product for the patient. Second, the process must market, there is a growing number of institutes engaging in CAR-T be robustly reproducible, which is a necessity to validate it and research and manufacturing. With the increased interest and to ensure quality. So, if the logistics are improved, yield increased cell therapy still largely being in the research and development and supply chain streamlined, then surely that would help reduce phase, standardization and characterization will become critical. the cost of these therapies? If the manufacturing problems can be solved and the production In order to increase the robustness and scalability of the process streamlined, consequently, the cost of these therapies bioprocesses to fit the therapy’s needs, companies such as should be reduced. Oxgene are accelerating the discovery and production of cell However, this is much easier said than done. It will take the and gene therapies by addressing key industry bottlenecks and cooperation between pharma, biotech academic institutes and facilitating new therapeutic approaches [11]. official bodies (FDA, EMA, and so on) to ensure that the path towards An automation equipment company, ThermoGenesis, is also safe and affordable therapies is accelerated. attempting to solve the abovementioned problems through the development of CAR-TXpress™, a platform designed to automate Written by Tristan Free and Joseph Martin many of the manual steps involved in cell processing. Platforms such as CAR-TXpress are helping strive towards more readily REFERENCES available CAR-T cell therapies by reducing processing time and 1. Zhao J, Lin Q, Song Y, Liu D. Universal CARs, universal T cells, and universal CAR T cells. J. Hematol. Oncol. 11(1), 132 (2018). increasing cell recovery, in order to significantly reduce the overall 2. Sadelain M, Riviere I, Riddell S. Therapeutic T cell engineering. Nature. 545, manufacturing time and cost, and increase the yield and efficiency 423–31 (2017). 3. Tang XY, Sun Y, Zhang A et al. Third-generation CD28/4-1BB chimeric antigen of high-value samples [12]. receptor T cells for chemotherapy relapsed or refractory acute lymphoblastic leukaemia: a non-randomised, open-label phase I trial protocol. BMJ Open. 6(12), Many people believe there are still some issues surrounding e013904 (2016). the production of cell therapies, but it is clear that the situation is 4. Lee DW, Kochenderfer JN, Stetler-Stevenson M et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and improving. Non-gene-edited allogeneic CAR-T therapy or ‘off-the- young adults: a Phase 1 dose–escalation trial. Lancet. 385(9967), 517–528 (2015). shelf’ technology could make CAR-T therapy significantly cheaper 5. Zhao L, Cao Y. Engineered T cell therapy for cancer in the clinic. Front. Immunol. by removing the need for individualized manufacturing, thus 10, 2250 (2019). 6. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. Case removing a big burden from healthcare systems. Do people need report of a serious adverse event following the administration of T cells transduc- ed with a chimeric antigen receptor recognizing ERBB2. Mol. Ther. 18(4), 843–851 to buy into the concept of an autologous therapy? (2010). The alloSHRINK Phase I trial, Celyad’s trial of CYAD-101 7. Minutolo N, Hollander E, Powell D. The emergence of universal immune receptor T cell therapy for cancer. Front. Oncol. 9, 176 (2019). combined with chemotherapy for the treatment of metastatic 8. Ramello MC, Benzaïd I, Kuenzi BM et al. An immunoproteomic approach to characterize colorectal cancer, has thus far yielded promising results, with no the CAR interactome and signalosome. Sci. Signal. 12(568), eaap9777 (2019). 9. Kassim SH. Toward an integrated model of product characterization for CAR-T cell thera- severe toxicity reported [13]. As promising as this technology is, py drug development efforts. Cell Gene Therapy Insights 3(4), 227–23 (2017). doubts still surround whether allogenic CAR-T can really be an 10. Whittington MD, Ollendorf DA, Campbell JD. Accounting for all costs in the total cost of chimeric antigen receptor T-cell immunotherapy. JAMA oncology 4(12), 1784–1785 off-the-shelf therapy and more work is still required to overcome (2018). challenges with regards to immunology as well as manufacturing. 11. OXGENE™. www.oxgene.com/ 12. Thermogenesis®. https://thermogenesis.com/ For the time being, schemes have been set up to ensure that 13. Prenen H, Rasschaert M, Hendlisz A et al. Results from the completed dose-escalation of patients can obtain these notoriously expensive treatments. the alloSHRINK Phase I study evaluating the allogeneic NKG2D-based CAR T-cell therapy CYAD-101 in metastatic colorectal cancer patients. Presented at: SITC 34th Annual Meet- The Advanced Therapy Treatment Centre (ATTC) project aims ing. National Harbor, MA, USA, 6–10 November 2019. Abstracts: https://sitc.sitcancer. org/2019/abstracts/titles/index.php?filter=Results+from+the+completed+dose-escala- to develop robust systems for the routine delivery of cell and tion+of+the+alloSHRINK+phase+I+study+evaluating+the+allogeneic+NKG2D-based+- CAR+T-cell+therapy+CYAD-101+in+metastatic+colorectal+cancer+patients gene therapies to patients around the UK in the fastest and 14. Advanced Therapy Treatment Centres. www.theattcnetwork.co.uk/ most cost-effective way possible [14]. For example, a number of payment schemes are being considered, including one that involves spreading the payment out over time to better share the risk of the therapies failing.

FUTURE PERSPECTIVE CAR-T therapy will continue to develop technically in a step-wise, almost evolutionary fashion. As issues such as toxicity, off-target effects and T-cell survival arise and are countered

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DEVELOPING NEW WAYS TO DIAGNOSE CANCER We explore the latest developments across cancer diagnostic techniques that are enabling researchers to improve accuracy in determining diagnosis and prognosis.

019 saw great strides being made in the field of cancer VOCs, which are linked to the early stages of six cancers (Figure 2). research, with not one but two of the 2020 US presidential Additionally, the trial is seeking to assess whether the breathalyzer 2 nominees promising to find a cure in the next 4 years. can differentiate between patients with and without different cancer Although the development of new treatments is vital, and advances types by comparing the breath biomarkers of patients with gastric, such as CAR-T will prove life saving for many, it has long been known esophageal, pancreatic, renal, prostate and bladder cancer from that the key to successful treatment is early diagnosis. If the high matched controls [4]. hopes of Trump and Biden are to come to light, then it is important Alongside this, liquid biopsies have advanced over the past year. to utilize the latest technology in order to improve diagnostic tests Of particular interest are the blood tests being developed for cancer and guide targeted treatments. diagnostics. A pilot study conducted by researchers based at the University of Nottingham (UK) developed a blood test for the early ALTERNATIVE BIOPSIES: BLOOD, URINE & BREATH detection of breast cancer. The research group used screening Alternative biopsies have generated significant interest in oncology technology to determine the presence of tumor-associated antigens, as they offer the potential to isolate and analyze biomarkers for which are indicative of breast cancer. The test could detect breast personalizing cancer therapy. cancer up to 5 years before any clinical signs appear [5]. A key advantage of alternative biopsies is that they bypass the A key study from the ASCO Annual Meeting (31 May–4 June need for invasive diagnostic procedures, which often have low uptake 2019, IL, USA) also centered around liquid biopsies, this time for the rates by patients [1]. A recent example of this is the development detection of lung cancer. Bob Li (Memorial Sloan–Kettering Cancer of an at-home urine test for prostate cancer. Traditionally, patient Center, NY, USA) presented his latest research investigating how ultra- urine samples were collected once a digital rectal examination of the deep next-generation sequencing of plasma circulating cell-free DNA prostate had been conducted, as it was thought this was necessary is being used in patients with advanced lung cancers [6]. to boost the levels of prostatic secretions in the urine. However, a In an exclusive interview he stressed that, in order to move liquid study published in BioTechniques demonstrated that urine collected biopsies from a research tool to part of routine clinical decision by patients in their own home was comparable in quality to the making, clinical utility studies are required. Li commented: “We traditional method whilst circumventing the inconvenience, cost, need to do clinical trials prospectively to demonstrate how this can discomfort and expense of patients having to visit the clinic [2]. help patients improve outcomes, such as treatment response, improve Along the same vein, researchers have developed a noninvasive disease control and overall survival end points.” [7]. and pain-free breathalyzer (Figure 1) to collect exhaled breath samples with potential biomarker volatile organic compounds GENETIC SCREENING & CLASSIFICATION (VOCs). Many diseases, including certain cancers, can potentially With attention turning away from the idea of a one-size-fits-all generate disease-indicative VOCs that can be a roadmap to their treatment and towards the notion of precision medicine, ensuring early diagnosis [3]. accuracy in diagnosis is of the upmost importance. Rather than The PAN Cancer trial for Early Detection of Cancer in Breath (Clini- differentiate patients by cancer type – breast, lung, brain, and so on calTrials.gov Identifier: NCT03756597) was launched to evaluate – focus has changed to classification based on tumor mutation and whether this breath biopsy technology could detect cancer biomarker other genetic factors. Utilizing the latest in biopsy techniques as

One issue with utilizing genetics in diagnostics is the difficulties existing sequencing methods have in detecting small-scale changes in the genome, leaving many types of genetic variation invisible to single-cell sequencing methods. However, hoping to have overcome this issue, a multicenter research group has developed a new method: single-cell tri-channel processing (scTRIP) [10]. scTRIP technology allows researchers to study genetic variation within the DNA of a single cell as well as identify new variations as they form in new cells. When testing the technique in patient-derived leukemia cells, they found four times more variants than could be detected by diagnostics currently in standard clinical use, including a clinically relevant translocation that was the driving force in the overexpression of the cancer-causing gene. “scTRIP combines signals from three distinct channels of infor- Figure 1. The Owlstone Medical ReCIVA Breath Sampler for breath mation from within the genomic code of the individual cell,” explained biopsy [19]. Jan Korbel (European Molecular Biology Laboratory, Heidelberg, Germany), one of the paper’s senior authors. “Doing so, our method described earlier, clinicians are now able to extract genetic material allows us to uncover the full spectrum of DNA rearrangements in from patients in minimally invasive procedures and can gain great individual cells.” [11]. insight into their individual cancer history from it. Using scTRIP, the researchers hope to gain a greater understanding The issue comes with the large amount of variation that occurs of how the DNA of one cell in the body can differ from another cell across oncology, making it difficult to compare like for like given all and, in doing so, develop the clinical utility of the technique. are unique. To overcome this, geneticists have worked on various However, the increase in data that researchers can gain from classification systems that allow those with similar, rather than a single patient’s DNA brings with it the issue of computational identical, mutations to be treated in similar ways. power. We come to the point where humans alone cannot process The latest of such classification systems comes from the Center the volume of data gained, at which point further technologies are for Genomic Regulation (Barcelona, Spain) where results from required to reduce the burden. Artificial intelligence (AI) and machine genome sequencing can be assigned to one of 16 groups, each of learning are becoming invaluable, not just in the field of genetics which defines the characteristics of the tumor type and origin. Ten but in diagnostics as a whole. of the classification groups have clinical relevance and can guide clinicians when making a diagnosis [8]. ARTIFICIAL INTELLIGENCE Mutations from over 2500 patients with 37 different types of cancer FOR MEDICAL DIAGNOSTICS were analyzed as part of the study. From this, 45 million mutations AI is a much-discussed topic in medicine, especially in the field of were detected, at least 1.2 million of which were non-unique, as defined diagnostics. In recent years, AI has become more accurate in identi- as a mutation found in the same location for two or more patients. This fying cancer and is therefore becoming an increasingly viable source means that, on average, 4% of mutations could be found in multiple of diagnostic information to complement current conventional patients [8]. Given that there are upwards of 3 billion potential mutation diagnostics [12]. sites in the whole of human DNA, the number of non-unique mutations One of the major tools in cancer diagnostics is medical imaging. was higher than what could be put down to chance. Although the current practice of radiography in cancer diagnostics “Cancer is a complex disease that requires a bespoke course of action is, in most cases, highly effective, it is not immune to human error to diagnose, manage and treat effectively,” commented Ivo Gut, senior and researchers are always looking for ways to improve methods. author of the study. “Currently doctors look for individual mutations at AI has been demonstrated to be a powerful potential tool for image specific locations in DNA, which has a limited view. Using whole genome analysis. A recent example of this is the development of AI models for sequencing provides a complete overview of the number of mutations breast cancer detection using x-ray images. Two studies, published in a tumor, allowing doctors to classify the cancer type and gain deeper in Radiology and Nature, respectively, revealed that AI technology, understanding of disease, which can have important implications for trained using mammography images, was able to predict breast the way they treat their patients.” [9]. cancer from scans to a similar standard as expert radiologists [13,14]. As well as guiding diagnosis, the classification system could help Furthermore, recent studies investigating the use of AI in the doctors in determining the most appropriate course of treatment diagnosis of prostate cancer have revealed the high level of accuracy for an individual. For example, those with tumors that fall into one that the technology can bring to this disease area. One such study, particular group are likely to respond better to immunotherapy conducted at the Karolinska Institute (Stockholm, Sweden) and treatments. Tampare University (Finland), used over 8000 digitized biopsies to The authors hope that these early insights into mutational classifi- train an AI system to discern which biopsies exhibited cancer. The cation are an important step towards a clinically viable, generic whole- results of testing were compared with expert uro-pathologists and genome sequencing test that is able to diagnose all forms of cancer revealed a comparable level of accuracy [15]. A similar study from and can replace the multitude of screening tests currently in use. the RIKEN Center for Advanced Intelligence Project (Tokyo, Japan)

Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 12 www.BioTechniques.com demonstrated the efficacy of unsuper- Complete blood cycle ~1 min vised learning by the AI technology Detecting low levels of VOCs ~10 min using deep neural networks to identify predictive features in pathology images Cancer from cancer patients, some of which Breathalyzer Bronchiole biomarker had not been noted by pathologists, VOCs VOCs suggesting a higher level of accuracy in predicting cancer recurrence [16]. Alongside pattern feature recognition from images, AI has also been utilized in the analysis of gene activity. Alveoli Acute myeloid leukemia (AML) is hard to detect from symptoms alone, and the Suspected cancer development of additional techniques Systemic and to aid early diagnosis for the acceler- pulmonary blood circulations ation of therapy is of vital importance. Researchers at the German Center for Figure 2. The breath biopsy process. Reprinted from [20]. Neurodegenerative Diseases (DZNE) and the University of Bonn (both Bonn, Germany) examined the genetic activity of both healthy and cancerous cells found in the Written by Jade Parker, Heather Jones & Jenny Straiton blood, derived from more than 12,000 blood samples. Algorithms searched the transcriptome for disease-specific patterns – a process REFERENCES known as machine learning. Based on this pattern recognition, 1. Zhao H, Bai Y. Liquid biopsy in tumors: opportunities and challenges. Ann. Transl. Med. 9(Suppl. 1), S89 (2018). further data were analyzed and classified by the algorithms. The 2. Webb M, Manley K, Olivan M et al. Methodology for the at-home collection of urine samples researchers observed a hit rate of 99%, with one algorithm being for prostate cancer detection. BioTechniques 68(2), 65–71 (2020). 3. Arasaradnam RP, McFarlane MJ, Ryan-Fisher C et al. Detection of colorectal cancer (CRC) particularly accurate [17]. by urinary volatile organic compound analysis. PLoS One 9(9), e108750 (2014). 4. ClinicalTrials.gov. PAN-study: Pan-Cancer Early Detection Study (PAN) (2018). https:// As AI technology continues to develop and emerge on the clinicaltrials.gov/ct2/show/NCT03756597 healthcare scene, it is important to recognize the benefits of using 5. Alfattani D. Clinical utility of autoantibodies in early detection of breast cancer. Presented at: NCRI Cancer Conference. Glasgow, UK, 3–5 November 2019. it alongside, not instead of, conventional human approaches. While 6. Li B, Janku F, Jung B et al. Ultra-deep next-generation sequencing of plasma cell-free DNA stressing the huge benefits in terms of cost and pressure reduction in patients with advanced lung cancers: results from the Actionable Genome Consortium. Ann. Oncol. 30(4), 597–603 (2019). for healthcare systems upon the introduction of AI, it should be 7. Oncology Central. The future of liquid biopsies: an interview with Bob Li (2019). www. concluded that the best tactic would be to view the two approaches oncology-central.com/spotlights/spotlight-on-liquid-biopsies/the-future-of-liquid-biopsies- an-interview-with-bob-li/ as synergistic. 8. Stobbe MD, Thun GA, Diéguez-Docampo A et al. Recurrent somatic mutations reveal new insights into consequences of mutagenic processes in cancer. PLoS Comput. Biol. 15(11), As an example, when highlighting the potential benefits of the e1007496 (2019). technology in AML diagnosis, Joachim Schultze (DZNE) commented: 9. Centre for Genomic Regulation. New classification system for tumours can guide diagnosis and treatment options for cancer (2019). www.crg.eu/en/news/new-classification-sys- “With a blood test, as it seems possible on the basis of our study, it is tem-tumours-can-guide-diagnosis-and-treatment-options-cancer conceivable that the family doctor would already clarify a suspicion of 10. Sanders AD, Meiers S, Ghareghani M et al. Single-cell analysis of structural variations and complex rearrangements with tri-channel processing. Nat. Biotechnol. doi:10.1038/s41587- AML. And when the suspicion is confirmed, the patient is referred to 019-0366-x (2019). 11. Mathias Jäger. Faster, cheaper and more detailed. EMBL etc Science. EMBL Heidelberg, a specialist. Possibly, the diagnosis would then happen earlier than it Heidelberg, Germany (2019). https://news.embl.de/science/new-method-cancer-diagnosis/ does now, and therapy could start earlier.” [18]. 12. Liu X, Faes L, Kale AU et al. A comparison of deep learning performance against healthcare professionals in detecting diseases from medical imaging: a systematic review and meta-analysis. Lancet Digital Health 1(6), E2710–E297 (2019). FUTURE PERSPECTIVE 13. Dembrower K, Liu Y, Azizpour H et al. Comparison of a deep learning risk score and standard mammographic density score for breast cancer prediction. Radiology 294(2), 265–272 When these techniques will make it into clinical practice is hard to (2020). 14. McKinney SM, Sieniek M, Godbole V et al. International evaluation of an AI system for predict. Breath biopsies are currently in clinical trials [4] and in time breast cancer screening. Nature 577(7788), 89–94 (2020). may become a regular occurrence at a primary care checkup. Genetic 15. Strom P, Kartasalo K, Olsson H et al. Artificial intelligence for diagnosis and grading of prostate cancer in biopsies: a population-based, diagnostic study. Lancet Oncol. doi 10.1016/ testing may currently be limited to the lab, but as sequencing S1470-2045(19)30738-7 (2020). techniques such as scTRIP develop, it seems inevitable that the 16. Yamamoto Y, Tsuzuki T, Akatsuka J et al. Automated acquisition of explainable knowledge from unannotated histopathology images. Nat. Commun. 10(1), 5642 (2019). jump from bench to bedside won’t be too far behind. As for AI, the 17. Warnat-Herresthal S, Perrakis K, Taschler B et al. Scalable prediction of acute myeloid leukemia using high-dimensional machine learning and blood transcriptomics. iScience integration of computational methods into all aspects of healthcare 23(1), 100780 (2020). is happening at a rapid pace. 18. German Center for Neurodegenerative Diseases. Artificial intelligence tracks down leukemia (2019). www.dzne.de/en/news/press-releases/press/artificial-intelli- Given the vast and complex nature of cancer, it seems unrealistic gence-tracks-down-leukemia/ to imagine the grand promises of Trump or Biden coming to light. A 19. Owlstone Medical. Breath Biopsy – VOC Biomarkers on Breath (2020). www.owlstonemed- ical.com/science-technology/breath-biopsy/ single cure for all cancers is likely a pipe dream; however, with more 20. Abderrahman B. Exhaled breath biopsy: a new cancer detection paradigm. Oncology Cen- sophisticated methods that can aid early diagnosis, cancer patients tral, London, UK (2019). www.oncology-central.com/subject-area/emerging-technologies/ exhaled-breath-biopsy-a-new-cancer-detection-paradigm/ can have a better chance of finding the correct treatment, specific to their unique form and presentation of the disease.

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FV3000_Expectmore_201912_16_8x10_B.indd 1 12/30/19 11:36 AM Advert template.indd 164 28/02/2020 10:24:16 News in Brief Jailing Jiankui: a barrier to progress or a vital verdict? He Jiankui, the researcher responsible for the CRISPR-edited babies, has been sentenced to 3 years in prison.

Tristan Free

When He Jiankui (formerly Southern University of Science and Technology; Shenzhen, China) announced that two babies had been born after the successful in vitro fertilization (IVF) of CRISPR-edited embryos, the scientific community was appalled and its outcry immediate. His experiments had breached the fundamental principles of ethical genetic practice and the slippery slope to ‘designer babies’ seemed to be receiving extra grease. Now just over a year later, after He’s suspicious absence from any public forum, the biophysicist and founder of multiple genetics companies has been sentenced to 3 years in prison, alongside

a hefty 3m yuan ($430,000) fine. The result GOOGLE.COM follows a trial kept private from the public He Jiankui in a court in southern China’s Shenzhen City. He was sentenced alongside fellow healthy individuals can avoid contracting recovery after stroke. The key takeaway from researchers Zhang Renli and Qin Jinzhou HIV and a few simple precautions could this is not that the twins will be super intel- (both of unnamed medical universities), have dramatically decreased the risk of ligent, but rather that – as any researcher who received charges of 2 years in prison the children catching the disease in later working on the translation of discoveries and and a 1m yuan fine and an 18-month life. Therefore, the CRISPR editing step of results in animal models into human thera- suspended sentence and a 500,000 yuan the process seems entirely unnecessary to peutics will know – this gene could poten- fine,respectively. protect the health of the mother or children tially impact human cognition but with varying But was this initial public outcry and the and instead adds in another step at which differences to the impacts in mice. subsequent sentencing justified? Is there complications could occur. What’s more, as discussed in a previous another angle to this story? The mutation induced by the He team BioTechniques article, mutations in the CRR5 He’s work was designed to enable in the children brings further issues to the gene have since been shown to bear a 28% couples with one partner infected by HIV fore. A key part of the HIV infection process increase in the risk of mortality before the to have children via IVF that were immune involves the virus binding to the protein age of 78. While this stat may not bear too to HIV and therefore would not be born with product of the CCR5 gene. This gene is much impact on the lives of the twins in China, the virus. The babies were born seemingly sometimes found mutated in some people, where the average life expectancy is 76, these healthy and two parents who would essentially inferring resistance to HIV. He examples demonstrate the fact that there are otherwise not have been able to have a child targeted this protein with CRISPR claiming a huge amount of unknowns in the effects of now had twins. On the face of it this seems to recreate this natural mutation. However, the mutations induced in these children. Who all great news, right? Fyodor Urnov of the University of California can tell what we will discover about the CRR5 Well… not quite. As the couples involved (CA, USA) states that this claim was a “delib- gene mutation next? in the experiments each consisted of erate falsehood” and that the mutation a healthy, HIV free mother and a father induced in the gene was entirely novel with HIV, whose sperm was “washed” to and was likely accompanied by additional remove HIV, there is no reason why this mutations at other locations in the genome. IVF treatment could not have been admin- At the time of the experiment, He should istered without a risk of the mother or the have been aware that mutations in CCR5 bear developing embryos contracting the virus. an impact on cognition in mice, making them …more on this story at Furthermore, there are many ways that more intelligent and also improving brain bit.ly/BTN_CRISPRbabies

There’s nothing Could genetic testing become a part of crabby about RNA routine healthcare? sequencing A new study aims to assess the feasibility of incorporating Researchers have utilized genetic testing into the primary healthcare setting. the neural networks of crabs to validate the Caitlin Killen use of RNA sequencing for the identification of single neurons.

Caitlin Killen

A team of researchers, led by David Schulz (University of Missouri, MO, USA), have demonstrated the ‘clawsome’ power of RNA sequencing for the anatomical and functional identification of single neurons. The use of RNA sequencing is

widespread for the identification of PIXABAY cells in the nervous system. However, previous classification studies have Led by Ros Eeles (Institute of Cancer is psychologically acceptable to patients, and been impeded by other features of Research, London, UK) and Michael can alter the way they are managed by their GP,” the cells. This work aimed to validate Sandberg (90 Sloane Street, London, UK), a explained Eeles. the accuracy of single-cell transcrip- pioneering study aims to evaluate the practi- “The project will give us crucial information tional profiling for the identification of cality and potential benefits of utilizing about whether genetic screening in primary individual neurons. genetic testing to assess an individuals’ risk care could be feasible, and how we should go Speaking to BioTechniques, Schulz of developing a given disease. about seeking to implement it within the NHS,” commented, “RNA sequencing is, and With the current popularity of direct-to- Eeles added. will be, used not only to understand how consumer genetic tests, such as 23andMe and The first 20 patients to join the study will neurons work under typical conditions – AncestryDNA, public interest in genetics is at an receive a psychological evaluation, to assess and what makes them distinct from one all-time high. Such tests are often inaccurate and the impact of genetic screening and its accept- another – but also to shed light on how make unsubstantiated claims, highlighting the ability to patients. All participants will receive development, growth, learning, injury, importance of integrating a properly controlled echocardiograms to ensure a full understanding and disease change neurons over the and verified genetic test into primary healthcare. of the patient’s health, but also to put those who lifetime of an individual.” The scheme, known as the 90S study, have demonstrated genetic risk of heart disease The team now hopes to utilize the launched on 6 December 2019, and aims at ease that their current health is good. knowledge gained from this study to to assess the suitability of whole-genome The study will be comprised of two groups, determine how neurons are affected in sequencing in healthy patients, with the hope half of which will have a family history of cancer spinal cord injuries. of aiding the early diagnosis of ailments such or heart disease; half will not. It is hoped that as cancer and heart disease. The study aims other GP practices will be incorporated into the to sequence the genomes of approximately study, widening the pool of study participants. 1000 general practitioner (GP) patients, who The primary aim of the study is to identify will be recruited from the private GP surgery, potential methods for the simplification and 90 Sloane Street. improvement of existing processes in order “Our new initiative takes cutting-edge to incorporate large-scale genetic screening science on the genetics of disease into a into the NHS. primary care setting, by sequencing patients’ “Working in partnership with experts at The entire genomes from samples taken at a GP Institute of Cancer Research and The Royal surgery and testing for the presence of 600 key Marsden (London, UK) means we can integrate genetic alterations. What we hope is that genetic whole-genome sequencing into screening in screening is practical as a way of picking up primary care with the genetic support that is

PIXABAY genes associated with cancer and heart disease, essential,” commented Sandberg.

Vol. 68 | No. 3 | © 2020 Future Science Ltd 16 www.BioTechniques.com Bioengineering protein assistants to help out CRISPR Custom-built ‘editorial assistants’ allow CRISPR to edit DNA previously hidden by chromatin, opening up new avenues for disease research.

Francesca Lake Here at BioTechniques, our editorial assistants to certain target sites in 2016. The Haynes CRISPR to edit a luciferase gene, whilst not are vital to ensuring our editors can maximize group has since been working on methods themselves affecting transcription. These their efficiency. Now, a new study from the to circumvent this blockage, along with the AAPs could, therefore, be harnessed as Karmella Haynes group at Emory University other challenges chromatin poses – its struc- editorial assistants and adapted to target (GA, USA) has applied that vital editorial tural complexity and variability can also pose different genes through switching out of the assistant–editor relationship to that famous problems for consistency in results. DNA-binding region. but as-yet imperfect DNA editor, CRISPR. In their latest study, the team demon- “The idea is that if CRISPR needs to bind In this case, the editorial assistants are strated that DNA-binding transiently in the middle of a gene but can’t bind close DNA-binding proteins co-delivered with expressed activation-associated proteins enough to edit the mutation, you could send CRISPR, which move chromatin packaging (AAPs) disrupted chromatin, enabling in our chromatin-opening protein to right out of the way and enable CRISPR to access outside that hard-to-bind region, rearrange genes of interest that have heretofore been The team the chromatin, and make the DNA across that inaccessible. These genes include those of gene more accessible for CRISPR to edit,” interest to researchers for developing gene- demonstrated that Haynes explained. based solutions for diseases. DNA-binding transiently The team now hopes to determine Chromatin is an RNA–DNA–protein expressed activation- whether effectiveness is dependent upon complex that epigenetically controls chromo- the type of the AAPs used or the target gene somal organization and gene expression, and associated proteins of interest, and whether combining proteins was highlighted as a block to Cas9’s access disrupted chromatin… might improve results.

Harnessing NGS to prevent HIV drug resistance An in vitro NGS assay for robust detection of HIV-1 drug resistance mutations has been granted FDA authorization.

Francesca Lake

HIV drug resistance is rising both in and decreased labor requirements of the developed and developing countries. With new assay also reduce likelihood of errors. 1.1 million currently infected and 40,000 “It’s all about being able to offer a quicker newly infected patients a year in the USA turnaround, better data, better efficiency and alone, there are ongoing efforts to stem it – better reach, so the disease can be better for example, the WHO began a 5-year global managed and controlled,” explained Sam action plan in 2017. Dajani, CEO of Vela Diagnostics. These efforts extend to the laboratory, In addition to now being able to apply ® and now the Sentosa SQ HIV-1 Genotyping PIXABAY the assay in the USA, it is also approved Assay, developed by Vela Diagnostics (NJ, in Europe, Australia and Thailand. Vela USA), has been approved in the USA. The Speaking to BioTechniques, Tim Baxter, Diagnostics is cooperating with various assay uses plasma from those infected with Executive Vice President of Vela Diagnostics, non-profits to help achieve access to the HIV-1 to detect mutations in the protease, explained that the test improves on current assay in developing countries. reverse transcriptase and integrase regions gold standards as it covers all three genes in of the POL gene. It is the first commercially one test and provides results immediately; available NGS assay to receive marketing the current standard test covers only reverse authorization from the US FDA (MD, USA), transcriptase and protease. What’s more, the Read more news at and is intended to help physicians treat and new test provides a shorter hands-on time www.biotechniques.com/news monitor HIV, thus attempting to reduce this of less than 2 hours, and a turnaround time rise of resistance. of around 2 days. The improved sensitivity

Vol. 68 | No. 3 | © 2020 Future Science Ltd 17 www.BioTechniques.com Top 5 From new protocols and biological understanding to improving lab best practice, here are the 5 most read items in Biotechniques in 2019.

same amount of time as we did when we ments across three independent laboratories. were younger. This is also evidenced in why They conclude: “Taken together, these babies move their eyes around so frequently results demonstrate that with modern – they are processing images much quicker methods pooling triplicate PCR reactions and so move their eyes more frequently to for 16S rRNA amplicon sequencing is more gain more information. expensive and does not provide improvement “The human mind senses time changing over single PCR reactions. This result was when the perceived images change,” explained confirmed in studies spanning three labora- Bejan. “The present is different from the past tories, hundreds of samples and numerous because the mental viewing has changed, not distinct environment types. However, although because somebody’s clock rings. Days seemed these results hold true for the range of condi-

PIXABAY to last longer in your youth because the young tions tested here, there are so many varia- WHY TIME FLIES SO FAST AS mind receives more images during one day than tions in PCR techniques that this type of WE GET OLDER the same mind in old age.” benchmarking effort should be validated for Do you ever feel like time is just See more online at www.biotechniques.com/cell-and- specific sample types and PCR protocols flying by so fast, with days and activities that development-biology/why-time-flies-as-we-get-older/ before a switch from established procedure seemed to last forever as a child going by so is implemented for specialized protocols. quickly now? Well, a researcher believes he For the general sample types tested here, we can now explain the phenomenon, answering recommend using single PCRs rather than the question: Why does time fly by so fast triplicate PCRs. Combined with other technical as we grow older? improvements in miniaturizing PCR reactions, this change in protocol will substantially Looking at the date and realizing we’re now reduce the cost and complexity of amplicon almost a quarter of the way through the year studies.” when the holidays seemed like just last week See more online at www.future-science.com/doi/10.2144/ can leave us dumbfounded – where did all btn-2018-0192 that time go? And, why do the days seem so much shorter than they did when we were

younger? PIXABAY According to Duke University (NC, TRIPLICATE PCR REACTIONS USA) J.A. Jones Professor of Mechanical FOR 16S rRNA GENE Engineering, Adrian Bejan, this apparent AMPLICON SEQUENCING ARE temporal discrepancy is due to our aging UNNECESSARY brains obtaining and processing images Conventional wisdom holds that PCR ampli- much slower. fication for sequencing should employ “People are often amazed at how much pooled replicate reactions to reduce bias due they remember from days that seemed to last to jackpot effects and chimera formation. Is

forever in their youth,” commented Bejan. “It’s that true? GOOGLE.COM not that their experiences were much deeper or NOT-SO IDENTICAL TWINS more meaningful, it’s just that they were being Modern amplicon data analysis employs Do identical twins have the same processed in rapid fire.” methods that may be less sensitive to such DNA? Despite the seemingly As we grow older, our nerves and artifacts. Here, Rob Knight and colleagues identical DNA coding sequence, early differ- neurons also grow, meaning signals must directly compare results from single versus ences in gene expression can make monozy- travel further along these pathways. These triplicate reactions for 16S amplicon gotic twins more different than you would pathways are also degrading as we age, so sequencing and find no significant impact think. the signals are experiencing more resistance. of adopting a less labor-intensive single- These factors lead to a decreased rate reaction protocol. There is a common misconception that of the acquisition and processing of mental They compared single PCR reactions identical twins have identical DNA and, images, resulting in the feeling that time is to pooled triplicate PCR reactions for 16S though they begin development with a passing more quickly because we cannot rRNA gene amplicon sequencing on nearly matching genetic blueprint, differences in view the same volume of images in the 400 samples from a diverse range of environ- gene expression throughout life means that

Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 18 www.BioTechniques.com they differentiate more and more as they immune systems. kDa age. Though still more similar than ordinary That is, of course, until recently when 170 siblings, they are no longer as identical as Claudia Stäubert (University of Leipzig; 130 one may believe. These differences mean Germany), leading a team of colleagues, α-Cas9 that despite their virtually identical identified the fact that humans and great 95 genotype, monozygotic (MZ) twins can still apes have cellular receptors that detect the be phenotypically different. metabolites of these lactic acid bacteria, With genetics often viewed as the be triggering the mobilization of immune cells, all and end all of what makes a person such as monocytes. 43 an individual, it can lead to questions The researchers were primarily focused such as why can variation occur in two on the study of hydroxycarboxylic acid α-Actin people originating from the same genetic (HCA) receptors, found in the cellular 34 material; what can make identical twins membrane. The study focused on the fact © 2019 VÉRONIQUE KRUYS different and when does this differen- that monocytes from humans and great 12 tiation start? You could also ask; how apes contain a HCA receptor type additional GUIDELINES FOR OPTIMIZED different can two twins get? Do MZ to the standard two variations present in GENE KNOCKOUT USING twins have to be the same gender? Do the majority of animals. This more coveted CRISPR/CAS9 identical twins have identical fingerprints? receptor was found to bind D-phenyllactic CRISPR/Cas9 technology has evolved as the Genetics, as with all fields of science, can acid, a lactic acid bacteria metabolite, which most powerful approach to generate genetic be complicated, and with thousands of signals the presence of the bacteria to the models both for fundamental and preclinical genes interacting with personal environ- immune system. research. This review provides a compre- ments, differences in development lead to The authors of the paper, published in hensive look at the latest tools and each individual having their own distinct PLoS Genetics, suggest that this interaction techniques. character, identity and look, even if that was established at some point in our evolu- distinction is only slight. tionary history between the evolution of Despite its apparent simplicity, the outcome See more online at www.biotechniques.com/omics/ hominids from other mammals in order to of a genome-editing experiment can be not-so-identical-twins/ signal the presence of lactic acid bacteria substantially impacted by technical param- in foods. This provides a clear signal for eters and biological considerations. Here, foods that are beginning to decay, and so we present guidelines and tools to optimize the activation of the immune system at CRISPR/Cas9 genome-targeting efficiency these times would impart a clear survival and specificity. The nature of the target advantage. locus, the design of the single guide RNA Discussing the implications of the and the choice of the delivery method should study results Stäubert stated that, “we all be carefully considered prior to a genome- are convinced that this receptor very likely editing experiment. Different methods can mediates some beneficial and anti-inflam- also be used to detect off-target cleavages matory effects of lactic acid bacteria in and decrease the risk of unwanted humans. That is why we believe it could serve mutations. Together, these optimized tools as a potential drug target to treat inflammatory and proper controls are essential to the diseases.” assessment of CRISPR/Cas9 genome- The next step for the research team is to editing experiments.

GOOGLE.COM examine D-phenyllactic acid and its inter- The authors conclude: “While several FERMENTED FOODS: actions with the immune system. Further solutions and guidelines to harness CRISPR- ACTIVATING THE IMMUNE study will also be required into the acid’s Cas9-based gene targeting have been provided, SYSTEM effects on other cell types that contain the it is expected that therapeutic, industrial and The mechanism by which lactic acid bacteria, third HCA type, such as adipocytes cells research applications will still place high frequently found in fermented foods, in lung and skin organs. Ultimately, the demand on improving the specificity and activates the immune system has been potential for these bacteria to provide a efficiency of the CRISPR/Cas9 system. As established in a recent study. clinically viable anti-inflammatory effect CRISPR-based gene targeting technology leads to the very exciting prospect of continues to become more sophisticated and A group of bacteria frequently involved in the possible changes in diet that could lead diverse, optimized procedures and quality fermentation of foods, known as lactic acid to a beneficial treatment for conditions of control guidelines should be established.” bacteria, have been associated with health chronic inflammation. See more online at www.future-science.com/ benefits when consumed. The mechanism See more online at www.biotechniques.com/microbi- doi/10.2144/btn-2018-0187 for these benefits, however, are poorly under- ology-virology/fermented-foods-activating-the- stood, particularly the association with our immune-system/

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The Life Science Business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the US and Canada. 60 Seconds BioTechniques caught up with Leslie Biesecker to discuss the issue of genetic exceptionalism and the importance of making genetics open to all

At ASHG 2019 you addressed genetic excep- something that just naturally occurred for you? tionalism. How do you think resolving the issue (LB) When I first started out, I did not have a clue of genetic exceptionalism will benefit the wider about genomics. I can remember going to that field of life science and society as a whole? first meeting in Detroit in 1982, as I mentioned in Leslie Biesecker (LB) My fundamental thesis, as the talk at ASHG. At that stage of my education, I briefly touched on yesterday, is that success in I did not understand a lot of what they were genetics and genomics is when it becomes saying, but it was clear that this was an exciting infused into every biologic discipline and every and dynamic field where people were doing cool facet of medical practice. I think that exception- stuff, and I wanted to be part of that. From this alism acts as a barrier between where we are meeting, as well as what I was seeing in my own now and that vision. I want to break down that medical training in the 1980s, I could see that Leslie Biesecker belief that we are somehow separate or apart or the issue of children with multiple congenital Clinical and molecular fundamentally different from other domains of anomalies (or ‘birth defects’) was a huge and geneticist from the National science or other practices of medicine and poorly understood problem and that this could Human Genome Research healthcare so that genetics diffuses out. I be an opportunity to do something interesting. Institute, MA, USA, and past believe that the more widely genomics and This led me to set out to do clinical genetics. President of the American Society of Human Genetics, genetics are used, in both science and medicine, I had a number of people who really helped MD, USA the more interesting work there will be for genet- me in clinical genetics such as Renata Laxova, icists to do. Other people might feel that the way who was a clinical geneticist at the University of to maintain their domain is to keep it held in. I Wisconsin–Madison (WI, USA), originally from have the opposite view, which is that if it is free The Czech Republic; Brian Hall, a clinical genet- and more widely used, more people will benefit icist at the University of Kentucky (KY, USA) and …exceptionalism from it and there is more interesting work to do. Mason Bar who was at the University of Michigan (MI, USA). These are people who prac- acts as a barrier Beyond the eradication of exceptionalism, what ticed purely clinical genetics because at that between where other challenges do you think are currently time, I thought I wanted to do something faced by genomics and genetics? clinical. Then, as I was exposed to them, they we are now and (LB) We often talk about it in terms of genetic helped me to recognize that there was a huge that vision. and genomic literacy, among the public, research opportunity there. That is when my non-genetics researchers and non-genetic career shifted from a clinical focus to a hybrid health care practitioners. I think we need to be clinical–research focus. Now, I am 100% careful about that, as these are groups of people research and I only see patients as a clinical who are doing great work in different domains. research activity. I do not do regular clinical Therefore, it should not necessarily be our genomics medical care. mission to make them all be geneticists – that The key thing that they showed me was that I is a fool’s errand. What we want to do is make could make a contribution to the scientific liter- genetics useful to them for what they want to ature by doing projects with them. That planted do. That is different from making them geneti- that seed of, “I can identify a question, figure out cists. What we have to do is make the how to address it and then I can write a paper technology, the approaches and the thinking; that will tell people what the answer to this user friendly, accessible and available to people question or problem is.” That is something so that they can do what they want with it and critical for any person who wants to get started we can help them do that. I think it is a very in this discipline. DISCLAIMER positive attitude towards getting genetics and The opinions expressed in this genomics out there and getting it broadly used. Read this interview 60 Seconds are those of the in full online interviewee and do not When you were starting out in your career, did bit.ly/BTN_LBiesecker60Sec necessarily reflect the views of you have any heroes or people that inspired you Future Science Ltd. to go into the genomics field? Or was it just

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Advert template.indd 164 28/02/2020 09:07:15 60 Seconds BioTechniques met with Robert Philibert, CEO of Behavioral Diagnostics, to discuss his new digital PCR test for addiction

Why use quantitative digital PCR in addiction? smoking cessation. Digital PCR allows this tech- Robert Philibert (RP) The challenge in behavioral nique to be done in any laboratory quantitatively, medicine is to be able to ‘measure’. All of quickly, precisely and reliably. psychiatry right now and a lot of medicine is based on self-report. The difficulty with this, Do you need to account for gene variants when particularly in addictions, is that patients are very using your test? reluctant to confide the depths of their despair. (RP) This is epigenetic. And the DNA methylation They wish to become favorably perceived by their is the same in everyone regardless of demo physicians and families, so they tend to under- graphic. So for instance in smoking your DNA report problematic substance use disorder. methylation is 86%, plus or minus about 2%. This This allows the substance use to grow and locus is not affected by genetic variation – all it is Robert Philibert fester to the point where it causes problems in affected is by your consumption. Thus it is a bias- CEO of Behavioral Diagnostics, their relationships, losses of their jobs, or severe free test. Better yet, it is a dynamic test – as you IA, USA impact for their health. The hard part here is once change, it changes. This gives us a chance to not that has happened, we have lost the strongest only tell how this is impacting your health, but predictor of recovery – strong familial support. show you how much you are benefiting by Early recognition of substance use is essential quitting. to good prevention and effective treatment. What quantitative digital PCR allows us to do is What do you need to advance this research in the What non-invasively take self-report out of it – to take it next 5 years? from the confession of guilt or a confession of (RP) I would say societal engagement. The quantitative failure into just another lab test. hardest thing to realize is smoking and drinking This allows us to treat smoking and drinking do not just affect me, and they do not affect digital PCR just like we do diabetes – as a number. This then doctors, but our entire society. I think we have to allows us to do allows prompt recognition and effective treat this as a bad personality trait and, too, as a treatment. More importantly, like in diabetes shared burden. is non-invasively where a person gets treatment, they get better, In other words, how do we incentivize and take self-report they consume less healthcare cost, and they can persuade people to do the pro-social thing? I can’t function better in their family unit; similarly when do that alone. This invention may lead to the out of it… a smoker quits smoking, everybody wins. They identification of better treatments. It may also win. Their health is better. Their insurance allow physicians and allied health professionals company wins because they pay less. The rest of to more effectively implement the treatments society wins because they have someone who’s they have, because now they can absolutely more productive in the work environment. Their determine smoking and alcohol consumption. employer wins because they have a more To do this, we’ve got to push this through the productive employee. FDA, and that isn’t going to come cheap. Doing So, this allows us not only to go from a clinical trials, obtaining CBT codes, persuading its mechanism of “well, you shouldn’t smoke” to, adoption and changing the clinical paradigm is a how can I persuade you to do a very pro-social tall order, but I think that it is the only pathway. thing and help all of us to quit smoking. This Failure is not an option anymore. We’ve got to cut allows us to use incentive-based methods, healthcare costs. Why not cut out the cigarette because we can absolutely tell how much you’re companies’ contribution to our excessive health smoking, absolutely tell how much you’re care costs? DISCLAIMER drinking, and we can reward you for doing it. The opinions expressed in this Study after study has shown that paying Watch this interview 60 Seconds are those of the people to quit works. It allows the person who has in full online interviewee and do not to do the heaviest lifting to receive a reward. The bit.ly/BTN_RPhilibert60Sec necessarily reflect the views of challenge in these approaches is to be able to Future Science Ltd. quantitate the amount of alcohol or cigarette or

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Biotechniques_Full Page Ad_March 2020_v2.indd 1 2/27/20 8:54 AM 60 Seconds BioTechniques sat down with the Broad Institute’s Anne Carpenter to discuss her work in cell imaging software and how AI is revolutionizing the field

Can you tell us about the exciting work you have considered looking for. You can look at so presented at ASCB 2019? many different combinations of features of cells Anne Carter (AC) I focussed on several really that it can go beyond what we are able to interesting biological stories where we have articulate and engineer an algorithm to specifi- uncovered more in images than biologists cally measure. Letting deep learning algorithms would have expected. For example, it’s difficult loose on images is an unbiased way of taking to tell what stage of the cell cycle cells are in the raw image data and converting it into a when looking at bright-field images. Aside from feature space that can go beyond what we can some very obvious telophase, you generally see by eye. can’t tell whether the cell is in G1 or G2 and whether it’s duplicated DNA or not. Is there information still locked away in cell Anne Carpenter We discovered that if you take the bright- and images that we can’t currently access due to Institute Scientist and Merkin dark-field images of unlabeled cells, there is software limitations? Institute fellow at Broad sufficient information to predict the amount of (AC) We’re definitely just scratching the surface Institute of MIT and Harvard, DNA that each cell has as well as some phases of what is possible to extract in images. For the MA, USA of mitosis. We can also tell the difference most part, biologists have moved from a between a cell that’s in G1 versus G2 versus microscopy experiment being purely qualitative prophase versus metaphase and onward. This and most people do quantify the phenomena …we should was pretty unexpected at the time. that they see. However, I would say the vast Originally, we did the experiment with majority only quantify the phenomena they be seeing a shift classical machine learning. Then we did it with already see by eye. I think we should be seeing towards more deep learning, to provide greater resolution. a shift towards more biologists using Now, we are going to test all kinds of different microscopy as a data source to look for things biologists using disease scenarios to see if this is true across that they can’t necessarily see, but are never- microscopy as the board. theless robust and reproducible. We want to look at whether we can detect, for Because we’re just starting to do this kind of a data source to example, leukemia in unlabeled cells. Leukemia an experiment, I suspect there are tremendous is an example where, typically, pathologists are improvements that can be made in the kinds of look for things required to use specific antibodies that label features that are extracted. Once a computer that they can’t specific biomarkers. It took years to figure out tells you that a set of patient cell lines A is which biomarkers you label in order to tell different from B, the next question that necessarily whether a patient’s leukemia is responding to biologists have is how are they different? That’s see… chemotherapy or not. a question that is actually a little difficult to Now we have this, we need to use these answer at times. antibodies to label the cells, and these panels There are various methods of taking the can be anywhere from four to ten different results of a deep learning model and antibodies. Once again, we discovered that if interpreting them and understanding the you just look at the bright-field and dark-field mechanism or the meaning of them. I think images alone, the cell’s internal structure is that’s a crucial area for improvement so that telling you enough about its state that we can when the computer does find interesting and distinguish leukemic from non-leukemic cells. unexpected things in images, we can translate that into some kind of deeper biological Can you tell us a bit more about the machine- understanding of the system. DISCLAIMER and deep-learning techniques you are using? The opinions expressed in this (AC) Deep learning is definitely causing a Read this interview 60 Seconds are those of the revolution in the computer vision field and in a in full online interviewee and do not lot of commercial applications, but now it’s bit.ly/BTN_ACarpenter60Sec necessarily reflect the views of coming to biology as well. Typically, deep Future Science Ltd. learning extracts features that humans may not

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Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 25 www.BioTechniques.com Call For Papers MEDLINE-indexed, peer-reviewed, open access journal that provides the life science research community with an Want to invaluable resource to access the latest methods, contribute? techniques and protocols. Get in touch today Speciﬁcally looking for the following: · Reviews of laboratory techniques, tools and methods +44 20 8371 6080 · Reports describing new methods, platforms and software · Benchmarks presenting updates to existing [email protected] techniques, or applications of existing techniques to new questions · Cautionary tales concerning experimental design, www.biotechniques.com methodology or analysis

Advert template.indd 164 28/02/2020 09:10:26 Interview

A peek behind the paper: Jeremy Clark on the new method for the at-home collection of urine samples for prostate cancer detection

Take a look behind the scenes of a recent report published in BioTechniques entitled ‘Methodology for the at-home collection of urine samples for prostate cancer detection’ as we ask lead author Jeremy Clark about his new urine test.

Our pilot study which was published earlier this Collection Kit has been really positive. year showed how useful we have found urine to be for diagnosing prostate cancer and predicting What led you to carry out which cancers will get bigger and nastier up to this research? 5 years later. PUR (prostate urine risk) signa- Current clinical tests for prostate cancer can have tures separated men with low-risk cancer into both false positives and false negatives. A better two groups, one of which had a risk of devel- means of detecting cancer, aggressive cancer in oping an aggressive form of the cancer eight particular, is needed. The position of the prostate Jeremy Clark times greater than the other. There is nothing in below the bladder and its secretory function led Norwich Medical School, University of East Anglia, clinical use at present that can do this. us to believe that the answer lay in the investi- Norwich, UK The new development is our ‘At-Home gation of urine. It’s non-invasive and has the Collection Kit’ which means that we can now potential to sample the whole prostate in one go. send out a urine collection kit to a man at home, he fills up a small pot with his first wee of the day What were the key conclusions; and posts it back to us for analysis. what impact do you foresee this This could The at-home collection part sounds like quite research having? “ a small innovation, but it means that in future the This could revolutionize the collection and revolutionize monitoring of cancer in men could be so much analysis of urine samples for prostate cancer less stressful for the patient and reduce the diagnosis and prognosis. The at-home collection the collection number of expensive trips to the hospital. from the first wee of the day has improved the and analysis of The idea behind it is as follows: the prostate is a sensitivity for prostate cancer biomarkers and secretory organ and these secretions carry cells has also improved the robustness of the test urine samples for and molecules from all over the prostate to the as it now does not rely on the efficiency of prostate cancer urethra which then get flushed out of the body on the digital rectal examination which is highly urination. If a cancer is present then tiny bits of variable between clinicians. diagnosis and the tumor are also carried with the secretions and prognosis. we can detect these in the urine. As the prostate What work are you hoping to do ” is constantly secreting the levels of biomarkers next in this area? in the urethra will build up with time. Collecting We have just been refunded for the next stage from the first wee of the day means that overnight of development of the at-home collection PUR secretions can be collected which makes the test by a Movember and Prostate Cancer UK analysis more sensitive and more robust. Innovation award – this will involve recruitment Before this, men would turn up at the hospital, of approximately 1500 men in the UK, Europe and nerves would inevitably mean that they would North America in a 3-year validation study. We have a wee before they saw the doctor and all hope that the at-home PUR test could be used the secretions would be flushed away. To get in hospitals in 5 years’ time. around this, urine was collected after a digital rectal examination of the prostate in the clinic whereby the doctor would stroke the prostate View online article here with their finger pushing prostate secretions bit.ly/BTN_JeremyClark into the urethra shortly before urination. The Interview KEYWORDS patients really don’t like this examination and cancer • diagnostics the feedback we have had from the At-Home

Ge Wang and Margarida Barroso on the HOTGEM imaging technology for breast cancer

BioTechniques spoke to Ge Wang and Margarida Barroso about their recent collaboration to develop a set of novel imaging techniques, known as HOTGEM.

Please describe the nature is ideal for performing material-decom- of your collaboration and the position-based optical heterogeneity Ge Wang Margarida Barroso aim of your study correction and contrast-enhanced cancer Rensselaer Albany Medical Wang: This project is a truly multi-inter- imaging. Optical molecular tomography Polytechnic College, NY, USA disciplinary partnership between RPI, can then be regularized by the background Institute, RPI, Albany Medical College and MARS Bioim- attenuation and tumor location priors. NY, USA aging (Christchurch, New Zealand), a tech In this project, we will customize the company led by Phil Butler. Barroso, Butler, photon-counting micro-CT system into Intes and I are leading investigators in our the HOTGEM system to upgrade whole- respective fields, which are complementary body preclinical imaging to the next level. for hybrid X-ray and optical tomography of Of relevance, our New Zealand collabo- small animal models, an area especially rators have reported that the X-ray photon- important for breast cancer treatment. More counting detection technology has been “The proposed specifically, the overall goal of this project scaled to humans. The fact that a large is to develop a hybrid X-ray and optical bore scanner based on the MARS photon- hybrid HOTGEM prototype for high-dimensional optical counting detection technology is ready system will be tomography guided by energy-resolved for clinical trial dramatically increases micro-CT (HOTGEM), which can be utilized to the potential of our proposed preclinical a breakthrough visualize and quantitate breast tumor hetero- HOTGEM system. for anatomical, geneity, the expression and dimerization of HER2 as well as therapeutic responses in How will the combination functional preclinical models. of these techniques help and molecular monitor the response of Can you describe the novel cancer cells to therapies? tomographic X-ray imaging methods and Wang: Optical imaging is unique in its imaging in small technologies developed for capabilities of providing structural, this study? functional and molecular contrasts from animals.” Wang: With over 12 million New Zealand a wide array of light-tissue interactions. dollars of government funding from 2014 Functional and molecular optical imaging through 2020, Butler’s group is developing generates quantitative information of photon-counting CT technologies for hemodynamics and multiple biomarkers preclinical and clinical applications. Wang simultaneously. Enhanced with lifetime is a major subcontractor of the current New imaging, we can monitor the microenvi- Zealand grant and has been closely collabo- ronment and protein–protein interactions. rating with Butler’s group for many years. Recently, one of the multiple principle Recently, the first photon-counting micro-CT investigators of this project, Intes, and his scanner has become commercially available, team members reported on a novel instru- developed by MARS Bioimaging. This novel mental and algorithmic approach, which technology generates multi-energy images combines modern optics components and with high spatial resolution for simultaneous compressed sensing techniques, to enable quantification of soft tissues, contrast the collection of high content 5D optical KEYWORDS agents, nanoparticles and pharmaceuticals. datasets (space, time and spectrum). This breast cancer • CT scans • imaging The scanner can differentiate up to six new imaging platform, which was featured • x-rays types of materials in a single scan, which in Nature Photonics, is based on two main

Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 28 www.BioTechniques.com innovations. First, comprehensive whole- These imaging receptor expression assessment. This is body imaging can be enabled via structured “ a critical issue in targeted drug therapy, light illumination to acquire hyperspectral technologies can as ex vivo receptor profiling is the main time-resolved data tensors so that method employed in tumor diagnosis and functional (total blood volume HbT [μM] and be applied to a wide therapy. Despite this, targeted drug delivery oxygen saturation StO2 [%]) and molecular variety of cancers…” in vivo may not always correlate with target (fluorescence-based multiplexing, effective expression levels. Our ability to measure quantum yield, and lifetime) parameters target engagement in tumors is crucial to can be quantitatively and tomographically receptor binding in breast cancer xenograft accelerate the prioritization of the most mapped in breast cancer mouse models. tumors in live intact mice. This approach is efficient targeted anti-cancer therapy. Second, a cutting-edge methodology has based on measuring FRET by estimating the Wang: A strong correlation between tumor been developed based on a state-of-the- reduction of the fluorescence lifetime of the heterogeneity, drug resistance and clinical art Monte Carlo forward model for fast and donor fluorophore when it is within 2-10nm outcome suggests that tumor heterogeneity accurate 3D reconstruction. to one or more acceptor fluorophores. Most is key for targeted therapeutics and precision Overall, our novel optical instrumen- importantly, MFLI-FRET can discriminate medicine. Analytical molecular tools, based tation acquires in vivo data over the whole soluble fluorescently labeled ligands from on biopsy or surgical excision, are widely animal body with high throughput. From those bound by dimerized receptors. Hence, utilized to characterize tumor heterogeneity. these datasets, quantitative images can be MFLI-FRET reports on molecular events at However, these provide an incomplete tumor reconstructed for functional parameters, the macroscopic level in live, intact animals. sampling, making in vivo imaging crucial. fluorescence probe distributions, and FRET MFLI-FRET imaging allows the use of any Importantly, the tumor heterogeneity signatures. ligand/homodimer receptor as well as in a subset of HER2-amplified cancers is therapeutic antibodies such as anti-HER2 associated with reduced survival and breast Which therapies will you drugs used currently in the clinic. cancer progression. In order to improve the be testing these imaging Importantly, our results have demon- clinical efficiency of targeted therapies, techniques with and why? strated that MFLI-FRET measurements there are major gaps in understanding the Barroso: Recently, Intes and I utilized a correlate with ligand-receptor binding interplay between HER2 receptor expression 1-2horiz-pushbuttonPRINT.pdfMFLI-FRET platform to quantify 1 2/25/20 ligand- 9:24in AM tumor cells but strikingly, not with level, targeted drug delivery and tumor

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response. Thus, we must simultaneously binding to tumor xenografts in live intact years X-ray photon-counting imaging acquire exquisite tumor’s features, physi- mice, using the novel imaging technologies techniques have been under active devel- ological states and pathological biomarkers, developed in this project. opment and achieved impressive results. which can currently only be, to a limited My expertise in receptor targeted therapy A specific aim of our project is to produce extent, obtained with histology/microscopy. in breast cancer will be utilized to demon- images in terms of attenuation coefficients Our innovative HOTGEM system will strate and validate HOTGEM applicability to as a function of X-ray energy, which enables characterize HER2 receptor expression HER2 cancer studies. We will demonstrate chemically specific material decomposition and dimerization, structural details such the utility of the HOTGEM multimodality and is more suitable for hybrid X-ray and as vasculature and functional param- imaging approach to characterize in detail optical tomography. The proposed hybrid eters including total blood volume and the correlation between receptor expression HOTGEM system will be a breakthrough tissue oxygenation levels in human tumor and dimerization, tumor morphology and for anatomical, functional and molecular xenografts using human cancer cell lines drug sensitivity in HER2-based tumor tomographic imaging in small animals. or patient-derived cells. After validation xenografts. Moreover, our expertise in the There are numerous preclinical imaging using histochemistry and microscopy, we area of tumor cell biology, HER2 cancer applications, including but not limited to will have early response parameters for and tumor heterogeneity will help guide the HER-2 related cancer imaging studies. targeted HER2 therapy. The HER2 receptor application of this novel platform to cancer Upon completion of this 5-year project, the dimerization heterogeneity mapping is a therapy and diagnostics. proposed HOTGEM system will have been novel feature that can only be extracted validated to offer 50μm X-ray resolution for non-invasively using HOTGEM. Therefore, How do you see these material decomposition, as well as 100μm HOTGEM may provide a high predictive studies and the imaging optical resolution for target localization value for HER2-targeted therapy in tumors techniques affecting the in co-registration within 30 minutes for carrying HER2 activating mutations. field over the next 5 years? each hybrid in vivo scan, demonstrated to Wang: In 2014, the first and only demon- be a breakthrough for tomographic HER2 Which therapies will you stration of fluorescence optical tomography imaging. By then, HOTGEM will be ready be testing these imaging guided by X-ray grating-based micro-CT – for technology transfer and commercial techniques with and why? a cutting-edge micro-CT technique that translation. Barroso: These imaging technologies can provides exquisite soft-tissue contrast – Barroso: Recently, a more complex view be applied to a wide variety of cancers since was reported with interesting results but of HER2 cancer has emerged due to they depend on the receptor-mediated major drawbacks, despite increased soft- widespread genomic sequencing and targeted therapies and not on the individual tissue contrast. increased understanding of tumor hetero- cancer location. We have focused on The system consists of separate geneity. Therefore, the combination of HER2-positive breast cancer because of subsystems, has a long acquisition time these imaging modalities into an integrated the HER2-targeted antibody drugs available (over 20 h), performs very limited optical non-invasive pre-clinical approach is crucial in the clinic. These drugs can be labelled measurements, and is only suitable for to further our knowledge of drug kinetics in using different near infrared dyes as well ex vivo X-ray imaging. In contrast to X-ray cancer microenvironments, as well as our as other contrast agents to visualize their grating-based imaging, over the past several ability to assess early drug responses of breast cancer. I am enthusiastic about the potential impact of this instrument that can integrate the ability to measure metabolic tumor Bioanalysis levels with HER2-targeted and vascu- ISSN: 1757-6180 Frequency per year: 24 lature imaging to reveal target availability and accessibility. HOTGEM is a significant breakthrough for tomographic HER2 INDEXING imaging in small animals and may provide high predictive value for HER2-targeted IMPACT FACTOR: MEDLINE/Index Medicus, EMBASE/Excerpta Medica, Chemical Abstracts, BIOSIS Previews, therapy in tumors. 2.321 (2018) BIOSIS Reviews Reports and Meetings, Journal Citation Reports/Science Edition, Science In association with Citation Index Expanded™ (SciSearch®), Scopus®

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Inscopix’s Jonathan Nassi and Kunal Ghosh on their cutting-edge neuroscience technology samples for prostate cancer detection

At SfN 2019, BioTechniques caught up with Kunal Ghosh and Jonathan Nassi from Inscopix to discuss their groundbreaking neurotechnology and how this is impacting the field of neuroscience.

Please tell us about interface that has paved the way to a cloud your technology future, which is important because neuro- Jonathan Nassi Kunal Ghosh Ghosh: The core invention was the miniature science is entering the age of big data sets. Senior Lead CEO at Inscopix, integrate microscope, or Miniscope, that A typical user in one of our customer labs Scientist at CA, USA myself and my collaborators at Stanford will generate a couple of terabytes of data Inscopix, CA, USA invented. We were inspired to build a head- in an afternoon from the Inscopix platform. mounted, wearable device for freely behaving We noticed that early on and have invested subjects that would provide researchers the in building up the platform and the data ability to look at large numbers of neurons acquisition in order to enable streaming with single-cell resolution. directly to a web-based interface, to transfer In neuroscience, electrophysiology and the data to the local network and in the future We are trying electrophysiology-based tools have been a do a cloud-based infrastructure for data “ mainstay in terms of trying to record brain storage analysis. to reduce and activity either through single cell, looking That’s a little bit on the platform itself and take down at a few cells at once or looking at average then of course there’s the entire workflow, recordings to determine brain state, as we which Jonathan has had significant contri- those barriers get with electroencephalography. However, butions in innovating; that has helped to get and expand the these methods do not make it possible to the prep more turnkey. At Inscopix, we are look at the individual neural activity of a large now manufacturing our own reagents and community to number of cells at once. we really incentivized this to try to provide We addressed this gap with the miniature customers collaborators with data as quickly allow for this integrated fluorescence microscope, which and routinely as possible. kind of parallel allowed us to look at the activity of hundreds There is analytics on the other end, which science. of cells with single-cell resolution and is where a lot of the magic happens. You ” also target specific cell types with geneti- might get amazing data sets but what does cally encoded fluorescent indicators. This it all mean? That is, of course, an area of technology differed to traditional electro- continued innovation for us. physiology-based recording technologies as now, not only can we look at the large popula- How are you making this tions of neurons with single-cell resolution, platform accessible to users? but we can also have cell-type specificity Nassi: We provide the full workflow solution by virtue of the labeling. There is an almost to carrying out these kinds of experiments, perfect confluence of traits and attributes including methods, analytics and the that make this technology truly enabling for technology itself. I think that is key, because mapping brain circuits. neuroscience has already moved into this That miniature integrated microscope is generation where these kinds of circuit- the core of the platform, but now it’s coupled level neuroscience experiments are very with a powerful data acquisition box based multidisciplinary. on a graphics processing unit (GPU), which Some of the big labs may already have all KEYWORDS enables streaming of the data directly to of the technology and expertise needed to, Inscopix • neuroscience • SFN19 any web browsers. It’s a network-based for example, express new genes in cells to

monitor their calcium activity by causing …so little is into drug development applications, is to them to be fluorescent. However, at Inscopix “ help to provide a much more predictive we are reaching well beyond those labs to, known about how path to clinical trial. Our technology allows perhaps, a lab that is just studying behavior researchers at the preclinical phase to in rodents and has seen our technology and the brain works … understand not only the disease phenotype related experiments and thought ‘I would that therapeutic at the circuit level, but also how the drugs love to do this, but it’s not that possible’. We work to restore the circuit. If they do not are trying to reduce and take down those development in restore the circuit, then that indicates the barriers and expand the community to allow neuro is really drug should not be advanced. for this kind of parallel science. challenging. Most preclinical models in the past have That requires streamlining the front end ” relied on behavior and the behavior has to make it an easy, one-step surgery and turned out to be a very poor predictor of the back end to provide powerful analytics. done to translate this from a rodent model clinical efficacy. Our overall goal is to try to That’s a key need that you sometimes don’t into a nonhuman primate model. provide a much more sophisticated, higher appreciate from the outside, but I think it has Other than that, the focus of the trans- dimensional, circuit-based readout across been one of our main motivators. lation work is developing some of the mental illness diseases. Ghosh: I totally agree. As we mature and surgical methods. The platform is so The Astellas collaboration is focused evolve our focus is accessibility and how we powerful as it is that we haven’t had to on understanding psychiatric disorders at can introduce this into every neuroscience make many innovations that are nonhuman the circuit level and then potentially devel- lab with an interest in understanding brain primate-specific. We are able to record from oping circuit-based screens to advance function or dysfunction irrespective of their over 100 neurons in the premotor cortex compounds with much higher predictive animal models. Today we are supporting of behaving macaque, which is an exciting value than anything else possible today. animal models from mice all the way through start to this important work. The collaboration with the Broad Institute to macaques. In this study, we were able to mount two (MA, USA) is related to Parkinson’s. This I think increasingly our focus is making microscopes on the head, which, given the involves both methods exploration and the things turnkey, thinking about the workflow larger size brain, means we can start to identification of potential new targets. The and making this platform as accessible as understand multi-area communication. The methods side of this involves combining possible so it empowers the entire neuro- complex behaviors that we are looking at in what we are doing to identify specific cell science community across the world to truly the macaques are represented by circuits types and implicating it in disorders like help decode the brain. widely distributed across the brain, so we Parkinson’s. Then, together with Broad, we are excited about the fact that we are able will perform single-celled transcriptomics You mentioned that the to put multiple microscopes on the head and with the goal being to try to identify new animals are freely behaving. really understand multiple brain regions in druggable targets. It’s a privilege to be able How easy was that to that context. to collaborate with them on this project. translate up into macaques? Nassi: We have over 100 publications now Nassi: In rodent models it’s completely Can you tell us more about and a lot of them are very fundamental, freely behaving. Currently in macaques the partnerships you’ve basic neuroscience discoveries advancing it’s a less restrained situation than typical recently entered into, our understanding of how the brain works. macaque experiments but not as free as in particularly the ones with More recently, though, we are seeing more the rodents. Most macaque experiments, Astellas and Broad? publications reporting translational neuro- especially those with any kind of benchtop Ghosh: With respect to Astellas (Tokyo, science discoveries that demonstrate how microscope imaging platforms, are done Japan), the overall goal here is to start to this kind of data could start to inform thera- with head fixation. It’s very challenging understand the neural circuits implicated peutic development. to align and to keep everything stable in different psychiatric disorders alongside For instance, we have a really nice study otherwise. where social or behavioral deficits stem out of Stanford University that involved In macaques, we do enable head-free from. The objective, of course, is to facilitate imaging in the striatum of the mouse, which behavior but we still have to have some a much more mechanistic process for drug is a key area for motor behavior and is a restraint because there is a cable on the development. key site in humans, as it is where we know microscope, so we can’t let the macaques Today so little is known about how the that dysfunctions lead to a lot of the motor roam free and potentially grab it. However, brain works, let alone how it doesn’t work deficits associated with Parkinson’s. we are working on future innovations to in the context of mental illness and brain With our Inscopix technology, they could overcome this. disease, that therapeutic development in image directly from specific pathways in The core platform is the same but there neuro is really challenging. a mouse model of Parkinson’s, where are some things, like the viruses that were Most clinical trials fail because of a lack dysfunction is known to be linked to the used to express these genetically encoded of efficacy. Our goal, as we think about disease, and for the first time be able to see calcium indicators, where work had to be translating the technology and the data and understand what’s happening in the

Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 32 www.BioTechniques.com disease. That paper found some interesting potentially lead to new druggable targets. not charge for this service so it’s our model neural circuit signatures of Parkinson’s It’s just the early days of doing that, but I of team science, if you will. disease dysfunction, including imbalances think a lot of people are seeing the power We provide talented scientists from between two important pathways. of the single-cell functional imaging that Inscopix as resources that our customers There are things that you would not have we are providing. There have also been and collaborators can talk to and get been able to pick up with electrophysiology, some amazing advances in single-cell RNA help from with methods troubleshooting, but because we’re now seeing for instance, profiling, and the Macosko lab at the Broad experimental design or data analysis. at a population level, spatiotemporal Institute has been one of the leaders in that. We believe that having someone next clustering of activity, we can discover inter- We are excited to start to look at these to you in the lab, working with you to solve esting neural circuit signatures, both in the two datasets together and explore ways that those problems is a huge catalyst in our normal and diseased state. Once you have together they can increase the value, in ways customers’ and collaborators’ success. But defined this, you can start to look at adminis- that they may not be able to individually. it also really humanizes the entire endeavor tering some compounds, such as dopamine and makes the journey fun. agonists, to see if these compounds can Is there anything else that All of this is an investment that Inscopix resolve the circuit level dysfunctions. you have ongoing at Inscopix is making in science. It’s our belief that One key thing that came out of that study that you are particularly this is key to making this field scientifically was that at the behavioral level you could excited about? successful. As a society, we seem to be administer almost any compound and, Ghosh: I think, in closing, the only thing I moving into a sharing-based economy and at the right dose, correct the abnormal will add is that as a company, we are now science is increasingly being less individual behavior that is the common readout in extremely focused on making the core driven and more team driven. I think the next these models. At the circuit level, though, platform as accessible as possible to the generation of scientists will be much more only L-Dopa – which is one of the most widest number of researchers across collaborative and driven more by shared clinically efficacious drugs out there – the world. A key part of this, of course, is goals than the desire to be the first to publish was able to completely rectify the circuit- making the technology easy to use and as and receive all the credit for a certain study. level dysfunctions. Of course, L-Dopa has streamlined as possible. We want to help catalyze that movement side effects and there are lots of efforts to because there are hard problems to solve replace it with better drugs. and we cannot solve them individually. We That case really highlighted the power As a society, we need to work in teams across stakeholders, of these circuit readouts in these kinds of “ companies, academics and governments. disease circuit-level experiments. That’s seem to be moving So, it’s really fun to be able to contribute to the kind of stuff that we are looking to do in this philosophy of team science. collaboration with Astellas in the context of into a sharing-based neuropsychiatric diseases and we are very economy and science USEFUL LINK excited about that. • Society for Neuroscience 2019 meeting Our collaboration with Broad is also is increasingly being highlights: https://www.biotechniques. very much in the context of that Stanford less individual com/opinion/society-for-neuroscience- paper on Parkinson’s. Stanford researchers 2019-meeting-highlights/ found these interesting, cell-specific differ- driven and more ential effects between these two different team driven. pathways. Then, of course, one of the ” questions that comes out is how can we effectively target one or both of those pathways? Another part of it is the human element There are lots of different therapeutic because, at the end of the day, science modalities out there; some are devices for is as much a lab-based exercise as it is a stimulating cells and maybe one can target human exercise. We strongly believe that those specific pathways. The pharmaceu- as a company we should also partner tical industry is thinking about creating with our scientist customer collaborators drugs to target specific receptors and and as such we have built a field scientific molecules or molecular pathways. consulting program. We can now make that transition from This is a team of almost 15 scientists, Read this interview these amazing functional datasets back to many of whom used Inscopix platforms in full online the molecular level through single-cell RNA when they were students themselves and bit.ly/BTN_NassiGhosh profiling and attempt to identify specific have published with the platform, who are Interview molecular pathways that relate to the now working hand-in-hand with users across measured circuit function. This could then the world to make them successful. We do

What are the off-target effects of CRISPR and why are they concerning? Gaetan Burgio of the Australian National University discusses the off-target effects of CRISPR, how they occur, how to avoid them and their impacts on the uses of the technique.

Written by Gaetan Burgio

RISPR gene editing technology has enzyme scans and unwinds the DNA to create been transformative in investigating an R loop, changing its conformation to C important biological questions or expose the nuclease domains and cleaving studying the function of genes. Importantly, the DNA to generate a double-stranded break the technology shows a lot of promise in of the DNA. The specificity of the guide RNA improving crop or food production, and in is critical to enable Cas9 to cut the right target detecting or treating diseases such as cancer, and to avoid cutting another piece of DNA (off infections or severe target). Therefore, the hereditary disorders. To combination specificity of date, 23 active clinical Ensuring safety the enzyme/guide RNA is trials at various stages of for gene editing crucial to avoid occurrence Gaetan Burgio progression using of off-target effects. A series Australian National University, CRISPR gene editing applications, of studies has demon- Canberra, Australia technology are regis- such as treating strated that Cas9 is sensitive tered in ClinicalTrials. to mismatches of the guide gov. Most of the diseases medical RNA, which in short means investigated are cancer conditions, is that Cas9 is a highly specific related, such as a enzyme. Additional recent recently published paramount for studies further refined Cas9 clinical trial on a patient a successful specificity and have shed with HIV and leukemia or light on how off-target severe hereditary condi- treatment. effects occur. Briefly, Cas9 tions such as sickle cell binds and hybridizes to anemia, highly prevalent in the African– off-target sites, but fails to generate a break American population. The use of CRISPR gene in the DNA. In short, this means that Cas9 is editing technology could potentially lead to highly sensitive to the nucleotide composition transformative changes, resulting in cure or at the target site, which means that off-target diagnosis of diseases, and improved food or effects are therefore predictable. fuel production. Ensuring safety for gene editing applications, such as treating medical HOW CAN WE PREDICT conditions, is paramount for a successful OFF-TARGET EFFECTS? treatment. Ultimately, it will improve lives. Considerable efforts have been undertaken Therefore, it is important to determine whether to better understand and predict how Cas9 off-target effects are a concern for gene off-target effects occur. Development of high- editing applications, the acceptable level for throughput sequencing technologies has gene therapy or food production, and how to largely contributed to improvements in the alleviate them. This short overview of the detection and prediction of off-target effects. question provides an overall picture of this Some of those are specifically dedicated to question and provides guidance on how to detecting off-target effects such as GUIDE- detect and minimize off-target effects. seq, Digenome-seq, Circleseq or DISCOVER- seq. In addition to whole-genome sequencing HOW DOES THE CRISPR-CAS9 ENZYME techniques, those techniques have estab- GENERATE OFF-TARGET EFFECTS? lished that Cas9 off-target effects are relatively Cas9 is a bacterial enzyme that uses a guide rare and detectable. In parallel, surveys on the RNA scaffold to direct the DNA cutting. The effect of Cas9 from over tens of thousands of

guide RNAs have defined and refined how organisms, the efficiency of the approach to predict efficiency and off-target effects will be prioritized over safety as the Ready for from a guide RNA nucleotide sequence on researcher has the possibility to eliminate a specific organism. These techniques, off-target effects in subsequent genera- combined with artificial intelligence such tions. Overall refinement of the gene- Any Assay as machine learning, now give an accurate editing technology and the introduction landscape of the frequency and the of novel techniques will enable us to fully likelihood of Cas9 off-target cutting. Today, eliminate off-target effects and ensure a this enables us to predict efficiency and safe approach in the use of gene-editing occurrences of off-target effects with high accuracy. The balance is HOW CAN WE MINIMIZE OR OVERCOME OFF-TARGET EFFECTS? to determine how Two factors are important to consider in acceptable CRISPR reducing off-target effects. First, controlling off-target effects are Combine cell imaging the timing of Cas9 delivery and reducing and multi-mode detection Cas9 availability is critical to avoid them. from mutations that For example, delivery of Cas9 in a form of in one instrument. ribonucleoprotein in a complex with the naturally occur in the 3D cell culture guide RNA has a short half-life compared genome. with plasmid delivery and, consequently, Colony counting Cas9 degrades rapidly in the cell, which in Nucleic acid quantification turn reduces the likelihood of generating technology for therapy or food production. Quantitative live cell imaging an off-target effect. Another important I would therefore argue that, given the Biochemical assays aspect in the reduction of off-target effects evolution of CRISPR technology, off-target Label-free cell counting is the choice of reagent. Some Cas9 effects are not such a concern. For a given Histology variants such as eSPCas9 or HFCas9 offer application, definition of the acceptability Calcium flux high fidelity and low-to-null frequency of level for off-target effects will be key to off-target effects. More importantly, the determine the suitability of using CRISPR Apoptosis & necrosis guide RNA design is critical in reducing gene-editing technology. Cell proliferation off-target effects. A good design using Cell migration & invasion online available guide RNA design tools DISCLAIMER Cell viability & toxicity such as CRISPOR, CCTOP or GtScan The opinions expressed in this Opinion are Confluence predicts quite well the frequency of those of author and do not necessarily Fast kinetics off-target effects for a given guide RNA reflect the views of Future Science Ltd. Genotoxicity sequence. Immunofluorescence ARE OFF-TARGET EFFECTS Microbiology REALLY SUCH A CONCERN? Phenotypic assays Accumulation of a large amount of Stem cell differentiation evidence suggests that Cas9 off-target Transfection efficiency effects are minimal and should be more Slide scanning or less considered for a given specific ELISpot imaging application. For gene therapy or food Whole organism imaging production, off-target effects are critical and good guide RNA design combined Normalization with the use of highly specific Cas9 and Phagocytosis cutting-edge sequencing technologies will Signal transduction ensure the safety of the approach and Translocation ultimately will prioritize safety over efficiency. The balance is to determine Read this opinion www.biotek.com/cytation how acceptable CRISPR off-target effects in full online are from mutations that naturally occur in bit.ly/BTN_BurgioOpinion the genome. However, for studying biology by the generation of genetically modified

2020 Cytation ad 0ne-third-Biotechniques.indd 1 2/21/20 11:54 AM Opinion

On high-throughput sample processing and laboratory techniques: concerns, pitfalls and more

Written by Mariana Salas Garcia and Jack Gilbert

enturies of scientific research and revolutionized microbiome studies, enabling development have led to techniques statistical associations between microbial C that have set the foundation for the phenotypes and disease states that were previ- future of automated research with high- ously invisible. Amplicon sequencing was throughput sample processing (HTSP). The enabled at scale purely through high-throughput world is facing challenges from climate change sequencing, providing an extremely low cost per to antibiotic resistance that require collaborative sample for analysis. Indeed, the development of research to find answers and potential solutions. highly curated platforms with robotic automation These large-scale collaborations can generate for generating high-quality sequencing libraries vast sample numbers, into the thousands or tens has enabled rapid turnaround, and improved of thousands, which require substantial infra- standardization and reagent use efficiencies. Mariana Salas Garcia structural innovation to enable efficient and HTSP in any laboratory requires a delicate University of California reliable processing. With evidence now mounting balance between speed, volume and quality. San Diego, CA, USA that longitudinal time-series studies are needed Many recent advances have substantially to understand microbiome dynamics, and to increased the speed and quantity of samples capture relevant microbial biomarkers of that can be processed, but to ensure quality relevance to human or environmental health, the requires advanced skills within the research number of samples required to test next- lab. Akin to having ‘green fingers’ many of the generation hypotheses will only increase. new robots and pipelines require expert handling. There are many known solutions to improve This leads to significant inter-lab variability, the throughput and cost of sample processing. reducing the beneficial impact of standard- HTSP requires the automation of experimental ization. The differences in sample processing components to ensure that large-scale studies quality are most obvious between research become feasible and reproducible, while requiring labs and service contract labs, primarily due less human effort and fewer resources (e.g., to the quality and quantity of the experience of consumables and reagents) to complete. As a the team. For example, if a student researcher Jack Gilbert result, HTSP also accelerates the development of needs to perform a task for which they have little University of California San Diego, CA, USA standard protocols which are essential for repro- experience and lack the appropriate equipment, ducibility and can aid democratization of technol- it can lead to spurious results that introduce ogies and techniques, providing the potential to substantial variability in data output across The hurdles improve the reliability and quality of data. Good studies. Service contract labs with extensive examples of this include the Human Microbiome technical staff typically already have a validated experienced Project and the Earth Microbiome Project, whose protocol ready and automation to handle in laboratory standard protocols have shaped an entire field. samples in a more controlled environment, thus Standardization also facilitates increases in data improving the success and standardization of techniques quality by enabling more robust statistical checks output. The key to success is consistency across and during on the larger datasets. To enable these advan- all protocols, and the use of standards, positive tages, it is essential that we continue to develop controls and quality checks during the process massive sample high-throughput approaches, which means to account for variance within and between large processing still overcoming hurdles, developing detailed records studies, which academic laboratories often lack. of processing steps, creating new collaborations Therefore, collaboration with an experienced indicate the especially across disciplines, and embracing research group or a service contract facility is importance modularity to improve the flexibility of processing necessary in order to avoid pipeline biases and pipelines to accept new technologies. increase the quality of sample processing and of existing HTSP has revolutionized sequencing, data generation. genotyping, cell biology, screening and In a semi-automated HTSP lab, samples are research imaging. The most visible success story is high- no longer manually transferred between 96- skills… throughput sequencing technologies which have and 384-well plates, but instead are processed

Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 36 www.BioTechniques.com using liquid-handling robotics, which can also handle PCR, amplicon quantification and DNA concentration for library preparation and sequencing (Figure 1). Academic labs often employ both manual and automated steps due to the cost of the equipment, as well as providing the flexibility to change steps when a pipeline has not been optimized for one process. Also, academic labs may not always need HTSP; when dealing with sample sizes of <1000 it is often easier to manually handle Figure 1. In the Gilbert Laboratory we have integrated manual labor (green arrows) and automated (blue many of the steps. The green arrows in Figure arrows) HTSP to maintain an open source for each research project within the lab and with external 1 represent manual processing steps, such as collaborators for a manageable quantity of samples and multiple projects at the same time. We are looking for ways to become more automated for HTSP, but the most quality-driven and cost-effective loading samples into a 96-well plate, preparing method is processing samples through a dedicated microbiome core with robotics calibrated for HTSP. reagents and plates, and transferring the plates between equipment. Loading and transferring exposure to the ubiquity of environmental on a single operation. Harnessing these data sample plates manually increases the level of microbes is probably unavoidable. Therefore, to predict when part of the equipment platform sample handling, which increases the risk of it is almost impossible to maintain a completely will fail would provide an opportunity to revolu- contamination due to human error. In Figure 1, sterile space for sample preparation, especially tionize access to these systems by reducing the blue arrows and equipment already include when the samples you are processing are a the dependency on a redundant system. automated steps. While pharma and biotech major source of potential contaminants. Machine learning and AI systems are helpful industries have already integrated automated It is essential that the community accepts for navigating the decision-making process pipelines into chemical and drug manufac- standards intended to better monitor and involved in cost vs benefit comparison in turing, the future of molecular biology and quantify these inevitable sources of noise in research outcomes. While it seems as if the microbiome research would benefit signifi- any given study, from the sample repository benefits outweigh the drawbacks, it is crucial cantly from the reproducibility, efficiency and to the sequencing platform. In addition to that the development of new automation incor- quality provided by full automation. It would external and cross-contamination, technical porates the user to reduce the likelihood that we reduce the tedium associated with massive hurdles such as DNA extraction efficiency and create an army of black boxes that could harm sample processing as well as mitigating molecular probe biases create noise that is also future innovation in pipelines. New protocols mistakes, allowing for quality control check essentially unavoidable and as such needs to must also include controls, quality checks points, and it would speed up data delivery be appropriately accounted for. and stop-go decision points to avoid errors for collaborators. For instance, in the fully However, increased automation creates throughout the pipeline. We must embrace automated microbiome core, only the robot specialized roles in the laboratory, so that a integration of computer scientists into the would interact with the processing equipment, laboratory technician with a broad molecular primary lab process to ensure that machine- reducing the risk of contamination, allowing skill set must now primarily oversee the readable data are produced and harnessed streamlining of each protocol. This will allow maintenance of complex robot platforms effectively to improve our ability to predict the technician to oversee and optimize the to ensure that the computational script and failures and refinements in the system. The process, rather than handling the samples mechanical operation runs as expected. This hurdles experienced in laboratory techniques throughout, significantly increasing the has the potential to lead to a generation of and during massive sample processing still likelihood of consistent and reproducible technicians who understand the robots, but indicate the importance of existing research results. The use of automation with specialized not the process the robots are mediating. skills, in addition to the new skills that the surge techniques and miniaturized assays reduces When the robots break down, lab activities of revolutionary technologies will require in the cost and the time required for processing, can grind to a halt as the system is now the future. allowing scientists to address biological dependent on these operations. Small labs questions that are otherwise unattainable cannot afford to hire specialized people, nor DISCLAIMER using conventional methods. to cover the cost of operational redundancies The opinions expressed in this Opinion are Despite the power of HTSP technology to avoid such downtime. Expensive robot those of author and do not necessarily reflect platforms to improve standardization, platforms require constant maintenance and the views of Future Science Ltd. minimize mistakes and streamline costs, the routine engineering. When the system breaks main pitfall remains cross-contamination down in the middle of an operation, it often between samples. Whether you are running means that those samples are lost, and the a protocol with single tubes, 96-well plates or whole system needs to be re-run. beyond, it is critical to have adequate labelling, A possible solution to ensure a reduction in Read this opinion online proper controls and to avoid contact between costly operation downtime could lie in the use bit.ly/BTN_GarciaOpinion samples. However, even with the most of machine learning techniques. A standard stringent preventative measures in place, robotic platform outputs megabytes of data

A hammer doesn’t need a nail

Who deserves more credit: the person that makes a hammer or the person that uses it to hit a nail in the wall? Joachim Goedhart recalls the lively discussion of this question between two professors (now both emeritus) of his department.

Written by Joachim Goedhart

y (easy) way out would be to answer methods that do not solve an existing problem. that we need both. A new tool is In several of those, not even a real application M more likely to be adopted if it is was included. Some of the methods that we applied to solve a pressing problem. That is have developed have been of interest to many the reason that PCR, GFP and CRISPR– [3], others are hardly cited [4]. The latter paper Cas9 are so famous that I don’t need to reported a new quantification method for a explain their abbreviation. These technol- bacterial molecule. It was actually based on ogies open new possibilities and they a relatively old paper that was poorly cited deserve(d) a Nobel Prize because of their [5]. This by itself shows that if papers are not demonstrated use in research. well cited, they can still be of use. Moreover, But the discussion about the hammer papers with new or improved methods may Joachim Goedhart touches upon another issue. Is the hammer be a source of inspiration, without being University of Amsterdam, by itself of interest? In other words, is a new cited. One thing is certain: if a method is not Amsterdam, The Netherlands technology relevant, if its application is not published, no one will know about it. demonstrated and does it deserve publication? Ideally, a new tool is reported in a paper I do think so. We have published a number that passes peer review, as peer-reviewed of papers ourselves with new or improved publications are the gold standard in scien- …is a new technology relevant, if its application is not demonstrated and does it deserve publication?

tific publishing. To this end, it is important to have platforms that have a strong focus on publishing methods and new technologies, even when there is no application demonstrated. In general, I would like to see more options for papers that focus on new methods. Sure, there are a couple of journals

A custom build confocal microscope made by Fred Brakenhoff and colleagues. This set-up is still present at Molecular Cytology (University of Amsterdam, The Netherlands) and is a modified version of the set-up that was described by van der Voort et al. [7]. Original photographs of the set-up are available at Zenodo: https://doi.org/10.5281/zenodo.3482606

Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 38 www.BioTechniques.com with a strong focus on technology and that publishing new methods, tools or 2. Nanninga N, Brakenhoff GJ, Meijer M, Woldringh CL. Bacterial anatomy in retrospect and prospect. Antoie methods, but the options are rather limited. technologies, regardless of the platform, Van Leeuwenhoek 50(5–6), 433–460 (1984). Quite often, I get the advice to publish a is valuable and that others can benefit 3. Goedhart J, Vermeer JEM, Adjobo-Hermans MJW, van Weeren L, Gadella Jr. TWJ. Sensitive detection of p65 methods paper in a (mega-)journal with from it. homodimers using red-shifted and fluorescent protein-based fret couples. PLoS One 2(10), e1011 (2007). Let’s keep on developing methods and 4. Goedhart J, Bono JJ, Gadella Jr. TWJ. Rapid Colorimet- technologies, even if the application is not ric Quantification of Lipo-chitooligosaccharides from Mesorhizobium loti and Sinorhizobium meliloti. Mol. New technologies, obvious. New technologies, new methods Plant Microbe Interact. 15(9), 859–865 (2002). and new tools will be key to break the bound- 5. Radin NS. Improved version of the Kean partition new methods and assay for cerebroside sulfate. J. Lipid Res. 25(6), aries of what is currently possible and that 651–652 (1984). 6. Goedhart J, Gadella TWJ. Jr. Photorelease of new tools will be is a core business of science. It is equally diacylglycerol increases the amplitude and duration important to publish results. It may inspire of protein kinase C-betall relocation in cyto. key to break the bioRxiv doi:10.1101/102657 (2017). others to make the method easier, cheaper 7. van der Voort HTM, Brakenhoff GJ, Valkenburg JAC, Nanninga N. Design and use of a computer controlled boundaries of what or better. And you may be surprised to confocal microscope for biological applications. learn that a seemingly useless hammer is Scanning 7(2), 66–78 (1985). is currently possible eventually applied in some unforeseen way. and that is a core DISCLAIMER business of science. The opinions expressed in this Opinion are It is equally important those of author and do not necessarily reflect the views of Future Science Ltd. Read this opinion to publish results. in full online REFERENCES bit.ly/BTN_GoedhartOpinion a broad scope, but this is not necessarily 1. Brakenhoff GJ, Blom P, Barends P. Confocal scanning light microscopy with high aperture immersion the best platform. I think that tools deserve lenses. J. Microsc. 117 (2), 219–232 (1979). specialized journals, which makes them easier to find. That is why I am a fan of journals that show a strong interest in the development of novel techniques. Clearly, BioTechniques is such a journal and that is why I did not take long before accepting their invitation to contribute. I strongly believe in open access publishing and I find that peer review usually improves a paper. However, there are alternatives. Let me explain this with a personal experience. When developing new methods for (cell) biology, we often are criticized for not showing any biological or physiological relevant experiments. Demonstrating the new tool is useful in a cellular context is not enough. Perhaps I’ve just been sending the manuscripts to the wrong journals. But, as I stressed above, it is important to get your work out there. This turned out to be extremely difficult in the case of a project that was part of CELL DISRUPTER my post-doc. We reported the synthesis • Fast Bead Milling in Microvials of a photo-activatable lipid and showed • Fast, E cient Cell Lysis that it can be used in cells. We failed to • Outstanding Value get it through peer review (believe me, we BioSpec Products, Inc. tried hard). Last year I decided to publish PO Box 788 the work as a preprint [6]. Only weeks Bartlesville, OK 74005, USA later, I was contacted by someone that 800-617-3363 biospec.com wanted to use the compound. For me, this was the ultimate reward. It proves

Vol. 68 No. 1–3 © 2020 Future Science Ltd www.BioTechniques.com | | BioSpec 4thpg39 Product Ads_BioTechniques.indd 2 9/18/19 11:32 AM Top Comments We enjoy seeing our content provoke discussion online. Here’s our favourite recent comments from our readers and contributors.

“I am trying to bring the scientific literature into the This season is notorious for 21st Century. It is amazing how many scientists still use struggles with #mentalhealth the term “DNA Fingerprint” when they really mean to say “DNA & #impostersyndrome. Bioprint”. The term bioprint should be used now and drop the So, here is my latest piece thanks to term fingerprint unless it is for the fingerprint. We no longer use @MyBioTechniques on my journey as the term horseless chariot when we are referring to a car or an someone who left his PhD & returned to automobile. We need to keep up with the times… Terms such as finish it – w tips on how to improve the DNA bioprint or molecular bioprint ARE so much more specific grad school experience! for the new technology than the 19th century fingerprint.” – @nakedcapsid – William Dettwyler, Codus Medicus, Inc. https://www.biotechniques.com/ opinion/breaking-the-silence-talking- about-mental-health-in-graduate- Thanks so much for the invitation @MyBioTechniques education/ – last year, I discussed the potential #stemcells have to treat challenging and debilitating diseases. This year, I share Replying to @NakedCapsid and what a dodgy #stemcell treatment might look like in the wild! @MyBioTechniques – @FreyaFSG Great essay. Something all graduate https://twitter.com/MyBioTechniques/status/1201886308508979200 students and their mentors should read more than once. – @eswillwalker A lot has been discussed ON #CRISPR off target effects & I’ve been asked a lot on whether these are concerning. So I decided to write a piece for @MyBioTechniques to discuss what are #CRISPR off target and should we be worried about 3D mapping of microbes and molecules in the human those. Comments welcome. body and environment and big data and health are – @GaetanBurgio some of the interesting topics I’ve been following on the https://www.biotechniques.com/crispr/what-are-off-target- #BTNPrecisMed online conference. Looking forward to the Q&A. effects-and-why-are-they-concerning/ –@IanaAmke

Replying to @GaetanBurgio and @MyBioTechniques An exciting new diagnostic technique for prostate cancer Great summary Gaetan. I agree that the biggest bottleneck for which could have a huge clinical impact! Great work clinical use is delivery, and not off-targets. Certain guides will @uniofeastanglia, @martynwebb93 and the rest of the research always have “strong off-targets”, that can only be solved with team! Practical, applicable science #cancer #cancerresearch proper guide RNA design. – @TristanFreeW1 – @MartinPacesa https://www.biotechniques.com/diagnostics/sample-shower- shave-the-at-home-urine-test-that-could-revolutionize- prostate-cancer-screening/

“Revealing the Kraken’s secrets: giant squid genome sequenced for the first time”. You know what? Good job @MyBioTechniques. You know how to subject an email. – @sciliz

discussed somatic mosaicism things CRISPR, from its exciting in the human brain; and Robert prospects to its morality. Catch Philibert, CEO of Behavioral up now on our Spotlight page. Diagnostics, who discussed a ■ Read more at www.biotechniques.com/ BIOTECHNIQUES.COM new PCR test for addiction. In Focus: DNA Sequencing Our roundup also presents spotlights-category/spotlight-on-crispr/ DNA sequencing has developed exclusive interviews with in leaps and bounds since the company experts who have completion of the Human been developing exciting Genome Project in 2003. new tech and software in the

PIXABAY This In Focus event included genetics space, including 10x Advancing Precision tips for next-generation seq Genomics, GenapSys, 54Gene Medicine 2019 uencing sample preparation, and Emedgene.

Over the last decade, precision the importance of genome PIXABAY medicine has taken leaps sequencing in precision med- ■ Read more at www.biotechniques.com/ Spotlight on: Cancer Research forward, with projects such as icine and the challenges still spotlights-category/ashg-2019/ Cancer Research is a highly the Precision Medicine prevalent in NGS today. Catch dynamic and vital aspect of life Initiative, All of Us and the up with the content on our In science research, with updates Human Cancer Models Initiative Focus page, which includes a and novel techniques in cancer putting a huge impetus behind podcast with Joanne Hackett, therapeutics and diagnostics the development of new Chief Commercial Office at continuously being developed. technologies to improve the Genomics England, among other To reflect this ever-evolving diagnosis and treatment of essential reading. topic, we ran a mini Spotlight on human disease. cancer research. This covered ■ Read more at www.biotechniques.com/ Our BioTechniques Online Spotlight on: CRISPR BIOTECHNIQUES.COM some of the latest updates in Event, Advancing Precision infocus-category/dna-sequencing/ CRISPR has demonstrated its cancer therapeutics and diag- Medicine 2019, was a free-to- ability to edit DNA in bacteria, nostics including a precision attend virtual symposium that viruses, plants and human cells. medicine podcast with Hans Kei- saw experts discuss the latest It is proving itself to have many rstead, CEO and Founder of Aivita technological advances in the advantages over other gene Biomedical; exclusive video inter- field of precision medicine editing techniques, including its views from attendees at AACR, – including microbiomics, simplicity, efficiency, easy cus- including Bodour Salhia (Keck wearables, organoids, software tomization of target DNA and the School of Medicine of the Uni- and more – and how they are ASHG 2019 ability to target multiple genes versity of Southern California) being translated from the bench The annual meeting of the simultaneously. Novel CRISPR and Bill Haney (director of ‘Break- to the bedside. If you missed American Society of Human systems and applications are through’ – the Jim Allison doc- it, catch up with the talks Genetics is always interesting, continuously being developed, so umentary), and more. Catch up on demand on our precision and 2019 was no exception. its potential is ever-growing. This on the latest advances, from medicine page, where you Check out our roundup of this Spotlight saw us hone in on the an exhaled breath biopsy for can also find other content event to see what was covered latest developments and chal- lung cancer to the new pre- including a discussion of the in the meeting. You can also find lenges in using CRISPR, what is cision immunotherapy currently common misconceptions of exclusive interviews – including on the horizon for CRISPR and undergoing phase II trials on our personalized medicine, and the with Leslie Biesecker (ASHG alternative applications in diag- Spotlight page. latest in precision medicines for President), who spoke to us nostics and modeling. cancer. about genetic exceptionalism; Of note, you can watch a ■ Read more at www.biotechniques.com/ Christopher Walsh (Chief of thought-provoking panel dis- spotlights-category/cancer-research/ ■ Read more at www.biotechniques.com/ Genetics and Genomics at cussion with two geneticists category/precision-medicine/ Boston Children’s Hospital), who and an ethicist who discuss all

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Learn how to acquire fluor escent high-speed 384-well plate images of live cancer organoids; optimizations for VIEW NOW optical clearing, refractive ON DEMAND The interplay between index matching and accuracy Rapid single-tube DNA Panel discussion: what does bacteria and bacteriophage of object detection; and how preparation from bacteria the future hold for CRISPR? Bacteria have evolved many the NoviSight™ HCA software for NGS workflows CRISPR has demonstrated its mechanisms to prevent viral generates True 3D models Whole-genome sequencing of ability to edit DNA in bacteria, infection. For each mechanism, with statistics across these microbes is a powerful tool for viruses, plants and human cells. it seems that bacteriophage samples. genomic studies, comparison, It is proving itself to have many have evolved a counter- mapping studies, microbial advantages over other gene measure. This webinar from ■ Watch this free webinar at bit.ly/ identification, and building editing techniques, including its Michael Weiner at Abcam (CT, BTNwebinar_3Dspatialanalysis reference genomes. The rate- simplicity, efficiency, easy cus- USA) reviews the immune limiting step in sequencing tomization of target DNA and systems of bacteria and how studies is the nucleic acid the ability to target multiple they are used to prevent extraction process. Currently genes simultaneously. Novel productive viral infection. The available methods of DNA CRISPR systems and applica- focus is on both lytic and extraction from bacteria are tions are continuously being lysogenic bacteriophage, and long and laborious and are developed, so its potential is how bacterial immune systems susceptible to cross-contami- ever-growing. relate to mammalian immune nation. In this webinar, the However, CRISPR is not systems. speakers discuss a novel currently perfect and does not method to extract DNA from come without its concerns. ■ Watch this free webinar at bit.ly/BTNwe- Cryo-ET contributes to bacterial cultures. The PDQeX it can often affect regions binars_strikecounterstrike bacterial–environment system, developed by Micro of DNA outside of its target, interaction understanding GEM, harnesses activities from hence continuous evaluation How are microbes able to a range of enzymes isolated and technique development actively seek out their preferred from extremophiles coupled is necessary. Therefore, there environmental niches, how can with thermoresponsive plastics are also huge ethical issues they effectively evade toxins, to extract DNA from samples with utilizing CRISPR editing on and how can they adapt to thrive without the use of centrifu- humans, on top of the question in their changing environments? gation or harsh solvents. The of whether gene editing on Tips for 3D spatial analysis of In order to gain insight into the protocol takes a fraction of the humans is moral in the first high-content drug screening structure and function of the time of existing methods place. High-content analysis during macromolecular complexes without the risk of cross- drug screening using 3D spher involved in these behaviors, contamination. The prepared ■ Watch our free panel discussion at oids and organoids requires ThermoFischer uses cryo- DNA can then be used directly bit.ly/BTNwebinars_futureCRISPR optimizations for speed, effective electron tomography (Cryo-ET). in whole genome sequencing sample imaging and clear and This technique allows them to workflows without any further efficient analysis. In this webinar, directly study microbes in their purification. imaging expert Brendan native state at resolutions Brinkman of Olympus discusses capable of visualizing individual ■ Watch for free at bit.ly/BTNwebinars_ live imaging of cancer organoids proteins. DNApreparationfrombacteria in 384 well plates, optimizations with optically cleared samples, ■ Watch this free webinar to learn how and the effects for efficient high- Cryo-ET works at bit.ly/BTNwebinar_cryo- content analysis. electrontomography

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In-depth Symposia, we can’t wait to attend. If you are attending, please stop by booth #1737 where our Editors will be looking to chat with you about your highlights of the meeting and work in microbiology, as well as answer any questions

BIOTECHNIQUES.COM you might have about BioTechniques Online Event: AACR 2020 BioTechniques.

SHUTTERSTOCK.COM Spotlight on: CRISPR The American Association for Catch up with our highlights BioTechniques Online Event: Over the past few years, the Cancer Research meeting is of ASM Microbe 2019 on our Spotlight on: 3D Cell Culture number of applications for back for 2020, this time running Spotlight page. 2D cell culture is generally insuf- CRISPR has grown rapidly as from 24 April in San Diego, ficient at reflecting the in vivo the technology has adapted California. The BioTechniques Date: 18–22 June 2020 behavior of an organ, and it can and advanced. Moving away team will be in attendance once Website: https://www.asm.org/ be difficult to translate research from single gene edits done again, and we hope that if you Events/ASM-Microbe/Home conducted in 2D cultures to the in vitro, CRISPR is making its are attending you will stop by Location: Chicago, IL, USA bedside. As a result, animal way into clinically viable thera- booth #1949, where our Editors models are often used; however, peutics and recently it entered would love to chat and hear your BioTechniques Online Event: differences remain, and thus its first clinical trial as a highlights of the meeting. Be In Focus: Reproducibility efforts to translate research to treatment for cancer. Further sure to watch the www.BioTech- Reproducibility remains a humans can still fail. 3D cell trials are lined up, aimed at niques.com website both during buzzword in the life sciences, culture techniques such as treating disorders ranging from and after the meeting for and there is now a large focus spheroids and organoids are inherited blindness to Duchenne exclusive news and views, both on ensuring that research is increasingly becoming the norm. muscular dystrophy. from our Editors and the experts reproducible and transparent. As part of this Spotlight As part of our second attending the meeting. This In Focus event will run event, running April through Spotlight on CRISPR event, through July 2020 and explore June 2020, we will explore the which will run from July through Date: 24–29 April 2020 the factors that contribute to biomaterials available to build September 2020, we will cover Website: https://2020aacr.com/ reproducibility issues, current relevant organoids, new imaging the latest advances in this field. Location: San Diego, CA, USA initiatives countering those approaches and techniques for In particular, we will explore the factors, and ongoing efforts to drug discovery and analysis use of CRISPR in diagnostics tackle the crisis – not just from using 3D cell cultures, and the and therapeutics, including the researchers, but also other key challenges facing organoid ethics and challenges faced in parties such as funding agencies construction and application, bringing CRISPR into the clinical and publishers. Head to our all whilst looking forward to the setting. website and subscribe for future of the field. Catch up with our first updates to ensure you don’t Head to our website to Spotlight on CRISPR event on miss out. access the Spotlight content, our Spotlight page, and be sure ASM Microbe or wait for the next issue for a to subscribe for updates on this The American Society for Micro- Date: July 2020 brief roundup. upcoming Spotlight. biology meeting will be Website: https://www.biotech- happening in Chicago this year, niques.com/in-focus/ Date: April–June 2020 Date: July–September 2020 from 18 to 22 June. With a Location: Online Website: https://www.biotech- Website: https://www.biotech- program chockablock with niques.com/spotlights/ niques.com/spotlights/ Meet-the-Experts sessions, Location: Online Location: Online Career Talks, Fireside Chats and

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Do you have a great image from your research that would look good on our cover? Enter it to our cover competition to feature on the December edition of BioTechniques!

Tech News: Tech News: From Sanger sequencing Tech News : Grow your to genome databases The unsustainable lab own brain and beyond

Bead-based assay for s patiotemporal gene Clarificationandconfocal Electrochemical methods for expression control in cell- imagingofthenonhuman probing DNA damage mechanisms free transcription–translation primate placental micro-anatomy and designing cisplatin-based systems combination chemotherapy Rapidquantificationofcellular A convenient method to proliferationandmigrationusing A method for rapid analysis of the root hydrotropic response in generate and maintain ImageJ poly(A)-encoding DNA Arabidopsis thaliana sequences required for in vitro transcription of mRNA

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Tech News: Antibodies: will their star continue to rise? Tech News: Genetically eeFit: a Microsoft Excel-embedded program for interactive analysis modified humans: and fitting of experimental the X-Men of dose–response data scientific research Single-step colony assay with autoinduction of scFv expression for the screening of antibody Engineering a minimal cloning libraries vector from a pUC18 plasmid backbone with an extended multiple cloning site

High-throughput Quant-iT PicoGreen assay using an ? automated liquid handling system

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Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 44 www.BioTechniques.com 2020First draft Expert Opinion submitted: 8 10 2019; Accepted for publication: 6 12 2019; Pub- lished online: 00 Step emulsification: high-throughput production of 00 0000 monodisperse droplets Linbo Liu1,2, Nan Xiang1, Zhonghua Ni1, Xing Huang2, Juanjuan Zheng2, Yunhua Wang2 & Xingcai Zhang*,2

tep emulsification is a promising with a small coefficient of variation (CV). The facial tension, rather than high-energy shear method for the production of use of the monodispersed emulsion droplets stress systems, the droplet size is S monodisperse droplets. Its main expands the applications of medicine [4], independent of the flow rates of both the advantage is that its geometry allows biotechnology [5–7] and biology [8,9]. continuous and the dispersed phases [14,28]. massive parallelization of multiple nozzles Monodispersed droplets are also applied in Therefore, there is no requirement for to achieve high-throughput production of the food [10], cosmetics [11] and chemical expensive gas regulators or precision pumps droplets. As the droplet generation process industries [12]. The CV of droplets is typically for preparing a monodispersed emulsion. in step emulsification is mainly driven by <5% in many step emulsification studies as The step emulsification devices can even be interfacial tension rather than high energy the standard CV [13–15]. Using droplets with operated by manual injection of the shear stress systems, the droplet size is a small CV as miniature test tubes can dispersed phase to produce droplets with a independent of the flow rates of both the improve the standardization and predict- small CV. Moreover, there are numerous continuous and the dispersed phases. ability of assays and increase the signal-to- papers on step emulsification with varied Therefore, the high productivity of droplets noise ratio. Another merit of step nozzle type and geometry. These nozzle (>100 l/h) and an excellent diameter coeffi- emulsification is that it is relatively easy to variations significantly influence the degree cient of variation (<5%) can be guaranteed parallelize to produce uniform droplets with and type of droplet size dependency on 2020 simultaneously, which makes step emulsi- high throughput [16–18]. At this time, a variety parameters such as volume flow rate. fication a potential tool for real industrial of encapsulated products can be produced Although in most cases the mechanism of © 2020 Xingcai Xhang applications. This article provides an using step emulsification, ranging from single the droplet breakup itself is independent of overview of step emulsification and to multiple emulsions, microcapsules, micro- dispersing and continuous volume flows, discusses the existing challenges that can spheres and many others [10,19–21]. the droplet size can be influenced by the potentially impact the practical use of this disperse flow rate. The dependency is tool; it concludes with perspectives and DROPLET GENERATION IN smaller compared with other microfluidic potential applications. STEP EMULSIFICATION droplet generation methods; however, it is In step emulsification, droplets are generated significant [13]. The degree of dependency STEP EMULSIFICATION by the spontaneous transformation of an varies with type and the geometry of the step The step emulsification was first proposed oil–water interface [22], with the flow of the (straight/trapezoid/terraced/millipede by Kawakatsu et al. in 1997 [1]. It is a droplet dispersed phase into the continuous phase etc.) [28,29]. Because of the importance of microfluidics technique that offers the through a rectangular microchannel [16–23], a stable and monodispersed droplet gener- detection and precise control of two incom- as shown in Figure 1. Microchannel arrays ation for industrial applications, these patible fluids at the microscale. Step emulsi- have been fabricated on a variety of phenomena must be taken into consider- fication can produce monodispersed substrates, such as a silicon-on-insulator ation when designing new microreactors. emulsion droplets, ranging from the and single silicon crystal, owing to the The droplet generation process in step 3 submicron range to around 1000 μm [2,3], surface properties that can easily be emulsification has been evaluated using modified using hydrophilic and hydrophobic a variety of simulation methods such as 68 treatments [24]. Microchannel arrays can computational fluid dynamics [30] and also be produced with materials such as Lattice Boltzmann methods [31,32] and KEYWORDS borosilicate glass, expanding the applica- using the experimental approach with high throughput • microchannel arrays • micro- bility to chemically aggressive high-speed microscopy [17]. They all fluidics • monodisperse droplets • spontaneous fluids [17,25,26]. Because of the sponta- conclude that the nozzle height deter- transformation • step emulsification neous droplet generation process, step mines the maximum production rate and

1School of Mechanical Engineering, & Jiangsu Key emulsification is highly energy efficient, with diameter of the monodisperse droplet in Laboratory for Design & Manufacture of Micro-Nano typical energy input. However, the mild step emulsification device. Even though Biomedical Instruments, Southeast University, Nanjing droplet generation in step emulsification, the nozzle height restricted the maximum 211189, China; 2John A. Paulson School of Engineering & Applied Sciences, Harvard University, Cambridge, with no involvement of energy, makes it a production rate and the diameter of the MA 02138, USA; *Author for correspondence: xingcai@ preferable system to prevent denaturation droplets, numerous scientific works have seas.harvard.edu of sensitive bioactive compounds [10,27]. already been done to address some of the BioTechniques 68: 114–116 (March 2020) 10.2144/ Because the droplet generation process in challenges in increasing the production btn-2019-0134 step emulsification is mainly driven by inter- rate. Xu et al. reported a high aspect

Third, the nanodroplets (diameter of the Step emulsification droplet is nanoscale) can be used as small containers that will encapsulate one molecule per droplet, forming a high- throughput robust tool for single molecular studies. Fourth, step emulsification should not be separated from biomedical research, such as drug delivery [45], biomedical imaging [46,47], bioregeneration [48,49] and biosensing [50,51]. It can make full use of the advantage of high throughput when the functional biomaterials are encapsulated in droplets by step emulsification. Additionally, they can be integrated to synergetically promote real industrial applications, especially in the biomedical field. Therefore, more collaborative effort is still needed from researchers and entrepre- neurs to achieve industrial-level high- throughput production of nanodroplet Figure 1. Step emulsification and parallelization: high-throughput production of monodisperse emulsions. We hope this article will droplets. stimulate researchers from different backgrounds to work on addressing the ratio (>3.5) step emulsification, which standards of step emulsification, to find problems described previously and to make can produce 15,000 droplets per second more detailed descriptions and discus- contributions to push step emulsification with 2000 nozzles, with a CV below 3% [33]. sions about droplet formation mechanism, forward to become a robust industrial Schuler et al. demonstrated a straight- device fabrication and various applica- technology. forward step emulsification system without tions, the reader is advised to study some any oil flow, which uses centrifugal forces of the excellent reviews and papers on the AUTHOR CONTRIBUTIONS to produce 500 droplets per nozzle (paral- topic [37–44]. X Zhang and L Liu conceived and wrote the lelization with 23 nozzles) with a CV paper. N Xiang, Z Ni and X Huang provided between 2 and 4% [34]. Recently, Schuler CHALLENGES & FUTURE the related references. J Zheng and Y Wang et al. demonstrated a step emulsification PERSPECTIVE provided constructive discussions for the system, which employed buoyancy in the Step emulsification can be useful for appli- challenges and perspective section. X Zhang centrifugal field to realize increased droplet cations that need large-scale production approved the final version of the manuscript. generation of 2800 droplets per second of monodispersed droplets or encapsu- and nozzle, with a CV below 5%. Droplet lating perishable samples. Despite the FINANCIAL & COMPETING generation rates are about a factor eight many compelling developments, there INTERESTS DISCLOSURE above the critical capillary number. The remains effort that needs to be put into The authors have no relevant affiliations or main advantage is that a single nozzle bringing step emulsification out of the financial involvement with any organization is used in which manufacturing toler- laboratory as an industrialized technology. or entity with a financial interest in or ances do not influence droplet generation First, researchers should focus on the basic financial conflict with the subject matter or rates [35]. Dangla et al. reported a droplet research of step emulsification. Although materials discussed in the manuscript. This microfluidics system driven by gradients droplet breakup dynamics have been includes employment, consultancies, of confinement, single droplet generation explored [13], to our knowledge, no model honoraria, stock ownership or options, as well as high throughput is demon- exists to figure out precisely the diameter expert testimony, grants or patents received strated. Although the methodology is not of droplets. More in-depth investigations or pending, or royalties. strictly the step emulsification, the gradient of the relationship between droplet No writing assistance was utilized in the confinement method is very close to it diameter and other parameters (flow rate, production of this manuscript. because droplet formation also depends on viscosity, microchannel geometry and the geometry and very weakly on the flow temperature) are still expected. Second, OPEN ACCESS rate. Droplet breakup is driven by surface mass production is a crucial issue. To lower This work is licensed under the Attribution- tension. The only difference is that no step the cost and to improve the throughput of NonCommercial-NoDerivatives 4.0 Unported is employed but rather an inclined surface the device simultaneously, the materials License. To view a copy of this license, visit to change the surface energy [36]. Because and the bonding approaches of the layers http://creativecommons.org/licenses/ of the profound engineering and scientific should be explored and prudently selected. by-nc-nd/4.0/

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nol. 42(10), 2195–2201 (2019). transition in a 2002001

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ReportsK. pneumonia in Brief Normalization Comparison Hi-C matrices with in of P. fluorescens six different methods multiple considerations different cell lines

ICE Heat map texture Statistical quality I HiCNorm S. marcesans Influence of resolution Cell line 1 Distance stratum chromoR TADs reproducibility

Summarized results KR P. mirabilis SCN HICNorm ICE KR chromoR multiHiCcompare Cell line 2 Model Implicit Explicit Implicit Implicit Implicit Implicit Sample combination Individual Individual Individual Individual Individual Across Distribution of IF - - - - + ++ SCN Correlation of replicates +/+/- +/+/- +/-/- +/+/- +/++/+ +/+/+ Comparability between contexts - - + - - ++ Distance stratum - - - - - ++ Comparison of TADs reproducibilityErythrosin++ + + B: ++a versa- - - At-home collection Detecting GPCRs in Language MATLAB R R, C MATLAB R R Implementation SCN_sum… HiTC/HiCN… HiTC/HiC-C… BNEWT.m chromoR multiHiCcompare normalization multiHiCcompare Runningtile time colorimetric++ -/+ +/++ ++ + and- of urine samples postmortem human Physical memory ++ +/+ +/++ ++ + - methodsCell line 3 for Hi-C fluorescent vital dye for prostate cancer brain with proximity data page 50 for bacteria page 63 detection page 71 ligation assay page 76

An efficient and novel technology for the extraction of parasite genomic DNA from whole blood or culture

David J Clark‡,1, Catherine M Moore‡,1, Marc Flanagan2, Katrien Van Bocxlaer3, Evangelia-Theophano Piperaki3, Vanessa Yardley3, Simon L Croft3, John Tyson2, Sam P Whitehouse2, Jonathan O’Halloran2, Sanjeev Krishna*,1 & Henry M Staines*,1

CITATION donovani amastigote culture was The DNA-XT method produced DNA detergent and enzymatic lysis step to BioTechniques 68: 79–84 (February extracted with DNA-XT and with lower PCR inhibition than release DNA from cells. Contami- 2020) 10.2144/btn-2019-0086 compared with that produced by a DNeasy. The new technique was also nating proteins and lipids are then commercial extraction kit (DNeasy®). twice as fast and required fewer bound to a matrix within a spin column ABSTRACT Eluates from large and small sample plastics and manipulations but had during a 1-min centrifugation step The aim of this study was to assess volumes were assessed by PCR and reduced total recovered DNA while DNA passes directly through. pathogen DNA extraction with a new spectroscopy. Using a small volume compared with DNeasy. spin column-based method (5 μl) of blood, the DNA-XT and KEYWORDS (DNA-XT). DNA from either whole- DNeasy methods produced eluates METHOD SUMMARY blood • diagnostics • DNA extraction blood samples spiked with with similar DNA concentrations DNA-XT, which is designed for small • Leishmania • malaria • PCR Plasmodium falciparum or Leishmania (0.63 vs 1.06 ng/μl, respectively). sample volumes, uses a 5-min • Plasmodium

DNA extraction is an essential starting point Sample nated in 1979 [4], uses silica matrices to for methodologies such as PCR, which is selectively bind DNA, allowing washing used in the laboratory for molecular biology (DNA-XT™ Prepare column before elution of the purified product. lysis buffer) (DNA-XT™ and clinical diagnostics. It was first achieved activation NanoMal was an EU-funded industrial– buffer) in 1869 by Friedrich Meischer [1], but a academic consortium that was brought routine laboratory procedure was not together with the aim of developing a simple- developed until 1958 [2]. Today, a variety of Tube Incubate to-use, affordable, handheld diagnostic (supplied) methodologies are available (reviewed by device to detect malaria infection and the Griffiths and Chacon-Cortes [3]); however, drug resistance status of the parasite by commercial spin column purification of DNA identifying associated genetic mutations. from blood and other tissues and fluids is continued online… the most common approach used in modern Pure DNA laboratories. The technique, which origi- Figure 1 DNA-XT protocol.

1Centre for Diagnostics & Antimicrobial Resistance, Institute for Infection & Immunity, St George’s University of London, Cranmer Terrace, London, SW17 0RE, UK; 2QuantuMDx, Newcastle upon Tyne, NE1 2JQ, UK; 3Infection & Immunity Department, Faculty of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, WC1E 7HT, UK; *Author for correspondence: [email protected] & [email protected]; ‡Joint first authors View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0086 article here

Yao Xu1,2, XueYu Ren1, HongBin Wang*,1, Mei Wang1 & GuoHong Li1

CITATION istics of DNA fragment distribution adult specimens) were different for on this model, this paper reveals the BioTechniques 68: 138–147 (March caused by external DNA damage different species. characteristics of DNA fragment 2020) 10.2144/btn-2019-0166 factors during specimen preser- distribution caused by external DNA vation. We found that the degree METHOD SUMMARY damage factors during specimen ABSTRACT of DNA degradation increased over Using one-way analysis of variance preservation. Millions of museum specimens time; DNA degradation of spread and and the likelihood ratio test, the are integral to biodiversity studies; dried adult specimens was signifi- differences in DNA fragment KEYWORDS however, DNA degradation may limit cantly higher than that in the folded distribution between different degradation analysis model • DNA the ability to obtain DNA sequences. and formalin-fixed larval specimens. Lepidopteran specimens were degradation • folding of wings In this study, a degradation analysis However, the effects of folding wings analyzed. Based on the random- • formalin • Lepidoptera specimens model for Lepidoptera specimens on DNA degradation and the effects breakage model, a degradation was established. Based on this of the preservation method/stage analysis model for Lepidoptera model, we revealed the character- (formalin-fixed larval vs air-dried specimens was established. Based

The combination of fresh and museum specimens has created incredible potential for the study of evolutionary 14 13 Experimental curve change [1–4]. Recent studies using these specimens have Predictive curve revealed some remarkable instances of phenotypic or 12 Calibrated curve 11 genotypic change over certain timescales in response to 10 strong selective pressures [5–12]. However, low concentra- 9 tions of extracted DNA and relying on small DNA fragments 8 may limit the ability to obtain DNA sequences from 7 specimens [13–15]. Traditional museums store collections 6 centage (%) in cool, dark environments to preserve specimens over long r 5 Pe periods. However, standard museum conditions for collec- 4 tions are inadequate to completely prevent the degradation 3 of genomic DNA. Not surprisingly, genomic DNA increasingly 2 degrades and often becomes difficult to use in genetic 1 studies [10,11,13,15]. 0 -1 Discussions about the factors that influence DNA degra- 0 90 180 270 360 450 540 630 720 810 900 dation will highlight important considerations and guidelines for The middle of the fragment interval processing and preserving specimens [13,14]. Previous relevant studies have investigated the effects of age on microsatellite Figure 1. DNA fragment size distribution for a Lymantria dispar (gypsy moth) amplification success rates and DNA barcode sequencing rates specimen obtained in 1957. of historic specimens in museum collections [16–19]. However, disparities in preservation methods and other treatments may because of the difficulties associated with DNA extraction from also lead to variations in DNA degradation. Regarding preser- formalin-fixed specimens [20]. vation methods, few studies have compared the degradation continued online… rate in formalin-fixed specimens and other types of specimens (such as air-dried specimens or those preserved in alcohol)

1Key Laboratory of Forest Protection of China State Forestry Administration, Research Institute of Forest Ecology, Environment & Protection, Chinese Academy of Forestry, No. 1 Dongxiaofu, Haidian District, Beijing 100091, China; 2Department of Entomology, China Agricultural University, Beijing 100193, China; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0166 article here

ABSTRACT GRAPHICAL ABSTRACT Hi-C has been predominately used to study the Normalization Comparison genome-wide interactions of genomes. In Hi-C Hi-C matrices in experiments, it is believed that biases originating of with six different methods multiple considerations from different systematic deviations lead to different cell lines extraneous variability among raw samples, and ICE Heat map texture affect the reliability of downstream interpreta- Statistical quality tions. As an important pipeline in Hi-C analysis, HiCNorm Influence of resolution normalization seeks to remove the unwanted Cell line 1 Distance stratum systematic biases; thus, a comparison between chromoR TADs reproducibility Hi-C normalization methods benefits their choice and the downstream analysis. In this article, a Summarized results comprehensive comparison is proposed to inves- KR SCN HICNorm ICE KR chromoR multiHiCcompare tigate six Hi-C normalization methods in terms of Cell line 2 Model Implicit Explicit Implicit Implicit Implicit Implicit Sample combination Individual Individual Individual Individual Individual Across Distribution of IF - - - - + ++ multiple considerations. In light of comparison SCN Correlation of replicates +/+/- +/+/- +/-/- +/+/- +/++/+ +/+/+ Comparability between contexts - - + - - ++ results, it has been shown that a cross-sample Distance stratum - - - - - ++ TADs reproducibility ++ + + ++ - - Language MATLAB R R, C MATLAB R R approach significantly outperforms individual Implementation SCN_sum… HiTC/HiCN… HiTC/HiC-C… BNEWT.m chromoR multiHiCcompare multiHiCcompare Running time ++ -/+ +/++ ++ + - sample methods in most considerations. Physical memory ++ +/+ +/++ ++ + - The differences between these methods are Cell line 3 analyzed, some practical recommendations are given, and the results are summarized in a table Hi-C technology allows for genome- nities and challenges. A series of ad hoc to facilitate the choice of the six normalization wide profiling of chromatin interactions algorithms, computational and statis- methods. The source code for the implemen- in space [1]. It is well known that spatial tical methods, as well as bioinformatics tation of these methods is available at https:// organization of chromatin is tools are available for the exploration github.com/lhqxinghun/bioinformatics/tree/ non-random and is crucial for and interpretation of Hi-C data. These master/Hi-C/NormCompare deciphering how the 3D architecture of pipelines cover all current aspects of METHOD SUMMARY DNA affects genome functionality and Hi-C analysis workflow, ranging from Six normalization methods for Hi-C data were transcription [2,3]. As a chromosome preprocessing of sequencing reads to compared comprehensively in terms of multiple conformation capture (3C)-based normalization and inference of genome considerations, including heat map texture, method [4], Hi-C provides a deeper structure [3,10]. The preprocessing statistical quality, influence of resolution, consis- insight into the 3D organization of pipeline consists of read mapping, tency of distance stratum and reproducibility of chromatin by comprehensive detection fragment assignment, filtering and topologically associating domain architecture. of spatial interactions between binning, and we are left with a symmet- Among these considerations, the quality of statistics was investigated in depth from three genomic regions [1,5]. Compared with rical contact matrix. Each entry in aspects, comprising distribution of interaction other 3C-based technologies, such as the matrix reflects the interaction frequency, correlation of replicates and compa- chromosome conformation capture- frequency (IF) observed between the rability of replicates between contexts. The on-chip (4C), chromosome confor- corresponding pair of loci (also known performance of these methods is compared. mation capture carbon copy (5C) and as bins). The two loci are separated by ChIA-PET, Hi-C technology combines a fixed size genomic interval, which is DNA proximity ligation with deep conveyed as resolution [11,12]. It is sequencing and is not dependent on found that the value of matrix entries associated proteins. These advances exhibits an exponential decay in give it the power to implement genome- signal as the distance between loci wide mapping of chromatin interac- increases [1], which is consistent with KEYWORDS tions [6–8]. the expectation that 3D interactions comprehensive comparison • Hi-C data • normal- Hi-C has predominately been used mostly occur within chromosomes ization methods to study the genome-wide interac- (cis) rather than between chromo-

1School of Electronic & Information Engineering, Xi’an tions of genomes [9]. Hi-C experi- somes (trans) [3]. Following prepro- Jiaotong University, Xi’an 710049, China; *Author for ments usually produce hundreds of cessing, normalization is carried out correspondence: [email protected]; millions of paired-end sequencing to correct systematic biases, making BioTechniques 68: 56–64 (February 2020) 10.2144/ reads, and this enormous amount of Hi-C samples more comparable and btn-2019-0105 genomic data presents great opportu- downstream analysis reliable [9]. The

Restriction enzyme Replicate Species Cell type Resolution Ref. HindIII MboI DpnII Samples (n) Human GM12878 ✓ ✓ ✓ 2.5M/1M/500K/250K/100K/50K/10K/5K 14 [8]

IMR90 ✓ ✓ 2.5M/1M/500K/250K/100K/50K 8 [8,13]

K562 ✓ ✓ 2.5M/1M/500K/250K/100K/50K 8 [8,25]

Mouse CH12-LX ✓ ✓ 2.5M/1M/500K/250K/100K/50K/10K/5K 10 [8]

Liver ✓ 2.5M/1M/500K/250K/100K/50K 2 [26]

mES ✓ 2.5M/1M/500K/250K/100K/50K 2 [13] inference of genome architecture can then in the sequencing coverage of each bin [9]. Thus, it is essential to comprehensively be investigated at different levels, such Typical methods are sequential component assess the performance of existing Hi-C as topologically associating domains normalization (SCN) [19], iterative normalization methods. (TADs) [8,13]. correction and eigenvector decompo- In this paper focusing on various Normalization is one of the most sition (ICE) [14], Knight-Ruiz (KR) [20] and datasets involving different species, cells important pipelines in Hi-C data chromoR [21]. This difference in model and experimental designs, six Hi-C normal- analysis [9]. Comparison between normal- assumption makes the two groups of ization methods including SCN, HiCNorm, ization methods benefits their choice as methods have their own characteristics in ICE, KR, chromoR and multiHiCcompare well as the downstream analysis. The raw terms of strategy and implementation. For were compared in terms of multiple consid- outputs of many genomic technologies explicit approaches, probabilistic models erations, including heat map texture, are influenced by technical biases and are commonly used and their implemen- statistical quality, influence of resolution, biological factors [14]. In Hi-C experiments, tation relies on a variety of additional infor- consistency of distance stratum and repro- the interaction frequencies of contact mation, such as restriction site, genome ducibility of TAD architecture. Among these matrix contain many unwanted biases sequence and mappability score. On considerations, the quality of statistics was that are derived from different systematic the contrary, the strategies of implicit investigated in depth from three aspects deviations in experimental procedures and approaches are dominated by matrix comprising distribution of IF, correlation of driven by DNA sequence and technical balancing, spatial transformation and local replicates and comparability of replicates variation, including library size, fragment regression, with fewer parameters and less between contexts. In regard to comparison length, GC content, sequence mappa- additional information. In terms of sample of results, the differences between these bility, copy number variations and other combination, normalization methods methods were analyzed, some practical unknown factors. It is believed that these can also be grouped into individual- recommendations were given, and the biases lead to extraneous variability among sample and across-sample approaches. itemized results for these considerations raw samples and affect the reliability of The difference between them is that the and representative implementations of these downstream interpretations [15,16]. In latter determines biases with the help methods are summarized in Tables 1 & 2 to Hi-C analysis workflow, normalization of fusion of data from multiple samples, facilitate comparison of the six Hi-C normal- pipeline attempts to remove the unwanted while the former does not. Thus far, most ization methods. systematic biases, so that the interaction of the existing normalization methods are frequencies reflecting the underlying individual-sample approaches, with only MATERIALS & METHODS architecture can be preserved as far as a few belonging to across-sample ones, Datasets possible [17]. A number of normalization such as multiHiCcompare [22]. With the A total of 44 real Hi-C samples involving methods for correcting Hi-C data are continuous accumulation of Hi-C data and different species, cells and experimental available. These methods can be roughly the emergence of various normalization design were used to investigate how well the grouped in different ways. According to methods, understanding how normal- various Hi-C normalization methods can model assumptions, they can be divided ization methods impact downstream correct for the system biases in them. Hi-C into explicit and implicit approaches [11]. analysis and how to choose them is a contact matrices were prepared in two steps: Explicit approaches assume that the valuable tool. However, there has been no systematic biases, such as fragment comprehensive comparison of Hi-C normal- 1. First, a highly compressed binary length, GC content and sequence mappa- ization methods. Recently, Forcato et al. format file .hic was created with Pre bility, are known and accounted for in the quantitatively evaluated the performance command provided by Juicer [24] based statistical model [9]. A representative of 13 Hi-C computational methods [3]. on the paired-end reads from multiple explicit method is HiCNorm [18]. Alterna- These methods cover the identification resources. Herein, the Hi-C data derived tively, implicit approaches assume that of TADs and interaction peaks [23], with from four Hi-C studies [8,13,25,26] were the cumulative effect of bias is captured normalization approaches not included. considered. A total of 44 .hic files were

Table 2. Summary of comparison results and representative implementations for the six Hi-C normalization methods. multiHiC SCN HiCNorm ICE KR chromoR compare Model Implicit Explicit Implicit Implicit Implicit Implicit

Sample combination Individual Individual Individual Individual Individual Across

Distribution of IF - - - - + ++

Correlation of replicates +/+/- +/+/- +/-/- +/+/- +/++/+ +/+/+

Comparability between - - + - - ++ contexts

Consistency of distance stratum - - - - - ++

Reproducibility of TADs ++ + + ++ - -

Language MATLAB R R, C MATLAB R R

Representative implementation SCN_sumV2.m HiTC /HiCNorm.R HiTC/Hi-Corrector BNEWT.m chromoR multiHiCcompare

Running time ++ -/+ +/++ ++ + -

Physical memory ++ +/+ +/++ ++ + -

‘–’ indicates that the method provided unsatisfactory results for the given consideration, while ‘+’ indicates satis- factory results. For the item of correlation of replicates, */*/* represents the satisfactions of results from low to high resolutions in the range of 2.5M to 50K, and for the last two items, */* indicates the satisfactions of results produced by the previous corresponding implementation. The running time and physical memory were obtained in the same computing environment (Supplementary Table S1). ICE: Iterative correction and eigenvector decomposition; IF: Interaction frequency; KR: Knight-Ruiz; SCN: Sequential component normalization; TAD: Topologically associating domain.

obtained, including 30 files for three hg19 attempts to make contact matrix double used to correct Hi-C contact matrices human cells (GM12878, IMR90 and K562) stochastic using a matrix-balancing as an implicit individual-sample normal- and 14 files for three mm9 mouse cells strategy. In SCN, all rows and columns of ization approach. In KR, a scaling vector (CH12-LX, Liver and mES). These files a contact matrix are successively scaled is calculated using an inexact Newton can also be downloaded from the Juicer by dividing by the corresponding sums. iteration with conjugate gradient, and data archive at https://bcm.app.box. This process is usually repeated until the matrix is made double stochastic by com/v/aidenlab/ and Gene Expression convergence. diagonal scaling. Omnibus [27] database at https://www. 2. HiCNorm is an explicit individual-sample 5. chromoR is an implicit individual-sample ncbi.nlm.nih.gov/geo/. approach for Hi-C normalization. It intro- approach for Hi-C normalization. It 2. Hi-C contact matrices were extracted duces a Poisson regression model to attempts to correct contact matrix by from these .hic files using the Dump correct contact matrix. In HiCNorm, the means of decomposition, de-noising command provided by a java-based systematic biases, including fragment and reconstruction procedures. In program juicer_tools [24]. length, GC content and sequence mappa- chromoR, IF is meant to be a Poisson bility, are estimated as a Poisson offset, random variable. Thus, Haar-Fisz Finally, the contact matrices for six cell and the residuals of the regression are transform can be applied to decompose contexts of human and mouse were prepared regarded as the normalized matrix. the Poisson distributed frequency into at eight different resolution levels, including 3. ICE is an implicit individual-sample Gaussian-distributed coefficients in 2.5M, 1M, 500K, 250K, 100K, 50K, 10K and approach for Hi-C normalization. It multiple scales, followed by a de-noising 5K. All the datasets above are summarized attempts to make all bins of contact procedure via wavelet shrinkage. A in Table 1. matrix equally visible using a matrix- corrected matrix is then reconstructed balancing strategy. In ICE, the systematic using the inverse Haar-Fisz transform. Normalization biases between two bins are considered 6. multiHiCcompare is an implicit across- Six different Hi-C normalization methods, as the product of their individual biases sample approach for Hi-C normal- including SCN, HiCNorm, ICE, KR, chromoR and the maximum likelihood solution for ization. It allows for data-driven joint and multiHiCcompare, were considered in the individual biases is obtained by an normalization based on locally weighted the comparative analysis. The categories iterative correction procedure, yielding regression (loess). In multiHiCcompare, and strategies are outlined as follows: a normalized matrix. the difference in IF between two matrices 1. SCN is an implicit individual-sample 4. KR is a fast algorithm for balancing of with respect to bin distance, named approach for Hi-C normalization. It square nonnegative matrices. It is widely difference versus distance (MD), is fitted

Vol. 68 | No. 1–3 | © 2020 Hongqiang Lyu 52 www.BioTechniques.com using loess on a log scale. This estimated underlying architecture can be preserved consistent decay pattern across samples. difference is equally segmented and as far as possible. Thus, before any other In addition, the stratum-adjusted corre- compensated into the two original investigation, it is important to verify that lation coefficient [28] within context is matrices in the opposite direction to these methods have not caused excessive compared with that across contexts, to remove systematic biases, and an damage to the biological structure repre- check the difference of variation between anti-log transformation is employed to sented by raw data. Since heat map is the normalized contact matrices of intra- obtain the final normalized matrices. primary means of overall graphical presen- context and intercontexts. Furthermore, tation of Hi-C data, an intuitive comparison this variation difference can also be During implementation, default parameter of texture between the heat maps of verified on each bin distance with the help options were used in all function calls unless raw and normalized contact matrices is of average deviation (AD) (Supplementary stated otherwise. The details of the imple- fundamental. Method 2). mentations of these normalization methods 2. Comparison of the statistical quality 5. It is assumed that the contact matrices are given in Supplementary Method 1. is necessary, as it is the guarantee of within the same context share similar TAD consistency and authenticity of the structures. For a comparison of TAD archi- logCPM transformation results of subsequent procedures, such tecture reproducibility between normal- The value of interaction frequencies of as architecture identification and differ- ization methods, the TADs of raw and contact matrices normalized by the afore- ential analysis. To do this, first the IF normalized matrices are compared using mentioned methods are obviously on distribution of raw and normalized TopDom [29] (Supplementary Method 2), different scales due to the various strategies contact matrices is examined. Second, and the Jaccard Index for concordance employed in these methods, even at the the correlation of replicates is scored of TAD boundaries between replicates is same resolution level. In order to adjust the by both coefficient of variation (CV) and computed as the measure of TAD repro- value to the same scale across different Spearman coefficient, where the CV is ducibility. Besides, the Jaccard Index of methods, a transformation of log counts per calculated per matrix element across repli- TADs boundaries between two different million (logCPM) was used before compar- cates. Finally, the comparability of repli- contexts is also calculated to check ative analysis. The definition of logCPM is cates between contexts is investigated whether the reproducibility of TADs across given by: via MA plot (Supplementary Method 2). contexts is lower than that within context There are some differences between after normalization. C X ij, 6 S (Equation 1) the three considerations. The first is Yij, log10D 10 s T E L U position independent, while the last two RESULTS & DISCUSSION are position dependent and can be further Heat map

Where Xi,j and Yi,j are the interaction distinguished depending on whether the To conduct an intuitive comparison between frequencies of row i and column j in contact comparison occurs within the same the results of these six different methods, matrices before and after the transfor- context or between two different contexts. heat maps of the raw and normalized mation, respectively, L denotes the library 3. Resolution may have a considerable contact matrices for chromosome 1 size estimated by the sum of the lower trian- impact on the performance of Hi-C normal- (0–100,000,000) and 18 (0–75,000,000) of gular matrix, and S was set to 1 to ensure ization methods, since it determines the the Hi-C sample GM12878–001 and that Yi,j is non-negative. dimension of contact matrix and is crucial CH12-LX-104 at resolutions of 1M, 500K and to matrix sparsity. In order to determine 100K were given (Figure 1 & Supplementary Comparative design the difference between these methods Figures S1–S11). It can be seen that, at the The Hi-C normalization methods mentioned in response to changes in resolution, three resolution levels, the matrices earlier were compared in terms of multiple an investigation of some concerns, normalized by the different methods considerations, including heat map texture, especially the considerations under which maintain a texture similar to the corre- statistical quality, influence of resolution, the comparison results are sensitive to sponding raw matrices, except for those by consistency of distance stratum and repro- resolution in the previous section, can be chromoR, whose details are not clear at 1M ducibility of TADs architecture. Herein, only further conducted at multiple resolution and 500K resolutions. This can be explained the cis contact matrix is taken into account, levels. by the de-nosing procedure in chromoR, since these methods have no ability to 4. It is known that the IF of contact matrix which helps to achieve higher correlation of handle asymmetric trans contact matrix, follows an exponential decay in signal as replicates but at the same time blurs the apart from ICE and HiCNorm, and chromo- bin distance increases. For the consis- details of contact matrix, especially at low somal matrix can be extended on a genomic tency of this type of distance stratum, resolutions. level to examine trans interactions in imple- the IF of raw and each normalized contact mentation. matrix is fitted against the distance with Statistical quality 1. As mentioned earlier, Hi-C normalization the help of loess on a log scale, that is, The statistical quality of normalized contact methods are devoted to the elimination loess(Yi,j ∼ log10(|i – j| + 1)). These loess- matrices was compared from three different of unwanted systematic biases so that fitted curves are then checked to determine aspects, including distribution of IF, corre- the interaction frequencies reflecting the which normalization method can ensure a lation of replicates and comparability of

replicates between contexts. This or similar after normalization. In most Resolution analysis was conducted based on the cases, the loess fitted line of multiHiC- The level of resolution has a more significant contact matrices for chromosome 1 and 18 compare runs much closer to the zero impact on the results of comparison in terms of GM12878, IMR90 and K562 contexts at line than those of the other methods. of replicate correlation. Thus, the average five resolution levels, including 1M, 500K The fitted line of ICE is usually better CV and Spearman coefficients of all the and 100K, 10K and 5K. than those of SCN, HiCNorm, KR and contexts with more than two replicates were 1. For distribution of IF, box plots of raw chromoR, and in some cases even computed at six resolution levels, including and normalized contact matrices across preferable than that of multiHiC- 2.5M, 1M, 500K, 250K, 100K and 50K, for contexts were presented without outliers compare (Supplementary Figure S16C). further analysis. The curves of the two coeffi- (Figure 2A & Supplementary Figures In addition, the line quality of chromoR cients versus resolution for chromosome 1 S12A–S16A). It can be seen that there varies greatly compared with the other and 18 of GM12878, IMR90, K562 and are differences in the IF distribution methods. Beyond the loess-fitted line, CH12-LX contexts were given (Figure 3 & between raw samples from multiple the median and IQR of the M value were Supplementary Figure S25). Obviously, the sources. These differences are expected also counted. The median value of multi- average CV generally increases and the to be removed by normalization proce- HiCcompare can be equal to zero in five Spearman coefficient decreases as contact dures. Considering the six normalization out of six cases due to its strategy of matrices step up to a higher resolution. That methods, multiHiCcompare aligns the differential segmentation and compen- is to say, the correlation of replicates median and two quantiles of IF much sation on a log scale. chromoR has the weakens with the increase in matrix better than any other method in all cases, smallest IQR in five out of six cases, dimension and sparsity. Considering the and the stabilization of distribution is which is consistent with the results different normalization methods, the significantly improved. Regarding the of previous distribution analyses. In changes of these curves are roughly similar other methods, chromoR usually has a addition, the remaining normalization to the raw, except for those corresponding smaller interquartile range (IQR) thanks methods including SCN, HiCNorm, to chromoR, multiHiCcompare and ICE. In to its de-noising procedure, leaving SCN, ICE and KR sometimes have smaller general, chromoR and multiHiCcompare can HiCNorm, ICE and KR with almost the medians than chromoR in absolute achieve a higher correlation of replicates same distribution as that of raw. value and lower IQRs than multiHiC- compared with the other methods. At resolu- 2. For correlation of replicates within compare. tions of 500K and 250K, the replicate corre- context, box plots of CV and Spearman lation by chromoR is usually significantly coefficient of contact matrices for In addition to human data, the contact higher. ICE has a slightly weaker correlation GM12878 replicates were shown without matrices for samples of three mouse than the other methods at resolutions of 1M outliers (Figure 2B, Supplementary Figure contexts, including CH12-LX, Liver and mES, and 500K in most cases. S12B–S16B & Figure S23). At resolutions were also involved in exactly the same way, of 1M and 500K, in most cases chromoR and consistent comparison results were Distance stratum and multiHiCcompare can achieve a received (Supplementary Figure S17–S22 & To investigate how well the different normal- smaller CV and larger Spearman coeffi- Figure S24). ization methods can produce a consistent cient than raw and the other methods, especially in comparison to ICE; that is, Raw SCN HiCNorm ICE 0 m the correlation of replicates by chromoR and multiHiCcompare is generally higher

at low-resolution levels while the corre- 50 m lation by ICE is weaker, even weaker than that of raw in some cases. As for 100K, 10K and 5K resolutions, no signif- 100 m icant difference can be observed in the KR chromoR multiHiCcompare box plots of CV and Spearman coeffi- 0 m 4 cient between these methods. Thus, 3 the results of the comparison between

replicate correlations vary at different 50 m 2 resolution levels. 1 3. For comparability of replicates between 100 m contexts, an MA plot between contact 0 matrices of GM12878 and IMR90 was Figure 1. Comparison of heat maps between different methods. These heat maps correspond to generated to detect and visualize the IF raw and normalized contact matrices for chromosome 1 (chr1: 0–100,000,000) of the Hi-C sample difference (Figure 2C & Supplementary GM12878–001 at 1M resolution. Figure S12C–S16C). Generally, the loess ICE: Iterative correction and eigenvector decomposition; KR: Knight-Ruiz; SCN: Sequential line fitted to the MA plot is improved component normalization.

2.5 2.5 2.5 1. 5 2.0 2.0 2.0

1. 5 1. 5 1. 0 1. 5

1. 0 1. 0 1. 0 LogCPM (IF) LogCPM (IF) LogCPM (IF) 0.5 LogCPM (IF) 0.5 0.5 0.5

0.0 0.0 0.0 0.0 1 4 1 4 1 4 1 4 on on on on K562-R1 K562-R2 K562-R1 K562-R2 K562-R1 K562-R2 K562-R1 K562-R2 2878-00 2878-00 2878-00 2878-00 2878-002 2878-003 2878-004 2878-005 2878-034 2878-035 2878-036 2878-037 2878-038 2878-039 2878-040 2878-041 2878-042 2878-002 2878-003 2878-004 2878-005 2878-034 2878-035 2878-036 2878-037 2878-038 2878-039 2878-040 2878-041 2878-042 2878-002 2878-003 2878-004 2878-005 2878-034 2878-035 2878-036 2878-037 2878-038 2878-039 2878-040 2878-041 2878-042 2878-002 2878-003 2878-004 2878-005 2878-034 2878-035 2878-036 2878-037 2878-038 2878-039 2878-040 2878-041 2878-042 K562-07 K562-07 K562-07 K562-07 K562-069 K562-070 K562-071 K562-072 K562-073 K562-069 K562-070 K562-071 K562-072 K562-073 K562-069 K562-070 K562-071 K562-072 K562-073 K562-069 K562-070 K562-071 K562-072 K562-073 IMR90-050 IMR90-052 IMR90-053 IMR90-054 IMR90-055 IMR90-056 IMR90-057 IMR90-050 IMR90-052 IMR90-053 IMR90-054 IMR90-055 IMR90-056 IMR90-057 IMR90-050 IMR90-052 IMR90-053 IMR90-054 IMR90-055 IMR90-056 IMR90-057 IMR90-050 IMR90-052 IMR90-053 IMR90-054 IMR90-055 IMR90-056 IMR90-057 IMR90-Dix IMR90-Dix IMR90-Dix IMR90-Dix GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1

KR chromoR multiHiCcompare

2.5 2.0 3.0

2.0 2.5 1. 5 2.0 1. 5 1. 0 1. 5 GM12878 1. 0 LogCPM (IF) LogCPM (IF) LogCPM (IF) 1. 0 IMR90 0.5 K562 0.5 0.5

0.0 0.0 0.0 4 4 4 on on on K562-R1 K562-R2 K562-R1 K562-R2 K562-R1 K562-R2 2878-0 01 2878-0 01 2878-0 01 2878-002 2878-003 2878-004 2878-005 2878-034 2878-035 2878-036 2878-037 2878-038 2878-039 2878-040 2878-041 2878-042 2878-002 2878-003 2878-004 2878-005 2878-034 2878-035 2878-036 2878-037 2878-038 2878-039 2878-040 2878-041 2878-042 2878-002 2878-003 2878-004 2878-005 2878-034 2878-035 2878-036 2878-037 2878-038 2878-039 2878-040 2878-041 2878-042 K562-07 K562-07 K562-07 K562-069 K562-070 K562-071 K562-072 K562-073 K562-069 K562-070 K562-071 K562-072 K562-073 K562-069 K562-070 K562-071 K562-072 K562-073 IMR90-050 IMR90-052 IMR90-053 IMR90-054 IMR90-055 IMR90-056 IMR90-057 IMR90-050 IMR90-052 IMR90-053 IMR90-054 IMR90-055 IMR90-056 IMR90-057 IMR90-050 IMR90-052 IMR90-053 IMR90-054 IMR90-055 IMR90-056 IMR90-057 IMR90-Dix IMR90-Dix IMR90-Dix GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1 GM1

0.98 icient

0.30 f

0.94

CV 0.15 man coef

Spear 0.90 0.00 e w e m w m KR KR ICE ICE Ra SCN Ra SCN omoR omoR chr HiCNor chr HiCNor multiHiCcompar multiHiCcompar Raw SCN HiCNorm ICE Median: 0.0211 Median: 0.0227 Median: 0.0217 1.5 Median: 0.0245 IQR: 0.216 IQR: 0.221 IQR: 0.236 IQR: 0.226 0.5 0.5 0.5 1.0

0.5 M M M 0.0 M 0.0 0.0 0.0

-0.5 -0.5 -0.5 -0.5

1 123 0 123 4 0 123 4 0 123 A A A A

KR chromoR multiHiCcompare

Median: 0.0229 0.6 Median: 0.0339 Median: 0 IQR: 0.221 IQR: 0.154 IQR: 0.172 0.4 0.5 0.5 0.2

Zero line M M M 0.0 0.0 Loess line -0.2

-0.5 -0.5 -0.6

0 123 4 0.5 1.5 2.5 3.5 0.0 1.0 2.0 3.0 A A A

Figure 2. Comparison of statistical quality between different methods. This comparison is conducted on raw and normalized contact matrices of human chromosome 1 at 1M resolution. (A) Box plots of the interaction frequency for replicates of GM12878, IMR90 and K562. (B) Box plots of CV and Spearman coefficient for GM12878 replicates. (C) MA plot between replicates of GM12878 and IMR90. CV: Coefficient of variation; ICE: Iterative correction and eigenvector decomposition; IQR: Interquartile range; KR: Knight-Ruiz; SCN: Sequential component normalization.

1.0 2.5 Raw SCN 0.9 HiCNorm 2.0 ICE KR 0.8 chromoR multiHiCcompare 1.5 0.7

0.6 1.0 Raw 0.5 SCN HiCNorm 0.5 ICE 0.4 KR Average Spearman coefficient Average coefficient of variance chromoR multiHiCcompare 0.0 0.3 2.5 m 1 m 500 K 250 K 100 K 50 K 2.5 m 1 m 500 K 250 K 100 K 50 K Resolution Resolution

Figure 3. Comparison of average coefficients of variation and Spearman coefficients of replicates between different methods at multiple resolutions. The comparison is based on the raw and normalized contact matrices for chromosome 1 of GM12878 at six resolution levels, including 2.5M, 1M, 500K, 250K, 100K and 50K. ICE: Iterative correction and eigenvector decomposition; KR: Knight-Ruiz; SCN: Sequential component normalization.

exponential decay pattern across samples, differ from each other in detail. In the not. This investigation was also conducted the IF of raw and each normalized contact reduction of such differences, the effec- on the three mouse contexts, including matrix within the three human contexts, tiveness of these normalization methods CH12-LX, liver and mES, in exactly the same including GM12878, IMR90 and K562, was involved in the paper is not significant, way, with identical comparison results fitted against the distance using loess on a except for multiHiCcompare, which presents received (Supplementary Figures S29–S32). log scale. The curves corresponding to a much better consistency of curve changes In addition, the SCC between replicates chromosome 1 and 18 at two resolution in all cases. It is no surprise that multiHiC- within GM12878 and IMR90 were compared levels, including 1M and 500K, were compare can achieve a much smaller with that across the two contexts. As shown visualized (Figure 4 & Supplementary variation between fitted curves, since it takes in box plots for chromosome 1 at resolutions Figures S26–S28). Although the raw loess into account the distance stratum by means of 100K and 50K (Supplementary Figure curves exhibit a similar decay pattern across of a novel MD plot concept in the normal- S33), it can be seen that ICE and multiHiC- replicates and even across contexts, they ization design, while the other methods do compare have a slightly higher correlation

Raw SCN HiCNorm ICE

3.0 3.0 3.0 3.0

2.0 2.0 2.0 2.0

LogCPM (IF) 1.0 LogCPM (IF) 1.0 LogCPM (IF) 1.0 LogCPM (IF) 1.0

0.0 0.0 0.0 0.0 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 Log10 (distance + 1) Log10 (distance + 1) Log10 (distance + 1) Log10 (distance + 1)

KR chromoR multiHiCcompare

3.0 3.0 3.0

2.0 2.0 2.0 GM12878 IMR90 K562

LogCPM (IF) 1.0 LogCPM (IF) 1.0 LogCPM (IF) 1.0

0.0 0.0 0.0 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 Log10 (distance + 1) Log10 (distance + 1) Log10 (distance + 1)

Figure 4. Comparison of loess-fitted curves of the interaction frequency versus bin distance between different methods. These curves are derived from raw and normalized contact matrices for chromosome 1 of GM12878, IMR90 and K562 at 1M resolution. ICE: Iterative correction and eigenvector decomposition; IF: Interaction frequency; KR: Knight-Ruiz; SCN: Sequential component normalization.

Vol. 68 | No. 1–3 | © 2020 Hongqiang Lyu 56 www.BioTechniques.com Jaccard Index for concordance of TAD 100 K 50 K boundaries within the contexts with more 0.8 0.7 than two replicates was calculated, and the corresponding box plots for chromosome 0.7 0.6 1 and 18 of GM12878, IMR90, K562 and CH12-LX at resolutions of 100K and 50K 0.6 0.5 were separately represented without outliers (Figure 5 & Supplementary Figure 0.5 0.4 S36). It was shown that SCN and KR had a Jaccard index Jaccard index similar Jaccard Index distribution in almost 0.4 0.3 all the cases, and their Jaccard Index was higher than that of any other method in nine 0.3 0.2 out of 16 cases, especially compared with chromoR and multiHiCcompare. Thus, SCN KR KR ICE ICE Raw Raw SCN SCN and KR had the best TAD reproducibility in comparison to chromoR and multiHiC- chromoR chromoR HiCNorm HiCNorm compare. For HiCNorm and ICE, the distribution of the Jaccard Index varies greatly

MultiHiCcompare MultiHiCcompare compared with the other methods – in some cases the Jaccard Index of ICE is even 100 K 50 K higher than that of SCN and KR. In addition, 0.7 the Jaccard Index of TAD boundaries 0.8 between two different contexts were also computed, and the corresponding box plots 0.6 0.7 for chromosome 1 of GM12878 and IMR90 at resolutions of 100K and 50K were given 0.5 0.6 (Supplementary Figure S37). Obviously, the reproducibility of TADs across contexts is 0.5 0.4

Jaccard index lower than that within context in all the Jaccard index cases. This is consistent with our expecta- 0.3 0.4 tions, since the normalized matrices in the same context should share a more similar TAD architecture compared with those KR KR ICE ICE Raw Raw

SCN SCN spanning different contexts. The present comparison deals with six chromoR chromoR HiCNorm HiCNorm Hi-C normalization methods to determine the difference between them. Although MultiHiCcompare MultiHiCcompare there is no method that can be considered the gold standard to correct for systematic Figure 5. Comparison of topologically associating domain architecture reproducibility of repli- biases of Hi-C data, there are still some cates between different methods. Box plots of Jaccard Index for topologically associating domains inferences that are beneficial to the choice between replicates are derived from raw and normalized contact matrices for (A) chromosome 1 and (B) chromosome 18 of GM12878 at 100K and 50K resolutions. of these methods. Different from the other ICE: Iterative correction and eigenvector decomposition; KR: Knight-Ruiz; SCN: Sequential methods, multiHiCcompare is an across- component normalization. sample approach and takes into account the decay pattern of IF versus distance. These advances give multiHiCcompare the level within contexts compared with the diagonal, and the AD ratio of intracontext to power to achieve an obviously better perfor- other methods. All the methods produce a intercontext is less than 1 at most bin mance in most considerations, including lower intercontext correlation level relative distances for all the methods; that is, the AD distribution of IF, comparability of repli- to their corresponding intracontext corre- variation within context is usually smaller cates between contexts and consistency of lation levels in all cases. Furthermore, this than that across contexts. This is in line with distance stratum. On the contrary, for such difference in variation was also checked for expectations. cross-sample approaches, larger memory each bin distance with the help of AD is needed in order to allow all the matrices (Supplementary Figures S34 & S35). TAD architecture to be loaded at the same time. Among the Generally, multiHiCcompare still shows a To compare the reproducibility of TAD archi- other methods that are individual-sample smaller variation, especially near the tecture between different methods, the approaches, SCN, KR and ICE are all

based on the matrix-balancing strategy, AUTHOR CONTRIBUTIONS 11. Lajoie BR, Dekker J, Kaplan N. The Hitchhiker’s guide to Hi-C analysis: practical guidelines. Methods 72, 65–75 and have been widely used in recent studies HL conceived and designed the study. HL (2015). due to their conceptual simplicity and and EL carried out acquisition of the data. 12. Le TBK, Imakaev MV, Mirny LA, Laub MT. High-resolution mapping of the spatial organization of a bacterial parameter-free nature. SCN and KR show HL, EL and ZW performed the analysis and chromosome. Science 342(6159), 731–734 (2013). 13. Dixon JR, Selvaraj S, Yue F et al. Topological domains in a better reproducibility of TAD architecture. interpretation of data. EL and ZW drafted the mammalian genomes identified by analysis of chroma- ICE usually produces a preferable compa- manuscript. HL revised it critically for tin interactions. Nature 485(7398), 376 (2012). 14. Imakaev M, Fudenberg G, Mccord RP et al. Iterative cor- rability of replicates between contexts than important intellectual content, and gave final rection of Hi-C data reveals hallmarks of chromosome the other methods except for multiHiC- approval of the version to be published. organization. Nat. Methods 9(10), 999–1003 (2012). 15. Callister SJ, Barry RC, Adkins JN et al. Normalization compare, but has a slightly weaker corre- approaches for removing systematic biases associated with mass spectrometry and label-free proteomics. J. lation of replicates at resolutions of 1M ACKNOWLEDGMENTS Proteome Res. 5(2), 277–286 (2006). and 500K in most cases. HiCNorm exhibits The authors would like to thank Wenjun Yang 16. Vandernoot VA, Langevin SA, Solberg OD et al. cDNA normalization by hydroxyapatite chromatography to good performance in all the consider- for his assistance in sample collection. enrich transcriptome diversity in RNA-seq applications. Biotechniques 53(6), 373–380 (2012). ations, although some additional genomic 17. Shavit Y, Merelli I, Milanesi L, Lio P. How computer features, such as genome sequence science can help in understanding the 3D genome archi- FINANCIAL & COMPETING tecture. Brief. Bioinformatics 17(5), 733–744 (2016). and mappability information, need to be INTERESTS DISCLOSURE 18. Hu M, Deng K, Selvaraj S, Qin ZH, Ren B, Liu JS. HiCNorm: removing biases in Hi-C data via Poisson specified during implementation since it This work has been supported by the regression. Bioinformatics 28(23), 3131–3133 (2012). is an explicit approach. As for chromoR, National Natural Science Foundation of 19. Cournac A, Marie-Nelly H, Marbouty M, Koszul R, Moz- ziconacci J. Normalization of a chromosomal contact its de-nosing procedure helps to achieve China under Grant 61602367. The authors map. BMC Genomics 13(1), 436 (2012). smaller IQR and higher correlation of repli- 20. Knight PA, Ruiz D. A fast algorithm for matrix balancing. have no other relevant affiliations or financial IMA J. Numer. Anal. 33(3), 1029–1047 (2013). cates, but at the same time blurs the details involvement with any organization or entity 21. Shavit Y, Lio P. Combining a wavelet change point and of contact matrix, especially at low resolu- the Bayes factor for analysing chromosomal interaction with a financial interest in or financial conflict data. Mol. Biosyst. 10(6), 1576–1585 (2014). tions. Thus, chromoR is preferably used with the subject matter or materials 22. Stansfield JC, Cresswell KG, Dozmorov MG. multiHiC- compare: joint normalization and comparative analysis for the normalization of Hi-C data at higher discussed in the manuscript apart from of complex Hi-C experiments. Bioinformatics 35(17), resolution levels. To facilitate selection of 2916–2923 (2019). those disclosed. 23. Cavalli G, Misteli T. Functional implications of genome one of the six Hi-C normalization methods, No writing assistance was utilized in the topology. Nat. Struct. Mol. Biol. 20(3), 290–299 (2013). the comparison results for these consider- 24. Sureka R, Wadhwa R, Thakur SS, Pathak RU, Mishra RK. production of this manuscript. Comparison of nuclear matrix and mitotic chromosome ations, the representative implementations scaffold proteins in Drosophila S2 Cells – transmission of hallmarks of nuclear organization through mitosis. of these methods and their consumption of OPEN ACCESS Mol. Cell. Proteomics 17(10), 1965–1978 (2018). computational resources are summarized 25. Naumova N, Imakaev M, Fudenberg G et al. Organiza- This work is licensed under the Attribution- tion of the mitotic chromosome. Science 342(6161), 948–953 (2013). in Table 2. NonCommercial-NoDerivatives 4.0 Unported 26. Rudan MV, Barrington C, Henderson S et al. Comparative License. To view a copy of this license, visit Hi-C Reveals that CTCF underlies evolution of chromosomal domain architecture. Cell Rep 10(8), 1297–1309 FUTURE PERSPECTIVE http://creativecommons.org/licenses/ (2015). In light of the comparison results in this 27. Barrett T, Wilhite SE, Ledoux P et al. NCBI GEO: archive by-nc-nd/4.0/ for functional genomics data sets-update. Nucleic Acids paper, it has been shown that the cross- Res. 41(D1), D991–D995 (2013). sample normalization approach demon- 28. Yang T, Zhang F, Yardimci GG et al. HiCRep: assessing REFERENCES the reproducibility of Hi-C data using a stratum-adjusted strates significantly better performance than 1. Lieberman-Aiden E, Van Berkum NL, Williams L et al. correlation coefficient. Genome Res. 27(11), 1939–1949 individual-sample methods in most consid- Comprehensive mapping of long-range interactions re- (2017). veals folding principles of the human genome. Science 29. Shin HJ, Shi Y, Dai C et al. TopDom: an efficient and de- erations. Regarding the pace of Hi-C data 326(5950), 289–293 (2009). terministic method for identifying topological domains 2. Ethier SD, Miura H, Dostie J. Discovering genome reg- in genomes. Nucleic Acids Res. 44(7), (2016). production and the lack of such approaches ulation with 3C and 3C-related technologies. Biochim. at present, it is expected that some new Biophys. Acta 1819(5), 401–410 (2012). 3. Forcato M, Nicoletti C, Pal K, Livi CM, Ferrari F, Bicciato competitive cross-sample normalization S. Comparison of computational methods for Hi-C data methods involving joint analysis of multiple analysis. Nat. Methods 14(7), 679–685 (2017). 4. Dekker J, Rippe K, Dekker M, Kleckner N. Capturing replicates in different contexts will be chromosome conformation. Science 295(5558), 1306–1311 (2002). designed in the near future. This will pave 5. Belton J-M, Mccord RP, Gibcus JH, Naumova N, Zhan the way for optimal selection of Hi-C normal- Y, Dekker J. 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Selection of reference genes suitable for normalization of RT-qPCR data in glioma stem cells

Weiqi Dang1,2, Xiang Zhang1,2, Qinghua Ma1,2, Lu Chen1,2, Mianfu Cao1,2, Jingya Miao1,2, Youhong Cui1,2 & Xia Zhang*,1,2

CITATION T98G, LN229, 090116 and 091214. reference genes, and validated the Normfinder and coefficient of BioTechniques 68: 130–137 (March Quantitative real-time reverse- geometric mean of these genes to variation methods to identify novel 2020) 10.2144/btn-2019-0098 transcription PCR was employed normalize the expression of stemness reference genes, with greater using 11 reference genes identified genes by GSCs. Therefore, it is expression stability in glioma stem ABSTRACT through a PubMed literature search, necessary to select novel cell-specific cells, for the study of gene expression. Considering the importance of gene and the assessment of stability reference genes with greater expression studies for understanding through the geNorm, Normfinder and expression stability for gene KEYWORDS the biology of glioma stem cells coefficient of variation methods was expression studies in GSCs. glioma stem cells • quantitative (GSCs), we aimed to identify the performed to select suitable reference real-time PCR • reference genes reliable reference genes in GSCs that genes. We found that HPRT1 and METHOD SUMMARY • stemness-related genes • tumor- were derived from the glioma cell lines RPL13A were the most suitable We employed the geNorm, initiating cells

Glioma is the most prevalent adult brain GRAPHICAL ABSTRACT tumor [1]. According to the 2016 WHO (Geneva, Switzerland) classification of CNS tumors, glioma is graded from I to IV, and the most malignant form of glioma (Grade IV) is referred GeNorm to as glioblastoma (GBM). GBM is featured by the remarkable cellular heterogeneity and le differential hierarchies of tumor cells [2], and GSC ence gene remains as an incurable disease. The average RT-qPCR NormFinder r survival time for patients with GBM is about fe

14 months [1]. nal re The current available treatments for er Obtain the most stab Coefficient of int patients diagnosed with glioma are surgery, variation chemotherapy and radiotherapy [3]; however, advanced stage of the disease is often difficult to treat owing to increased chemo- and radio- Monolayer resistance by tumor cells, glioma recurrence and poor survival outcome prevail [4,5]. Culmi- nating evidence has indicated that a subset of tumor cells, referred to as glioma stem cells radiation and chemotherapy, and contribute cable for the improvement in the current patho- (GSCs) or glioma-initiating cells, is critical for to therapeutic resistance and tumor reiniti- logical diagnosis and clinical therapeutics GBM progression and recurrence, however, ation [5]. The pivotal role of GSCs in promoting for GBM. the cellular origin of these cells still remains malignant progression and tumor recurrence Gene expression analysis using quantitative debatable [2,4]. GSCs are known to possess in GBMs indicates that this subpopulation of real-time reverse-transcription PCR (RT-qPCR) some features of embryonic or somatic stem cells is a critical target for GBM treatment, is a promising approach to identify novel genes cells, such as self-renewal capacity and multi- and targeting GSCs could overcome the thera- and molecular biomarkers [6]. lineage differentiation potential, and resistance peutic resistance. Therefore, it is necessary to continued online… to chemotherapy and radiotherapy [4]. GSCs analyze the etiology and development of GSCs are often found to be enriched in GBM after and search for new biomarkers that are appli-

1Institute of Pathology & Southwest Cancer Center, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, China; 2Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing 400038, China; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0098 article here

ELIMU-MDx: a web-based, open-source platform for storage, management and analysis of diagnostic qPCR data

Silvan Krähenbühl1,2, Fabian Studer1,2, Etienne Guirou1,2, Anna Deal1,2,3, Philipp Mächler1,2, Salome Hosch1,2, Maximilian Mpina1,2,3,4, Sarah Mswata4, Claudia Daubenberger1,2 & Tobias Schindler*,1,2

CITATION RDML data standard. This platform The global market of in vitro biological analysis [3]. Quantitative BioTechniques 68: 22–27 (January was designed as an open-source diagnostics, comprising tools to PCR (qPCR) is a well-established 2020) 10.2144/btn-2019-0064 software tool and can be accessed detect, diagnose or monitor method for the detection, quantifi- through the web browser on all diseases, is estimated to be worth cation and typing of bacteria, ABSTRACT major operating systems. US$40–45 billion [1]. One of the viruses, fungi and protozoa in the The Electronic Laboratory Infor- fastest growing areas is the identi- areas of clinical and veterinary mation and Management Utensil METHOD SUMMARY fication of infectious diseases diagnostics, as well as food for Molecular Diagnostics (ELIMU- ELIMU-MDx is an open-source using molecular diagnostics, which safety [4]. Among the biggest MDx) is a user-friendly platform web-application developed using is becoming an integral part of advantages of qPCR-based designed and built to accelerate PHP to analyze, manage, validate medical practice and public health diagnosis are the universality in the turnaround time of diagnostic and store user-provided qPCR data worldwide [2]. Molecular diagnostic designing and developing new qPCR assays. ELIMU-MDx is in a MySQL database. tools, especially nucleic acid ampli- assays as well as the widespread compliant with the MIQE guide- fication techniques, provide faster, availability of the technology. lines and has extensive data- KEYWORDS more sensitive and often more continued online… import capabilities for all major diagnostic • ELIMU-MDx • infectious cost-effective diagnoses than tradi- qPCR instruments by using the diseases • MIQE • qPCR • RDML tional culture methods for micro-

ELIMU – MDx accelerate your qPCR analysis

Sample types Sample inventory Sample metadata Lab journal Instruments Virtual freezer biological type of storage and e.g., identifier and Modules Repository for lab Repository for instruments Inventory of reagents, specimen shipment logs collection date protocols and SOPs and maintenance logs oligos, controls and qPCR programs Central node

Components

Parameters

Samples Experiments Assays

fully compatible with the MIQE guidelines

Automated quality control, Assay definition file Data analysis and interpretation of data. Parameters for analysis and interpretation of data

Results

Quality control parameters Interpretation parameters Documentation parameters Reporting of – Acceptable Cq values for – Cut-offs of Cq values for – Assay-specific values such qPCR results positive, negative and qualitative interpretation of as limit of detections, non-template controls qPCR data qPCR efficiencies, qPCR RDML data qPCR melt data Non-qPCR data Statistical – Thresholds for acceptable – Slope and intercept for reaction volumes and qPCR RDML with melt Data formatted based on analysis and Data in xlsx file format temperature data number of replicates and quantification of qPCR qPCR target genes RDML standard visualization of attached qPCR data standard devitions result – MIQE compliant

Figure 1. Structure of ELIMU-MDx.

1Department of Medical Parasitology & Infection Biology, Swiss Tropical & Public Health Institute, Basel, Switzerland; 2University of Basel, Basel, Switzerland; 3Equatorial Guinea Malaria Vaccine Initiative, Malabo, Republic of Equatorial Guinea; 4Bagamoyo Research & Training Centre, Ifakara Health Institute, Bagamoyo, United Republic of Tanzania; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0064 article here

Korena K Mafune*,1, Bruce J Godfrey2, Daniel J Vogt1 & Kristiina A Vogt1

CITATION varying ratios of 16 taxa. The data METHOD SUMMARY and 1D ligation sequencing BioTechniques 68: 72–78 (February were processed through our selected Genomic DNA from 16 diverse protocols, respectively. The raw 2020) 10.2144/btn-2019-0072 pipeline. The MinION recovered all fungal taxa was extracted from data were basecalled through mock community members, when fungal sporocarps and fungal Oxford’s Albacore software, and ABSTRACT mixed at equal ratios. When a taxon cultures isolated directly from roots then the data were processed The Oxford Nanopore Technologies was represented at a lower ratio, it and leaves. The identities of species through a pipeline that calculates MinION™ sequencer holds the was not recovered or decreased in were confirmed using Sanger a highly accurate consensus capability to generate long amplicon relative abundance. Despite high sequencing, and then mixed sequence from operational reads; however, only a small amount error rates, highly accurate consensus together at varying ratios to create taxonomical units. of information is available regarding sequences can be derived. This three fungal mock communities. methodological approaches and the methodological approach identified The mock communities were KEYWORDS ability to identify a broad diversity of all mock community taxa, demon- amplified using tagged primers fungal communities • fungal fungal taxa. To assess capabilities, strating the MinION can be used as ITS1f-Kyo and LR3-I, and then diversity • fungi • high-throughput three fungal mock communities were a practical alternative to profile processed through library prepa- sequencing • MinION™ nanopore sequenced, each of which had fungal communities. ration following Oxford’s barcoding sequencer • mock library

High-throughput sequencing (HTS) provides However, the ongoing improvements of third- The Oxford Nanopore Technol- the ability to multiplex and profile fungal generation HTSs hold promise for diversity ogies (ONT) MinION™ sequencer is a communities across environmental analyses of environmental samples. For small third-generation HTS platform that samples, including but not limited to leaf example, they provide longer sequence became commercially available in 2015 [14]. tissue [1], soil [2] and roots [3,4]. Despite reads, and despite the higher error rates It has the ability to generate longer reads these capabilities, established HTS methods (5–13%, [6,7]), high-quality reference data and is efficient in determining bacterial (e.g., Illumina and Pyrosequencing) are can be generated and species-level identifi- microbiomes [9,15]. limited in taxonomic resolution because of cation can be inferred by averaging out the continued online… their shorter sequence reads [5] and the need errors with a consensus from multiple for expensive analytical technology. reads [8–13].

Table 3. Number of sequences passed through built-in Albacore quality filtering and subsequent filtering at q9, the total amount of bases, mean read length and mean read quality (q-score), and the percent of sequences remaining. Mean read Mean read Sequences Percent Library Number of reads Total bases length quality passed at Q9 remaining Mock A 46,216 66,187,339 1446 8.4 12,657 27.39%

Mock B 59,688 83,342,273 1381 8.4 15,196 25.46%

Mock C 25,761 35,891,285 1375 8.4 6832 26.52%

On average, 26.36% of sequences were recovered, with a high consistency among the samples with a standard deviation of 0.008. Average read quality was determined by NanoPlot, and reads were filtered through NanoFilt. Both were provided by [28]. Mock A was mixed at equal ratios, and Mock B and C had staggered ratios of select species.

1School of Environmental & Forest Sciences, College of the Environment, University of Washington, Box 352100, Seattle, WA 98195-2100, USA; 2Department of Environmental & Civil Engineering, University of Washington, Box 355014, Seattle, WA 98195-2100, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0072 article here

A simplified method to calculate telomere length from Southern blot images of terminal restriction fragment lengths

Lisa F Lincz*,1,3,4,5, Fiona E Scorgie1,4,5, Madhu B Garg2,4,5, Jayne Gilbert2,4,5 & Jennette A Sakoff2,4,5,6

CITATION method for analyzing telomere METHOD SUMMARY of each telomere smear to enable BioTechniques 68: 28–34 (January smears. Basic 2D gel imaging Here we demonstrate a new method the software to calculate net 2020) 10.2144/btn-2019-0082 software was used to automatically of analysis to calculate mean intensity and MW at each position subtract background, generate telomere length from Southern blot (i). Then, the data were batch ABSTRACT standard curves and calculate net images of DNA terminal restriction exported into Microsoft Excel for Southern blotting of DNA terminal intensity and MW at each position (i) fragment lengths using basic final calculation of mean telomere restriction fragment lengths is the along the telomere smear. Our imaging software. ImageQuant was length. gold standard for measuring mean method required no statistical used to automatically subtract telomere length. Analysis of the final software or major data manipulation background, generate standard KEYWORDS image is a crucial step in this process, and correctly classified >80% of 18 curves and set MW across the width cancer cell lines • mean telomere however, current techniques are samples as having short, medium or of the image. A series of 31–60 length • net intensity • peripheral cumbersome and prone to error. Here long telomeres compared with small (5 pixel) boxes were blood • Southern blot • terminal we present a simple and accurate 33–72% using other methods. manually inserted along the length restriction fragment length

Human telomeres are protective nucleoprotein of the image to account for any gel curvature, describing the methodology and analysis structures consisting of 5–15 kb of tandem and then background removal is performed procedures have been published and are still TTAGGG repeats that cap the ends of chromo- manually for each NI at every MW position extensively referenced [10,11]. However, calcu- somes to help maintain genomic stability [1,2]. before summing the required data for use lated MTL does not always reflect the apparent Telomeres progressively shorten by approxi- in the MTL calculations. nd visualized by size distribution of telomeric smears depicted mately 50 bp with each cell division in normal hybridization to a labeled telomere-specific in hybridized images [9,11–13]. cells [3,4]; however, they can also be negatively oligonucleotide probe. The intensity and Although the laboratory procedures for affected by environmental, genetic and lifestyle size distribution of the resulting telomeric generating and visualizing telomeric TRFL are factors [5]. Once a critical telomere length is smear are used to calculate the MTL of the generally straightforward and reproducible by reached, cells are no longer able to divide and cell population. The first publication of this a skilled molecular scientist, the manual image senescence ensues [6,7]. Accordingly, telomere technique used traditional Southern blotting analyses described in current protocols are length is often referred to as a mitotic clock methodology. Based on the assumption of cumbersome, laborious, require knowledge, and used as a biomarker of cellular aging and equal efficiency of DNA transfer at all areas use of statistical software and fraught with risk of related diseases [8]. of the gel, and with the number of telomere potential for human error [10–12]. All strategies Because telomere length varies between repeats per DNA fragment being proportional require conversion of distance (in pixels) to chromosomes, cells and tissues within any to the DNA length, calculated MTL by the MW, using data that must be extracted from the individual, obtaining an accurate measurement Equation 1: image analysis and transferred to a separate can be challenging. Although advanced software package to generate a standard ∑∑NI //NI MW molecular techniques have been developed, ii( i ) curve. This must be repeated for each side the original method of terminal restriction where NIi is the net intensity at position (i) and of the image to account for any gel curvature, fragment length (TRFL) analysis remains MWi is the MW at position (i) [9]. Later publica- and then background removal is performed the gold standard for quantitation of mean tions using in-gel hybridization revised the manually for each NI at every MW position telomere length (MTL) [3,9]. The method uses calculation to Equation 2 [3]: before summing the required data for use in restriction enzymes to digest gDNA and to the MTL calculations. NI MW / NI leave behind intact telomere repeats, which ∑×( ii) ∑ i continued online… are then resolved by agarose gel electropho- However, both equations remain in use today. resis a This must be repeated for each side In the last 10 years, detailed protocols

1Haematology Department, Calvary Mater Newcastle, Australia; 2Medical Oncology Department, Calvary Mater Newcastle, Australia; 3School of Biomedical Sciences & Pharmacy, University of Newcastle, Callaghan, Australia; 4Hunter Medical Research Institute, New Lambton, Australia; 5Hunter Cancer Research Alliance, NSW, Australia; 6School of Environmental & Life Sciences (Chemistry), Faculty of Science, University of Newcastle, Callaghan, Australia; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0082 article here

Vol. 68 | No. 1–3 | © 2019 Liza Lincz 62 www.BioTechniques.com Reports Erythrosin B: a versatile colorimetric and fluorescent vital dye for bacteria Josef D Franke*,1, Ann L Braverman1, Alison M Cunningham1, Erin E Eberhard1 & Greg A Perry2

ABSTRACT Determining cell viability is fundamental to of cells are spread on agar plates and the Rapidly assaying cell viability for many cell biological studies. Due to their number of colonies are counted after an diverse bacteria species is not always ease of use, low cost and rapid results, incubation period and compared to control straightforward. In eukaryotes, cell numerous dyes are commonly used for cell- plates. This method has two significant viability is often determined using colorimetric dyes; however, such dyes viability determination in eukaryotes. drawbacks. First, the proportion of dead have not been identified for bacteria. Membrane-exclusion dyes (e.g., trypan blue cells in a population can be overesti- We screened different dyes and found and propidium iodide) are rapid and enter mated because the method requires that that erythrosin B (EB), a visibly red dye cells with compromised plasma all viable cells undergo sufficient rounds with fluorescent properties, functions membranes, such that dye-positive cells of division to form a visible colony. Living as a vital dye for many Gram-positive are scored as dead [1,2]. Other dyes, such cells unable to divide, or dividing slower and -negative bacteria. EB worked at as fluorescein diacetate (FDA) or methylene than controls, will be incorrectly scored as a similar concentration for all bacteria blue, indicate a cell’s viability based on a dead if they fail to divide sufficiently and studied and incubations were as short particular metabolic activity [3,4]. For these form a colony in the appropriate amount of as 5 min. Given EB’s spectral properties, dyes, the initially incubated dye is chemi- time. Second, the CFU method is particu- diverse experimental approaches are cally altered by cellular enzymes. This larly time consuming when working with possible to rapidly visualize and/or quantitate dead bacterial cells in a results in a modified dye with new spectral slow-growing bacteria in which colony population. As the first broadly appli- properties that indicates living cells with formation takes several days, resulting in cable colorimetric viability dye for metabolic activity. This diversity of undesirable waiting periods between exper- bacteria, EB provides a cost-effective eukaryotic dyes allows for cell viability to iments. To date, there are no colorimetric alternative for researchers in academia be determined based on the characteristics bacterial vital dyes available to researchers. and industry. of either living cells or dead cells, and using Such a dye would save time and allow either colorimetric or fluorescence-based bacterial viability to be determined by METHOD SUMMARY methods of detection. light microscopy. It could also be used in Incubation of erythrosin B with bacteria Unlike eukaryotes, no rapid colori- experiments ranging from routine viability specifically stains membrane-compro- metric vital dyes are routinely used to assays on a specific bacterial species to mised dead cells. Live and dead cells assay cell viability in bacteria. Propidium high-throughput screens identifying novel can then be quantified via bright-field iodide is often used as a vital dye in combi- bactericidal compounds and analyzing microscopy or any number of instruments nation with other dyes that stain all cells multi-species samples. that quantitate fluorescence. (e.g., SYTO9), forming the basis of different Here, we show that erythrosin B (EB), live/dead assays such as the widely used a dye with colorimetric and fluorescent BacLight™ kit [5]. There are downsides to properties, functions as an indicator of the BacLight kits, in that they are costly bacterial viability. EB staining is rapid and and require expensive fluorescence quanti- works at a single concentration for diverse tation equipment. Also, in these assays species of bacteria. The dye is inexpensive cells do not always fall into discrete live or and allows for live/dead determination dead categories as intermediate popula- in both colorimetric and fluorescence- KEYWORDS tions are possible [6,7]. Depending on the based assays for low, medium and high- bacteria • cell viability • flow cytometry bacterial species being investigated, the throughput experimentation. • membrane-exclusion dye • microscopy concentration of each dye and the relative • vital dye ratio of the two dyes often require optimi- METHODS & MATERIALS 1Department of Biology, Creighton University, zation [8,9]. As a result, these kits are not Organisms & reagents 2500 California Plaza, Omaha, NE 68178, well suited for field work, research involving The following bacteria were used in this USA; 2Department of Medical Microbiology & Immunology, 2500 California Plaza, Omaha, multiple species or adherent cells in study: Bacillus cereus (Carolina Biological, NE 68178, USA; *Author for correspondence: biofilms [10,11]. NC, USA 154870), Escherichia coli (MG1655), [email protected] Alternatively, bacterial cell viability can Klebsiella pneumoniae (Carolina Biological, BioTechniques 68: 7–13 (January 2020) be determined by the colony-forming units 155095A), Pseudomonas fluorescencs 10.2144/btn-2019-0066 (CFUs) method [4]. Here, dilute solutions (Carolina Biological, 155255), Serettia

marcesans (Carolina Biological, 155450), Untreated + EB Heat shocked + EB Ethanol treated + EB CaCl2 treated + EB Mixture + EB Streptomyces albus (Ward’s Science, NY, USA, 470179-180), Streptococcus mutans (Ward’s Science, 470179-582), Proteus E. coli mirabilis (Ward’s Science, 470179-138), Staphylococcus aureus (Carolina Biological, 155554) and Enterococcus faecalis (Ward’s Science, 470179-184). K. pneumonia EB (Sigma, MO, USA, 200964-5G) was prepared by dissolving powder in 10 mM Tris pH 7.5. Published values of the solubility of EB in aqueous solutions vary and may result from different prepa- P. fluorescens rations of the dye and from which supplier it is purchased. A small amount of undissolved dye was present when preparing a 0.1% (w/v) solution. This stock solution was filtered through a 0.22-μm filter to remove S. marcesans undissolved dye. To determine the concentration of this stock solution, a fresh dilute EB solution was prepared in which all dye fully dissolved after 10 min of stirring. This P. mirabilis dilute solution was serially diluted and A530 readings (the wavelength of maximal absorbance for EB) were collected, generating a reference relating EB dye concentration to A . The filtered stock solution of EB Figure 1. Erythrosin B functions as a vital dye specifically staining dead or membrane-compro- 530 mised Gram-negative bacteria. Bacterial cells were incubated with EB after heat shock, ethanol was then serially diluted, and absorbance treatment or CaCl2 incubation. Images in the ‘Mixture’ column were taken of cell suspensions values of the dilutions were compared to containing equal amounts of live, untreated cells and dead cells (either heat shocked or ethanol the reference absorbance values for the treated). Mixtures were incubated with EB and images show the ability to distinguish between living dilute solution. The stock EB concentration (dye-negative) and dead (dye-positive) cells. An arrowhead in the untreated control image (Klebsiella was determined to be 0.08% and is used pneumoniae) indicates a clearly distinguishable dye-positive dead cell among dye-negative cells, further demonstrating the visible difference in staining between dye-positive and dye-negative throughout this work. cells. The scale bar in the bottom right panel is 5 μm and applies to all panels. EB: Erythrosin B. Growth & treatments of bacteria To prepare cells for viability studies, 3-ml were centrifuged and the pellet was resus- For plate reading assays, a total of four overnight bacterial cultures were grown in pended in 1 ml of ice-cold 100 mM CaCl2 optical densities (ODs) of log-phase cells Luria broth. Fresh 5-ml cultures were inocu- and left on ice for 45 min. After this initial were collected and evenly split between two lated from the overnight cultures and grown incubation, the cells were centrifuged and tubes. Cells were centrifuged as described for 1–4 h until reaching an OD600 of 0.4–1.1. resuspended in 100 μl of 100 mM CaCl2. above. One tube was resuspended in 1 ml Cells were harvested by centrifugation of PBS (live cells), and the other tube was (15,000×g for 4 min) and then resuspended Assaying bacterial viability incubated at 70°C for 30 min (dead cells) in phosphate-buffered saline (PBS). A For bright-field microscopy assays, 10 μl of before being resuspended in 1 ml of PBS. portion of these cells were kept at room each cell suspension (untreated, heat A total of 100-μl cell mixtures were then temperature (untreated controls) while the shocked, ethanol treated and CaCl2 treated) prepared in triplicate by mixing appropriate remainder were transferred into separate was mixed with 10 μl of 0.8% EB and volumes of live and dead cell suspensions microfuge tubes for different treatments. incubated for 5 min at room temperature. to generate the following: 100% live cells,

For heat shock treatment, microfuge tubes For CaCl2-treated cells, mixtures were 75% live cells/25% dead cells, 50% live were placed at 70°C for 30 min before placed at 42°C for 60 s before returning to cells/50% dead cells, 25% live cells/75% returning to room temperature. For ethanol room temperature for the remainder of the dead cells and 100% dead cells. A total treatment, 100% ethanol (Decon Labs, Inc., incubation. A total of 5 μl of the incubated of 100 μl of 0.8% EB was added to all cell PA, USA, 2716) was added to cell suspen- dye:cell mixtures were taken to prepare wet mixtures and incubated for 5 min. Control sions to a final concentration of 30%. Cell mounts. Imaging was performed on a Zeiss cell mixtures containing 100 μl of live cells mixtures were incubated at 37°C for 30 min, Olympus BX61 microscope using an or dead cells were incubated with 100 μl centrifuged, and then the pellet was resus- UPlanSApo 100X/1.14 NA oil objective lens of PBS instead of EB. All mixtures were pended in PBS. For CaCl2 treatment, cells with an Olympus SC10 camera. centrifuged and the supernatant removed.

Vol. 68 | No. 1–3 | © 2020 Josef D Franke 64 www.BioTechniques.com The tubes were briefly spun again and Untreated + EB Heat shocked + EB EtOH treated + EB CaCl2 treated + EB Mixture + EB any residual supernatant was removed. Cell pellets were resuspended in 100 μl

PBS, vortexed and 75 μl of each mixture B. cereus was transferred to a clear 96-well plate (Nunc™, 80042LE). Absorbance readings

(530 nm; A530) were taken using a Synergy

H1 hybrid plate reader. Control A530 values E. faecalis were subtracted from the experimental A530 values prior to calculations. For flow cytometry assays, different EB cell suspensions were incubated in the presence of EB for 5 min at room temperature, centrifuged, and the cell pellets were S. aureus resuspended in 1 ml PBS. To remove excess unbound EB, the cells were washed twice in 1 ml PBS prior to analysis. For the sodium azide time course, a 5 ml S. marcesans S. albus culture was grown to an OD600 of ∼0.2 in Luria Broth at 37°C. Sodium azide was added to a final concentration of 0.07% and 0.5 ml samples were taken at different times points. Each sample was then immedi- S. mutans ately centrifuged to pellet the bacteria and stained with EB as described above to prepare samples for flow cytometry. A YETI flow cytometer (Propel Labs, Inc., CO, USA) Figure 2. Erythrosin B functions as a vital dye specifically staining dead or membrane-compro- was used to analyze cells (excitation with mised Gram-positive bacteria. Bacterial cells were incubated with EB after heat shock, ethanol a 561 nm laser/emission collected using a treatment or CaCl2 incubation. Images in the ‘Mixture’ column were taken of cell suspensions 583/30 filter). containing equal amounts of live, untreated cells and dead cells (either heat shocked or ethanol treated). Mixtures were incubated with EB and images show the ability to distinguish between living (dye-negative) and dead (dye-positive) cells. The scale bar in the bottom right panel is 5 μm and applies to all panels. EB: Erythrosin B.

Figure 3. The relative amount of dead cells in a bacterial suspension can be determined using erythrosin B absorbance values. Mixtures -EB +EB +EB +EB +EB +EB 1.2

) E. coli containing different ratios of cells (0% dead/100% 1.0 53 0 live; 25% dead/75% live; 50% dead/50% live; 75% 0.8 dead/25% live; and 100% dead/0% live) were 0.6 prepared and then similarly incubated with EB. In 0.4 these assays, dead cells were prepared by heat

P. mirabilis 0.2 shock. All mixtures were prepared in triplicate, and Absorbance (A unbound EB was removed after the incubation. (A) 0.0 0 25 50 75 100 Equal amounts of all Proteus mirabilis cell mixtures 0025 50 75 100 Control Percent dead and controls (cells not exposed to EB: -EB) were Percent dead transferred into separate wells of a 96-well plate and imaged to show the colorimetric variation 1.2 1.2 K. pneumonia

) P. mirabilis ) of these different mixtures. (B–D) Compa- 1.0 1.0 53 0 53 0 rable experiments were done with Escherichia 0.8 0.8 coli and Klebsiella pneumoniae, and A values 530 0.6 0.6 were collected for all three organisms and their 0.4 0.4 mixtures. Control values (from boxed wells) were 0.2 0.2 subtracted from each experimental value. The Absorbance (A Absorbance (A means and standard deviations were calculated 0.0 0.0 for each mixture condition and plotted. Linear 0 25 50 75 100 0 25 50 75 100 trends were observed for all species with R2 values Percent dead Percent dead as follows: E. coli (0.992), P. mirabilis (0.987) and K. pneumoniae (0.997). EB: Erythrosin B.

Figure 4. Distinguishing between live cell

5.0 K populations and dead cell populations via E. coli 4.0 K S. albus erythrosin B fluorescence. Clear differences in erythrosin B fluorescence intensity were 4.0 K observed when live (green plots) and dead cells (red plots) were analyzed by flow cytometry. 3.0 K In these assays, dead cells were prepared by

3.0 K ethanol exposure. Both Gram-negative bacteria (left panels) and Gram-positive bacteria (right 2.0 K 2.0 K panels) exhibited these differences. All flow cytometry plots indicate the number of cells analyzed on the y-axis and the fluorescence

1. 0 K 1. 0 K intensity (em. 583/30; log10 scale) on the x-axis. A minimum of 100,000 cells were analyzed for each species under each condition. 0 0 10 2 10 4 10 6 10 8 10 2 10 4 10 6 10 8

K. pneumoniae S. mutans

4.0 K 5.0 K

4.0 K 3.0 K

3.0 K

2.0 K 2.0 K

1. 0 K 1. 0 K

0 0 10 2 10 4 10 6 10 8 10 2 10 4 10 6 10 8

4.0 K 5.0 K S. marcesans S. aureus

4.0 K 3.0 K

3.0 K 2.0 K

2.0 K

1. 0 K 1. 0 K

0 0 10 2 10 4 10 6 10 8 10 2 10 4 10 6 10 8

6.0 K P. mirabilis E. faecalis 5.0 K

4.0 K 4.0 K

3.0 K

2.0 K 2.0 K

1. 0 K

0 0 10 2 10 4 10 6 10 8 10 2 10 4 10 6 10 8

Vol. 68 | No. 1–3 | © 2020 Josef D Franke 66 www.BioTechniques.com RESULTS & DISCUSSION Figure 5. Monitoring cell viability in cultures 4.0 K In eukaryotes, EB is known to function as a Cells -EB exposed to a toxin. A Serettia marcesans culture was incubated with sodium azide and membrane-exclusion vital dye such that assayed for cell viability using EB at different 3.0 K dead cells with compromised plasma time points. Prior to sodium azide addition, membranes are dye positive [12]. Despite two samples were taken from the culture certain advantages over other traditional 2.0 K and analyzed directly. One sample was not colorimetric membrane-exclusion dyes [13], incubated with EB (top panel) while the other one was incubated with the dye (0 min panel). the application of EB as a vital dye in 1.0 K At the indicated time points, samples were eukaryotes is not as widespread as other taken from the culture, incubated with EB and dyes with a comparable mechanism of prepared for flow cytometry analysis. Over 0 time, the relative amount of dye-positive cells action (e.g., trypan blue). We were searching 102 104 106 108 for a practical vital dye for bacteria and in the culture increased. All flow cytometry plots indicate the number of cells analyzed tested EB due to its intense red color and 0 min. +EB on the y-axis and the fluorescence intensity spectral properties. The intense color would 3.0 K on the x-axis. 100,000 cells were analyzed for allow for a clear distinction between live each plot. and dead cells by bright-field microscopy EB: Erythrosin B. for small cells like bacteria, and the spectral 2.0 K properties would allow a single dye to be used for cell-viability assays measuring 1.0 K either absorbance or fluorescence. A variety of differently shaped Gram- negative and -positive bacteria were 0 102 104 106 108 screened. Three different treatments were performed and designed to induce cell 5 min. +EB death (heat shock and ethanol exposure) 3.0 K or affect membrane permeability (CaCl2 exposure) across diverse bacterial species. For all species examined in this study, EB 2.0 K specifically stained dead, or membrane- compromised, Gram-negative (Figure 1) 1.0 K and -positive bacteria (Figure 2). For heat- shocked and ethanol-treated organisms, virtually all treated cells were dye positive. 0 102 104 106 108 For CaCl2-treated cells, not all treated cells were dye positive. CaCl2 treatment was 30 min. +EB designed to affect membrane permeability 3.0 K and not cause cell death, and resulted in a variable amount of dye-positive cells for 2.0 K different species. This variability likely reflects the inherent species-specific

effects of CaCl2 exposure. Increased 1.0 K

CaCl2 concentration, incubation time and/or prolonged heat shock would likely have resulted in a greater proportion of 0 102 104 106 108 dye-positive cells.

For all species examined in this study, 2.5 K 60 min. +EB <3% of healthy cells were dye positive by bright-field microscopy when incubated 2.0 K with EB (untreated columns in Figures 1 & 2), demonstrating that EB does not readily 1.5 K stain living bacterial cells. EB-containing 1.0 K cell suspensions were generally viewed after 10–15 min of dye incubation, although 500 clear differences between dye-negative and dye-positive cells were visible within 0 5 min. Longer incubations, up to 30 min, 102 104 106 108

did not affect the ability to distinguish amount of EB-positive cells over time. versatile bacterial vital dye could signifi- dye-positive from dye-negative cells. Faint These results show that EB can detect cantly reduce the cost and time associated staining of live bacteria was sometimes changing cell viabilities rapidly and poten- with conducting viability studies on observed in three species (S. albus, S. tially be used to assay cell viability when diverse bacterial species as well as experi- mutans and E. faecalis) after typical incuba- screening possible antimicrobial agents. ments involving multiple species. Optimi- tions. However, the amount of staining For completeness, our research group zation of dye concentrations for different in dye-positive dead cells was clearly primarily studies members of the Plancto- species is likely unnecessary, as this is an increased compared with live cells, making mycetes phylum, a diverse group of bacteria individual dye that works well at a single it easy to distinguish between living and with evolutionarily unusual cell biological concentration. dead cells. Importantly, EB can also spatially features (reviewed in [14]). In heat shock resolve heterogeneities in the viability of trials, EB functioned as vital dye for some, FUTURE PERSPECTIVE different compartments in filamentous but not all Planctomycetes. In the excep- Unlike existing methods, EB’s colorimetric bacteria (Supplementary Figure 1), similar tions, 20–30% of healthy cells were dye properties allow for rapid, straightforward to the BacLight™ system [9]. Lastly, visual- positive at the concentration used in this live/dead determination by bright-field ization by bright-field microscopy allows for study (>90% of dead cells were dye positive), microscopy with a single dye. Importantly, a rapid and accurate quantitation of dead ruling this concentration of EB out as a its sensitivity allows for low rates of death cells in a sample with a low incidence of viability indicator. It is possible that lower EB to be accurately quantified. EB has diverse dead cells. concentrations will work as a viability dye, commercial and academic applications We examined the ability of EB to function or that some of these organisms have cell such as studies screening for new antimi- as a general bacterial cell-viability indicator biological features making live cells more crobial compounds and determining the in different high-throughput assays permeable to the dye. concentration of these needed for bacte- based on its colorimetric and fluorescent All cell-viability assays have limitations. ricidal effects. EB staining may be partic- properties. Mixtures of different ratios of The CFU method can overestimate the ularly useful for field studies looking at living and dead cells were prepared for a proportion of dead cells because any viable diverse microbiomes, in field hospitals in variety of bacteria. These mixtures were cells that are unable to divide, or dividing the developing world where equipment incubated with EB for 5 min, followed by the more slowly than wild-type, will fail to form and resources are limited, and in studying removal of unbound dye and the transfer visible colonies and will be scored as dead. or identifying pathogenic bacteria that of cells to a 96-well plate. A530 measure- Membrane-exclusion dyes, such as EB and have entered a viable but nonculturable ments were collected and plotted for propidium iodine, can underestimate the state [15]. Last, while this study examined these different bacteria, with each exhib- proportion of dead cells, as recently dead EB’s utility as an individual dye, future iting a linear trend based on EB absor- cells may not have sufficiently compro- studies may identify important uses for bance (Figure 3). EB also functioned as a mised membranes to allow the dye to it in bacterial viability studies as part of fluorescent bacterial cell-viability indicator enter the cell. The sodium azide time dye mixtures. by clearly distinguishing between living and course (Figure 5) suggests that EB stains dead cells for diverse bacterial species cells soon after dying. How other viability SUPPLEMENTARY DATA using flow cytometry (Figure 4). The extent methods score dying cells also varies, and To view the supplementary data that of the fluorescence shift between live cells it is often difficult to accurately quantitate accompany this paper please visit the and dead cells did vary depending on the or distinguish a dying cell population [4]. journal website at: www.future-science. bacterial species (e.g., compare E. coli to For membrane-exclusion dyes, a concern com/doi/suppl/10.2144/btn-2019-0066 S. aureus). These differences are likely due is whether the dye enters cells with mildly to either the relative cell size (larger cells compromised membranes that may be AUTHOR CONTRIBUTIONS will bind more dye) or inherent properties dying but not dead. Chemical treatments JDF conceived and designed the experi- in the way in which EB binds to cellular that can mildly affect membrane perme- mental study, collected data and wrote the materials. Together, these results demon- ability in bacteria (e.g., 20 mM EDTA, manuscript; ALB, AMC and EEE performed strate that it is possible to rapidly estimate 0.1–0.5% Triton X100, 0.12 M guanidine HCl experiments and collected data; and GAP the amount of dead cells in a population or 0.1% SDS) did not result in an observable designed experiments and provided based on EB absorbance (Figure 3) and/or increase in dye-positive live cells [unpub- technical assistance. fluorescence (Figure 4) in different high- lished observations]. The results suggest throughput assays. that EB stains dead cells and not living cells ACKNOWLEDGMENTS To examine the ability of EB to assay with mildly compromised membranes. The authors would like to thank Mackenzie changing cell viabilities in a bacterial culture, EB is the first broadly applicable colori- Taylor for use of her microscope for imaging we treated an S. marcesans culture with a metric vital dye for assaying viability in and Charles Deutch for providing the known toxin to Gram-negative bacteria – both Gram-positive and -negative bacteria. bacterial strains used in this study. The sodium azide (Figure 5). Samples taken EB’s spectral properties allow for its use in authors would also like to thank Jill from the treated culture and examined by diverse experimental approaches with low-, Blankenship, Charles Deutch and Soochin flow cytometry showed an increase in the medium- and high-throughput assays. This Cho for comments and suggestions on the

Vol. 68 | No. 1–3 | © 2020 Josef D Franke 68 www.BioTechniques.com manuscript. Creighton University has Unported License. To view a copy of this complement-mediated killing of bacteria by viability staining and bioluminescence. Appl. Environ. Microbiol. applied for a provisional patent in the United license, visit http://creativecommons.org/ 64(2), 515–519 (1998). 8. Stiefel P, Schmidt-Emrich S, Maniura-Weber K, Ren Q. States for using this dye as a live/dead licenses/by-nc-nd/4.0/ Critical aspects of using bacterial cell viability assays bacteria indicator. with the fluorophores SYTO9 and propidium iodide. BMC Microbiol. 15(1), 36 (2015). REFERENCES 9. Stocks SM. Mechanism and use of the commercially 1. Crowley LC, Marfell BJ, Christensen ME, Waterhouse available viability stain, BacLight. Cytometry A 61(2), FINANCIAL & COMPETING NJ. Measuring cell death by trypan blue uptake and 189–195 (2004). INTERESTS DISCLOSURE light microscopy. Cold Spring Harb. Protoc. 2016(7), 10. Rosenberg M, Azevedo NF, Ivask A. Propidium iodide (2016). staining underestimates viability of adherent bacterial The authors have no relevant affiliations or 2. Crowley LC, Scott AP, Marfell BJ, Boughaba JA, cells. Sci. Rep. 9(1), 6483 (2019). financial involvement with any organization Chojnowski G, Waterhouse NJ. Measuring cell death 11. Netuschil L, Auschill TM, Sculean A, Arweiler NB. by propidium iodide uptake and flow cytometry. Cold Confusion over live/dead stainings for the detection of or entity with a financial interest in or Spring Harb. Protoc. 2016(7), (2016). vital microorganisms in oral biofilms – which stain is 3. Altman SA, Randers L, Rao G. Comparison of trypan suitable? BMC Oral Health 14, 2 (2014). financial conflict with the subject matter or blue dye exclusion and fluorometric assays for mam- 12. Bhuyan BK, Loughman BE, Fraser TJ, Day KJ. Compar- materials discussed in the manuscript. This malian cell viability determinations. Biotechnol. Prog. ison of different methods of determining cell viability 9(6), 671–674 (1993). after exposure to cytotoxic compounds. Exp. Cell Res. includes employment, consultancies, 4. Kwolek-Mirek M, Zadrag-Tecza R. Comparison of 97(2), 275–280 (1976). methods used for assessing the viability and vitality 13. Krause AW, Carley WW, Webb WW. Fluorescent eryth- honoraria, stock ownership or options, of yeast cells. FEMS Yeast Res. 14(7), 1068–1079 rosin B is preferable to trypan blue as a vital exclusion expert testimony, grants or royalties. (2014). dye for mammalian cells in monolayer culture. J. 5. Boulos L, Prevost M, Barbeau B, Coallier J, Desjardins Histochem. Cytochem. 32(10), 1084–1090 (1984). No writing assistance was utilized in the R. LIVE/DEAD BacLight: application of a new rapid 14. Wiegand S, Jogler M, Jogler C. On the maverick staining method for direct enumeration of viable and Planctomycetes. FEMS Microbiol. Rev. 42(6), 739–760 production of this manuscript. total bacteria in drinking water. J. Microbiol. Methods (2018). 37(1), 77–86 (1999). 15. Oliver JD. Recent findings on the viable but noncultur- 6. Berney M, Hammes F, Bosshard F, Weilenmann HU, able state in pathogenic bacteria. FEMS Microbiol. Rev. OPEN ACCESS Egli T. Assessment and interpretation of bacterial 34(4), 415–425 (2010). viability by using the LIVE/DEAD BacLight Kit in com- This work is licensed under the Attribution- bination with flow cytometry. Appl. Environ. Microbiol. 73(10), 3283–3290 (2007). NonCommercial-NoDerivatives 4.0 7. Virta M, Lineri S, Kankaanpaa P et al. Determination of

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Noninvasive cell counting of adherent, suspended and encapsulated mammalian cells using optical density

Ayesha Aijaz1, Dylan Trawinski1, Scott McKirgan1 & Biju Parekkadan*,1

CITATION mammalian cells achieving a maxima of the polymer. These develop an analytical method for BioTechniques 68: 35–40 (January minimal detectable cell count of studies provide feasibility for cell counting. A multiwavelength 2020) 10.2144/btn-2019-0052 62,500 at 295 nm. Light absor- optical density as a simple end spectral analysis was conducted bance was insensitive to culture point to indirectly quantify from 200 to 800 nm with a 5-nm ABSTRACT volume, giving an absolute cell mammalian cell number for step size to detect peak and In situ measurement to determine count rather than a concentration. continuous monitoring of cell minimal detectable cell absor- mammalian cell number in a The activation state of cells was cultures. bance. The optical density of cell- noninvasive, non-destructive and also considered. The study was laden polymeric microcapsules reagent-free manner is needed to extended to quantification within METHOD SUMMARY was also evaluated. enable continuous cell manufac- polymeric microcapsules as an Cell type and anchorage- turing. An analytical method is advanced substrate for mammalian dependent cells versus suspension KEYWORDS presented for noninvasive cell cell growth in bioreactor formats cells, as well as cell state (activated biomanufacturing • bioprocess • cell counting by conducting multiwave- and resulted in an offset directly vs unactivated), were assessed for therapy • optical density • optical length spectral analysis of correlating with the absorbance changes in optical density to sensor

Rapid, noninvasive and accurate determi- destructive assays that can result in imaging technique that can be applied nation of mammalian cell growth is critical contamination and enzymatic cell flexibly to closed-system cell cultures of for monitoring during many industrial detachment, as in the case of anchorage- different formats could enable real-time bioprocesses. Several viability tests are dependent cells. They also increase quantification and process control feedback, used, such as Trypan Blue exclusion, to operator time and effort and instrument-to- an important criterion for realizing the monitor cell density and viability, and the instrument variability [4] and have low potential for continuous biomanufacturing. equipment required to automate this throughput, all of which result in an unmet continued online… process is commercially available [1–3]. need for the development of inline modules However, these tests are generally to monitor cell density. A noninvasive

1 × 106 cells 1 × 106 0.5 × 106 cells 0.5 × 106 1.4 6 1.4 0.25 × 106 cells 0.25 × 10 6 0.125 × 106 0.125 × 10 cells 1.2 1.2 0.0625 × 106 cells 0.0625 × 106 1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

Optical density (AU) 0.2 Optical density (AU) 0.2

0.0 0.0 340 260 265 280 285 295 300 305 310 315 320 325 330 275 335 290 260 265 270 275 280 285 290 295 300 305 310 315 320 325 330 335 340 Wavelength (nm) Wavelength (nm)

Figure 1 Multiwavelength spectra of (A) anchorage-dependent cells and (B) suspension cells.

1Department of Biomedical Engineering, Rutgers University, New Brunswick, NJ 08854, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0052 article here

Martyn Webb1, Kate Manley1,2, Mireia Olivan4, Ingrid Guldvik5,6, Malgorzata Palczynska2, Rachel Hurst1, Shea P Connell1, Ian G Mills7, Daniel S Brewer1,3, Robert Mills2, Colin S Cooper1 & Jeremy Clark*,1

CITATION tions in the urine. We herein describe expense of patients having to visit from historic frozen urine samples, BioTechniques 68: 65–71 (February methodology that allows urine to be the clinic. and allowed the harvest of sRNA. 2020) 10.2144/btn-2019-0092 collected by patients at home and Comparisons between digital rectal then posted to a laboratory for METHOD SUMMARY examination (DRE) and non-DRE ABSTRACT analysis. RNA yields and quality The use of a commercial preser- urine RNA yields and RT-PCR Urine from patients with prostate were comparable to those for post vative allowed samples to be expression levels have demon- cancer (PCa) contains gene digital rectal examination urine, and maintained at room temperature strated that the collection of transcripts that have been used for there was improved sensitivity for without loss of RNA quality. Harvest non-DRE urine by men at home is a PCa diagnosis and prognosis. the detection of TMPRSS2:ERG of cell-free RNA using a novel high- viable and simple option. Historically, patient urine samples transcripts by RT-PCR. The At-Home volume vacuum extraction method have been collected after a digital collection protocol has opened up increased total RNA yields, improved KEYWORDS rectal examination of the prostate, the potential to perform large-scale the detection sensitivity of prostate- biomarker • cancer • diagnosis • home- which was thought necessary to PCa studies without the inconve- cancer-specific transcripts by screening • prognosis • prostate • urine boost the levels of prostatic secre- nience, cost, discomfort and RT-PCR, enabled extraction of RNA nucleotide

Figure 5 Contents of the at-home collection kit. (A) Invitation to participate. (B) Study information sheet. (C) Two urine collection tubes (30 ml) containing dried Norgen preservative. (D) Two consent forms (one for the patient to keep and the other to return with the samples). (E) Disposable non-allergenic glove. (F) Pen (to write the time and date on the tubes). (G) 1-h frog timer. (H) Sealable plastic bag with wadding. (I) Preaddressed postage-paid SafeBox for returning the samples. I continued online…

1Norwich Medical School, University of East Anglia, Norwich, UK; 2Norfolk & Norwich University Hospitals NHS Foundation Trust, Norwich, UK; 3Earlham Institute, Norwich, UK; 4Group of Biomedical Research in Urology, Vall d’Hebron Research Institute & Universitat Autònoma de Barcelona, Barcelona, Spain; 5Prostate Cancer Research Group, Centre for Molecular Medicine Norway, University of Oslo & Oslo University Hospital, Oslo, Norway; 6Department of Tumour Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 7School of Medicine, Dentistry & Biomedical Sciences, Institute for Health Sciences, Centre for Cancer Research & Cell Biology, Queen’s University Belfast, Belfast, UK; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0092 article here

A proof-of-concept analysis of carbohydrate-deficient transferrin by imaged capillary isoelectric focusing and in-capillary immunodetection

Jiaqi Wu*,1, Charles H Haitjema1, Christopher D Heger1 & Annegret Boge1

CITATION apparent isoelectric points and abuse. Here, we developed a GRAPHICAL ABSTRACT BioTechniques 68: 85–90 (Feb- relative percentages were deter- technique for the detection of ruary 2020) 10.2144/btn-2019- mined. The relative CDT values carbohydrate-deficient trans- 0058 (percent of total transferrin) ferrin by isoelectric focusing matched expected values for followed seamlessly by Serum ABSTRACT both healthy and alcoholic immunodetection directly in the Carbohydrate-deficient trans- samples. Because the method capillary. The method offers ferrin (CDT) is a reliable leveraged the sensitivity of an several advantages over 10K biomarker for chronic alcohol immunoassay, CDT was existing techniques, including abuse. We developed a method measured when serum samples small sample size (15 μl), speci- EDTA/PBS exchange for CDT analysis by capillary were diluted up to 1200-fold, ficity and automation, while also isoelectric focusing, followed by reducing the volume of serum providing a complete charge- immunodetection directly in the required. Finally, the process is based separation profile of capillary, in an automated fully automated, with up to 96 transferrin. ++- fashion and on a single platform samples analyzed per batch. - + - Carrier ampholytes (Peggy Sue™; ProteinSimple, KEYWORDS + - + CA, USA). Transferrin glyco- METHOD SUMMARY carbohydrate-deficient trans- - forms in serum samples, Carbohydrate-deficient transferrin • imaged capillary including disialo-transferrin, ferrin is a common biomarker isoelectric focusing • immuno- were separated and their for diagnosing chronic alcohol detection • Simple Western Sample plates

Transferrin (Tf) is an important iron-trans- elevated levels of carbohydrate-deficient Tf porting protein in human serum. With two (CDT), which refers collectively to asialo-Tf available N-linked glycosylation sites, several (nonsialylated) and disialo-Tf [2]. This makes glycoforms of Tf exist, depending on the CDT a reliable and specific biomarker to attachment of zero–two N-glycans. These identify chronic alcohol abuse. In addition to Peggy Sue N-glycans display a varying number of Tf glycoforms, other isoforms of Tf exist terminal sialic acid residues, and this number owing to genetic polymorphisms caused by is used to name each glycoform. For example, one or more amino acid substitutions to the in healthy individuals, the major Tf glycoform primary structure of the protein. Therefore, contains four sialic acid residues (tetrasialo- the monitoring of Tf glycoforms and genetic Tf) with minor glycoforms of disialo-Tf (two isoforms is important in clinical and forensic 50,000 sialic acid residues), trisialo-Tf (three sialic analysis of chronic alcohol abuse, the inves- 40,000 30,000 acid residues), pentasialo-Tf (five sialic acid tigation of genetic variants and the 20,000 residues) and hexasialo-Tf (six sialic acid assessment of congenital disorders of 10,000 residues) [1]. By contrast, chronic alcohol glycosylation [1,2]. 0 Chemiluminescenc e 4.5 5 5.5 676.5 abusers, defined as those that consume more continued online… pI than 50 g of alcohol a day for greater than seven consecutive days, typically have

1ProteinSimple, a Bio-Techne Brand, 3001 Orchard Parkway, San Jose, CA 95134, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0058 article here

Chao Chen‡,1,2, Jun Sun‡,1, Yun Yang2, Lu Jiang1, Fengyu Guo1, Yaping Zhu1, Dan Li3, Renhua Wu1, Rong Lu3, Mei Zhao4, Fang Chen5, Peixiang Ni1, Zhihui He**,3 & Zhiyu Peng*,5

CITATION genotypes of the mothers and shown to be accurate and reliable. cell-free DNA of maternal plasma are BioTechniques 68: 117–121 (March probands. Then, fetal haplotypes With further validation in a large analyzed by targeted sequencing 2020) 10.2144/btn-2019-0113 were deduced using a maternal patient cohort, this haplotype-based and maternal pathogenic haplotypes haplotype-assisted hidden Markov approach could be feasible for the deduced using the genotypes of ABSTRACT model. Finally, the NIPD results were NIPD of HA and other X-linked mother and proband. Then, fetal Aim: We aimed to demonstrate nonin- further confirmed by invasive single-gene disorders. haplotypes were inferred using a vasive prenatal diagnosis (NIPD) of prenatal diagnosis. Results: Two fetal hidden Markov model and Viterbi hemophilia A (HA) using a haplotype- genotypes were successfully METHOD SUMMARY algorithm. based approach. Methods: Two inferred, with one normal fetus and We propose a haplotype-based families at risk for HA were recruited one carrier fetus. The NIPD results noninvasive prenatal diagnosis KEYWORDS for this study. First, maternal haplo- were confirmed by invasive prenatal approach for hemophilia A using a factor VIII • haplotype • hemophilia A types associated with pathogenic diagnosis, with a 100% consistency small target region. The genomic • noninvasive prenatal diagnosis variants were constructed using the rate. Conclusion: Our test has been DNA of the parents and proband and • targeted sequencing

Hemophilia A (HA) is an X-linked recessive trate is the most common approach used in the clinic at present focuses on detecting bleeding disorder affecting approximately clinical practice at present. However, the ability de novo or paternally inherited variants [7]. 1/5000 male live births [1]. HA is caused by a to manage bleeding episodes is greatly The high percentage of maternal cfDNA pathogenic variant in the F8 gene, resulting in impaired in patients who develop alloimmune present interferes with direct observation of a qualitative or quantitative deficiency in factor antibodies (inhibitors) [4]. Because of the a fetal allele inherited from the mother, which VIII (FVIII). More than 2000 sequence variants severity of the disease, pregnant carriers of presents significant technical challenges for in F8 have been collected in the HA severe HA can benefit from prenatal diagnosis. NIPD of recessive disorders [8]. Few studies databases [2]. The F8 intron 22 inversion However, invasive prenatal diagnosis have reported NIPD of HA using fetal cfDNA. (Inv22) is found in approximately half the (IPD) approaches such as chorionic villus Tsui et al. first developed a NIPD method for patients with severe HA [3]. Without prophy- sampling and amniocentesis pose a small risk the detection of F8 sequence variants using lactic treatment, patients with severe HA suffer of miscarriage and infection [5]. The discovery digital PCR and relative mutation dosage from spontaneous or traumatic bleeding in of cell-free DNA (cfDNA) in maternal plasma analysis [9]. several organs. Intracranial hemorrhage can makes noninvasive prenatal diagnosis (NIPD) continued online… occur after even mild head trauma and lead to possible, which carries no risk to the pregnant death. Replacement therapy with FVIII concen- woman or her fetus [6]. The NIPD available in

chrX 153063964 Upstream 1 M chrX 154063964 chrX 154251098 Downstream 0.7 M chrX 154929412

239SNPs F8 206SNPs

26E 252423 22 21201918171615 14 13 12 11 10 98 7 6 5 4 3 2 1

EXON

Figure 1 Target region used for haplotyping.

1Tianjin Medical Laboratory, BGI-Tianjin, BGI-Shenzhen, Central Avenue 55, Airport Business Park East Building E3, Tianjin 300308, China; 2Wuhan BGI Clinical Laboratory Co., Ltd, BGI-Wuhan, BGI-Shenzhen, Wuhan 430074, China; 3Department of Obstetrics & Gynecology, The First Affiliated Hospital of Guangzhou Medical University, 151#, Yanjiang Road, Guangzhou 510120, Guangdong, China; 4BGI-Guangzhou Medical Laboratory, BGI-Shenzhen, 22#, Qinglan Street, Panyu District, Guangzhou 510006, Guangdong, China; 5BGI Genomics, BGI-Shenzhen, Bei Shan Industrial Zone, Yantian District, Shenzhen 518083, Guangdong, China; *Author for correspondence: [email protected]; **Author for correspondence: [email protected]; ‡Authors contributed equally View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0113 article here

A cautionary tale of cross-contamination among plasmids from commercial suppliers

Jinli Sun1,2, Yaping Tian3, Yingying Du2, Zhenzhen Wang4, Guodong Zhao*,2, Yong Ma*,2 & Minxue Zheng*,2

CITATION proved that this contamination METHOD SUMMARY amplified by overnight culture for BioTechniques 68: 14–21 (January occurred during the production Cross-contamination among plasmid repurification. To minimize 2020) 10.2144/btn-2019-0018 process, which was also shown for commercial plasmids was the possibility of cross-contami- another two sets of commercial discovered by performing real-time nation, all equipment used for ABSTRACT plasmids. Our experience indicates quantitative PCR reactions specific plasmid purification was thoroughly Many researchers have switched to that the absolute purity of plasmids to individual plasmids on all cleaned both before and after purchasing their desired plasmids obtained from external sources plasmids, and the identities of the purifica. from commercial suppliers to save cannot be guaranteed. Extreme suspected contamination deter- time and resources, as we did for 17 caution should be exercised, mined by Sanger sequencing of the KEYWORDS high-risk human papillomavirus especially when such plasmids are PCR products and subsequent biotechnology • cross-contami- plasmids. To our surprise, they were used for human gene therapies BLAST searches with the sequencing nation • plasmid • purity • quanti- shown to be cross-contaminated and DNA vaccines, where even a results as query sequences. The tative PCR with one another. Comparison minute amount of contamination contaminated plasmids were trans- between the production schedule may pose significant risks to formed into Eschericia coli competent and the pattern of contaminations patients. cells and single transformants were

Plasmids are a class of transmissible, extra- scientific community. in gene therapy vectors or plasmid vaccines chromosomal genetic elements that were first Like every other reagent, absolute purity could pose significant risks to the health and identified in bacteria [1]. Naturally occurring is desired for plasmids from commercial safety of patients [15,16]. It has been shown plasmids mediate a variety of biological sources as well as other research organi- that, despite the extremely low likelihood, rare functions including bacterial conjugation, zations. Even though, depending on the events have occurred in clinics [20]. Therefore, transfer of disease-causing virulence genes goals, some research projects may tolerate the approaches to detect and minimize and transfer of resistance genes to various reagents with very low levels of contamination nucleic acid contamination for these appli- detrimental environmental factors, such as by unintended components such as buffer cations have been extensively discussed in antibiotics, radiation and heavy metals. With solutions, protein preparations and even the literature [21–23]. However, except for the discovery of restriction enzymes [2,3] and plasmids, absolute purity is necessary for double-transformation artifacts [24], to our subsequent development of recombinant other applications. The areas where nucleic knowledge no published studies have touched DNA technology [4], plasmids have become acid contamination may cause serious conse- on the possibility that plasmids used for these versatile tools in basic biological research and quences include molecular diagnosis, gene applications may be contaminated by other biotechnology development for biomedical, therapy and plasmid vaccines developed unwanted plasmids. agricultural and industrial applications [5–11]. to detect, treat and/or prevent human In this study, we reported multiple With the advent of de novo DNA synthesis [12] diseases [15,16]. PCR technology has been incidences of proven cross-contamination and the decreasing cost and rising efficiency widely used for the molecular diagnosis among the same batch of plasmids purchased of relevant technologies, more and more of various diseases, where its high sensi- from commercial suppliers, which calls for researchers have switched to de novo tivity is desirable [17]. However, high sensi- more attention to be paid to plasmid purity synthesis of their desired plasmids to save tivity of PCR could also lead to false-positive for applications where absolute purity is resources and time required for traditional diagnostic results as a minute amount of paramount. cloning methods [13,14]. In addition, nearly nucleic acid contamination may result in continued online… every supplier of biological research reagents misdiagnosis [18,19]. On the other hand, worldwide is now providing this service to the even extremely low levels of contamination

1School of Life Sciences, University of Science & Technology of China, Hefei, Anhui 230027, China; 2Suzhou Institute of Biomedical Engineering & Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163, China; 3The First Hospital of Jilin University, Changchun, Jilin 130021, China; 4School of Medical Technology, Xuzhou Medical University, Xuzhou, Jiangsu 221004, China; *Authors for correspondence: [email protected], [email protected], [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0018 article here

Tomo Kondo*,‡,1 & Shigehiko Yumura1

CITATION limited. Recently, we reported that tively screened E. coli colonies screening method using green BioTechniques 68: 91–95 (February translational enhancement by a harboring the desired plasmid fluorescent protein (GFP) 2020) 10.2144/btn-2019-0127 Dictyostelium gene sequence (TED) functions in a prokaryote (Magne- expression. No expression inducer boosted protein expression even tospirillum gryphiswaldense) or is required for GFP expression. In ABSTRACT without an expression inducer in eukaryote (Dictyostelium addition, this method is easily During molecular cloning, Escherichia coli. Here, we demon- discoideum). Thus, our system applied to any vector by insertion of screening bacterial transformants strate a generally applicable represents a user-friendly a module related to GFP expression. is a time-consuming and labor- molecular tool using the expression technique for cloning. intensive process; however, of green fluorescent protein KEYWORDS tractable tools that can be applied enhanced by TED. By inserting a METHOD SUMMARY Dictyostelium • E. coli • GFP • Magneto to various vectors for visual confir- module related to TED into the This work presents a simple, reliable spirillum • molecular cloning mation of desired colonies are cloning site in advance, we effec- and cost-effective bacterial colony • screening

Molecular cloning is a technique used to create DNA constructs for studies in various biological fields, TED module Negative screening SD Spacer 100 including molecular cell biology, biotechnology and CAP site lacO 90 synthetic biology. Because of extensive studies using Plac mlcR25 gfp 80

t (%) 70 Escherichia coli, many versatile methods are available for 60 molecular cloning [1]; however, screening methods for 50 40 ed colonies bacterial transformants that contain the desired plasmid st 30

Te 20 are limited. with the inser 10 A popular color-based screening method is blue– LB agar w/o IPTG 0 0 10 20 30 40 50 60 70 80 90100 white screening using the pUC vector series [2,3]. The GFP-negative colonies (%) vector contains the α-peptide gene [4] in the multi-cloning 760 bp Positive screening site (MCS). After its translation, followed by complemen- SD Spacer 100 90 tation of the β-galactosidase, blue pigments are produced mlcR25 gfp 80 through hydrolysis of the colorless lactose analog 70 60 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal). GOI 50 α CAP site lacO 40 When the -peptide gene is disrupted by insertion of a Plac 30 Colonies (%) DNA fragment into the MCS, the colony appears white. 20 10 Because α-peptide expression is controlled by the lac 0 AmpR ori operon, it can be upregulated by the addition of isopropyl GFP-positive Insert-positive β-d-thiogalactopyranoside (IPTG) [5]. Although this method is simple, various factors cause false-positive Figure 1 GFP fluorescence-based colony screening. results, including the presence of an intact α-peptide gene without corresponding product synthesis [1]. continued online…

1Graduate School of Sciences & Technology for Innovation, Yamaguchi University, 753-8512 Yamaguchi, Japan; *Author for correspondence: tomokondo@ bio.c.u-tokyo.ac.jpl; ‡Present address: Department of Life Sciences, Graduate School of Arts & Sciences, The University of Tokyo, Tokyo 153-8902, Japan View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0127 article here

Detecting G protein-coupled receptor complexes in postmortem human brain with proximity ligation assay and a Bayesian classifier

Ying Zhu1,2, József Mészáros1,2, Roman Walle3, Rongxi Fan1,2, Ziyi Sun1,2, Andrew J Dwork2,4,5, Pierre Trifilieff3 & Jonathan A Javitch*,1,2,6

CITATION complexes have been detected in the possible existence of D2R-A2AR machine learning method (Bayesian BioTechniques 68: 122–129 (March rodent brains by proximity ligation heteromers. These methods are optimized PLA signal sorting) was 2020) 10.2144/btn-2019-0083 assay; however, their existence in the applicable to the relative quantifi- developed to automatically quantify human brain has not been demon- cation of proximity of two proteins, Brightfield proximity ligation assay ABSTRACT strated. In this study, we used Bright- as well as the expression levels of data. Despite the controversy regarding field proximity ligation assay, individual proteins. the existence and physiological combined with a systematic sampling KEYWORDS relevance of class A G protein- and a parameter-free naive Bayesian METHOD SUMMARY adenosine • adenosine A2A coupled receptor dimerization, there classifier, and demonstrated Brightfield proximity ligation assay receptor • dopamine • dopamine is substantial evidence for functional proximity between the D2R and the was used to assess the expression receptor type 2 • G protein-coupled interactions between the dopamine A2AR in the adult human ventral of G protein-coupled receptors and receptor • proximity ligation assay D2 receptor (D2R) and the adenosine striatum, consistent with their their proximity in postmortem adult • machine learning • naive Bayesian A2A receptor (A2AR). A2AR-D2R colocalization within complexes and human brains. A novel automated classifier

The dopamine receptor type 2 (D2R) is an their relevance to receptor physiology or patho- systems, and it is important to study these extensively studied class A G protein-coupled physiology remain unclear and a topic of active complexes in native mammalian brain. receptor (GPCR) that has been shown to play study and debate [22–24]. The signaling continued online… a critical role in various brain functions and has properties of putative D2R-A2AR heteromers been implicated in a variety of neuropsychiatric have been mostly studied in heterologous disorders, including schizophrenia [1,2], Parkin- son’s [3,4], Alzheimer’s [5] and Huntington’s Perfusion Direct immersion disease [7] as well as addiction [8]. In addition, the D2R is the common target of all current antipsychotic medications [9], which has led to extensive publication of literature regarding its pharmacological and signaling properties [10]. Previous studies in cell lines suggest that D2Rs may function as a part of heteromeric complexes, in which D2R activity and signaling can be modulated by another receptor subunit [11–14]. The putative heteromer formed by the D2R with the adenosine A2A receptor (A2AR) is one of the most studied among the class A GPCRs [15–19], and it has been hypothesized that pharmacological targeting of A2AR could be an efficient strategy to modulate D2R activity [20,21]. However, the structural properties of class A Figure 1 The effect of fixation on the nonspecific nuclear signal in single-recognition PLA-FL in receptor heteromers, their existence in vivo and mouse brain tissue.

1Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY 10032, USA; 2Department of Psychiatry, Columbia University, New York, NY 10032, USA; 3Université de Bordeaux, INRA, Bordeaux INP, NutriNeuro, UMR 1286, F-33000, Bordeaux, France; 4Department of Pathology & Cell Biology, Columbia University, New York, NY 10032, USA; 5Division of Molecular Imaging & Neuropathology, New York State Psychiatric Institute, New York, NY 10032, USA; 6Department of Pharmacology, Columbia University, New York, NY 10032, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0083 article here

Patrick A Kates1, John J Tomashek1, David A Miles1 & L Andrew Lee*,1

CITATION effective on, and scalable for, an BioTechniques 68: 148–154 (March automated platform (INTEGRA Batch extraction Fixed-bed pipette extraction 2020) 10.2144/btn-2019-0140 ASSIST). The results suggest that high-throughput biomolecular ABSTRACT workflows will benefit from the Automation gives researchers the integration of INtip separations. ability to process and screen Load Centrifuge Decant orders of magnitude higher METHOD SUMMARY Spin-column chromatography numbers of samples than manual We have developed a pipette tip experimentation. Current biomac- containing nickel-affinity resin romolecule separation method- that utilizes dispersive solid- ologies suffer from necessary phase extraction to purify manual intervention, making their histidine-tagged proteins at high dSPE (INtip) translation to high-throughput yields and purity. This tip has been automation difficult. Herein, we employed on an automated device Load Centrifuge Elute present the first characterization and can be scaled for high- Functional magnetic beads of biomacromolecule affinity throughput applications. The purification via dispersive solid- efficiency, capacity and affinity of phase extraction in a pipette tip these tips were tested with three (INtip). We use commercially proteins in bacterial cell lysates. available resin and compare efficiency with batch and spin KEYWORDS column methodologies. Moreover, automation • chromatography Load Separate Elute we measure the kinetics of • dispersive solid-phase extraction binding and evaluate resin binding • high-throughput screening capacities. INtip technology is • INtip chemistry • purification Figure 1 Graphical representation of five separation techniques.

Over several decades, microscale protein and elution of captured protein. The second DNA [1], more recently this approach has purification techniques have been sorted into method traps resin between filters in a spin become popular for antibody purification [2]. five basic approaches (Table 1), all premised column and passes liquid samples, washes These technologies vary in their processing on the same fundamental steps (Figure 1). and eluents through the resin by centrifu- mechanics, necessary hardware, and Protein targets with an affinity tag are mixed gation. Less cumbersome than batch investment of operator time and expertise. In with a functionalized surface to encourage extraction, spin columns reduce the carryover some instances, automated HTP systems can specific binding. A wash step then removes of resin into subsequent steps. A third method, reduce operator input or minimize centrifu- nonspecifically bound or weakly bound used mostly for high-throughput (HTP) appli- gation, but to maintain consistent flow rates contaminants. Finally, an elution step disrupts cations, employs functionalized magnetic backpressure must be kept low, and to achieve the tag–surface interaction, allowing for beads to separate the bound proteins from high purity carryover between sample wells or collection of a purified protein. The first solution. A fourth approach, using resin wash steps must also be controlled. method, batch extraction, involves mixing packed in a pipette tip, is a miniaturized version continued online… loose resin with liquid sample in a single of (bi)directional fast protein liquid chroma- vessel, followed by sedimentation, washing tography. Used for over 30 years to purify

1Integrated Micro-Chromatography Systems, Inc., Irmo, SC, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0140 article here

An improved method for the rescue of recombinant Newcastle disease virus

Pheik-Sheen Cheow1, Tiong Kit Tan2, Adelene Ai-Lian Song1, Khatijah Yusoff1,3 & Suet Lin Chia1,4,*

CITATION helper plasmids into a T7 RNA plasmids. The virus progenies produce viral progenies from the BioTechniques 68: 96–100 (February polymerase-expressing cell to produced were quantitated with flow transfected mammalian cell line, 2020) 10.2144/btn-2019-0110 produce viral progenies, poses a cytometry analysis of the infectious which improved the titer of virus great challenge. We have modified virus unit. The modified transfection progenies. The method of viral titer ABSTRACT the standard transfection method to method increased the titer of virus quantification by infecting the Reverse genetics has been used to improve the transfection efficiency progenies compared with that of the cancer cell line was simple and time generate recombinant Newcastle of the plasmids, resulting in a higher standard transfection method. saving. disease virus with enhanced titer of virus progeny production. immunogenic properties for vaccine Two transfection reagents (i.e., METHOD SUMMARY KEYWORDS development. The system, which Lipofectamine and polyethylen- The standard transfection method Newcastle disease virus • polyethyl- involves co-transfecting the viral imine) were used to compare the was modified by changing the time enimine • reverse genetics • trans- antigenomic plasmid with three transfection efficiency of the four to perform the co-transfection to fection • virus titration

Newcastle disease virus (NDV) is a single- plasmids (one for full-length cDNA and three knowledge, no transfection reagents other than stranded, negative-sense, nonsegmented RNA for RNP produced collectively by individual Lipofectamine have been used to rescue rNDV. virus [1]. A recombinant NDV (rNDV) can be plasmids encoding the NP, P and L genes) into Therefore, it is unclear whether this has not been generated from a cloned cDNA using reverse a cell. Several attempts have been carried out done or because it was not successful, failed to genetics. The cDNA of the full-length antig- to either generate cells that constitutively be reported. Nevertheless, PEI has been used to enome of the virus, with plasmids encoding for express the RNP or to reduce the number of transfect plasmids for the rescue of influenza ribonucleoprotein (RNP), are co-transfected into plasmids to be transfected [3,4]. Both efforts virus, a segmented negative-sense RNA virus [5]. T7 RNA polymerase-expressing cells [2]. The are complicated and time-consuming. Here, our aim was to compare the transfection transcription and translation of the RNP in the Lipofectamine, a cationic lipid, is commonly efficiency of Lipofectamine™ 3000 (Invitrogen, cells will result in the production of viral used for the transfection of the plasmids into MA, USA) and PEI with optimized transfection progenies, which can then be propagated in cells. Other transfection reagents, such as protocol for the rescue of rNDV. embryonated eggs. cationic polymers (polyethylenimine [PEI]) and continued online… The success of the rNDV rescue depends inorganic compounds, have also been used on the efficiency of the transfections of four for this purpose. However, to the best of our

Table 1. Summary of successful viral rescue attempts from co-transfection of BSR T7/5 with either forward or reverse transfection and different total mass of pDNA. Transfection Forward Reverse 22.5 μg of total pDNA

Lipofectamine™ 3000 4/9, 44.4% 3/9, 33.3%

PEI 2/9, 22.2% 1/9, 11.1%

1.8 μg of total pDNA

Lipofectamine 3000 9/9, 100% 8/9, 88.9%

PEI 0/9, 0% 0/9, 0%

1Department of Microbiology, Faculty of Biotechnology & Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS, Oxford, UK; 3Malaysia Genome Institute, Jalan Bangi, 43000 Kajang, Selangor, Malaysia; 4Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0110 article here

Isolation of DNA-free RNA from human bone marrow mononuclear cells: comparison of laboratory methods

Tawakalitou Alabi1, Sweta B Patel2, Smita Bhatia1, Julie A Wolfson1 & Purnima Singh*,1

CITATION either are labor intensive (requiring method achieves optimal quantity Reagent with RNeasy® Protect Cell BioTechniques 68: 159–162 (March the use of toxic organic solvents and and high-quality RNA for sequencing Mini Kit (Qiagen, Hilden, Germany) 2020) 10.2144/btn-2019-0093 separate DNase treatment) or require and RT-PCR while remaining efficient was superior in purity and quality. automation (with extensive setup and and cost effective. This kit does not use biohazardous ABSTRACT startup costs). To optimize both the reagents and was cost effective, and RNA quality (purity and integrity) and quality and quantity of RNA from METHOD SUMMARY RNA free of genomic DNA was quantity are of critical importance to bone marrow, we recommend stabi- Using mononuclear cells from obtained without the need for a ensure reliable gene expression lization and storage of bone marrow human bone marrow specimens, we separate DNA removal kit. analysis, reproducibility of RNA mononuclear cells in RNAprotect® evaluated two commonly used RNA sequencing and microarray data and Cell Reagent, followed by extraction extraction methods. RNA extracted KEYWORDS validation by RT-PCR. Currently using the RNeasy® Protect Cell Mini from bone marrow mononuclear RNA • RNeasy protect cell mini kit available methods for isolating RNA Kit (Qiagen, Hilden, Germany). This cells stored in RNAprotect® Cell • TRIzol

Genomic and transcriptomic analyses have and susceptible to rapid degradation. Few specific large-scale equipment required for proven crucial in identifying molecular studies have done a comparative analysis automation available, our aim was to examine mechanisms associated with cancer recur- of commonly used RNA isolation kits; budget-friendly options, including mecha- rence and survival. Nucleic acids from white however, our objective was to simultane- nisms of freezing cells for later nucleic acid blood cells are most commonly examined [1]. ously obtain DNA, RNA and viable cells isolation [11,12]. Unlike DNA, RNA biomarkers provide dynamic for xenograft from bone marrow aspirates continued online… insights into cellular states and regulatory and to explore only manual extraction kits processes [2]. However, the success of for RNA isolation [9,10]. Moreover, without downstream molecular applications, including cDNA preparation, quantitative RT-PCR, microarrays and RNA sequencing, 2.5 260/280 260/230 relies on the integrity and purity of the RNA (e.g., free of gDNA and organic contami- 2.0 nants) [3]. The reproducibility of time- consuming, expensive and labor-intensive 1.5 downstream applications can be undermined by poor-quality RNA [4,5]. Contamination of 1.0 RNA with gDNA leads to an overestimation

of RNA yields, which consequently impacts 0.5 the selection of library preparation platforms that have different input requirements [6]. If 0.0 proper controls are not included in the RT reactions for cDNA preparation, RT-PCR will 1 TriZol 2 TriZol 3 TriZol 4 TriZol 5 TriZol amplify gDNA, especially if the target is an

intronless gene, with possible overestimation 1 RNA Protect 2 RNA Protect 3 RNA Protect 4 RNA Protect 5 RNA Protect of the transcript levels [7,8]. In light of its single-stranded nature Figure 1 The 260/280 and 260/230 ratios for RNA extracted using the TRIzol/precipitation versus and ubiquitous RNases, RNA is unstable RNAprotect/RNeasy/column method.

1Institute for Cancer Outcomes & Survivorship, University of Alabama at Birmingham, Birmingham, AL 35233, USA; 2Division of Hematology/Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35233, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0093 article here

Ultra-fast conductive media for RNA electrophoretic mobility shift assays

Samantha Z Brown1, Lebaron C Agostini1, Henry L Thomsett1 & Jonathan R Brody*,1

CITATION electrophoretic techniques. Nucleic conditions and crisp band efficiency, traditional Tris-based BioTechniques 68: 101–105 (February acid fragments are end-labeled with resolution (i.e., optimized results). conductive running media are 2020) 10.2144/btn-2019-0111 fluorescent tags, as opposed to the replaced with low-molarity, lithium radioactive or biotin tags. The METHOD SUMMARY borate-conductive media. These ABSTRACT fluorescent probes may be detected We present an improvement in improvements to the methodology The use of RNA electrophoretic directly from the electrophoresis resolution, speed and ease of RNA significantly reduce assay time, as mobility shift assays (REMSAs) for gel, eliminating the need for electrophoretic mobility shift well as improve the quality and analysis of RNA–protein interac- cumbersome membrane transfer assays. First, sensitive and quanti- overall utility of this technique in the tions have been limited to lengthy and immunoblotting. Modifying the tative detection of gel shifts can be study of RNA–protein interactions assay time and qualitative REMSA protocol to include improved with near-infrared tagged in vitro. assessment. To vastly improve low-molarity, lithium borate RNA oligos, as opposed to more assay efficiency, feasibility and conductive media and near- toxic and cumbersome labeling KEYWORDS quality of data procured from infrared-labeled probes allows for methods, such as radioisotopes or conductive media • ELAVL1 • EMSA REMSAs, we combine here some of a reduction assay time, quantitative biotin tags. Second, for • HuR • IR-oligonucleotides • LB • the best-known labeling and comparison between experimental improvement of resolution and RBP • REMSA

Gel electrophoresis mobility shift assay (EMSA) is a long-established biochemical technique for the qualitative assessment of nucleotide–protein complexes [1–4]. This method combines the COX-2: 3’UTR - ++ - ++ - ++ - ++ principles of protein and oligonucleotide electrophoresis to [62.50 nm] determine biochemical relationships between these species. HuR [2.39 µg] --+ --+ --+ --+ Ribonucleotide-based EMSAs (REMSAs) are a modified version TBE LB TBE LB of this technique to evaluate ribonucleotide–protein complexes. Most often, REMSAs are employed for the validation of RNA-binding Bound proteins (RBPs) to their regulated transcripts. COX-2: 3’UTR Although the current methodology of this application is infor- mative, its output is largely qualitative, with a variety of resolution and sensitivity issues. Earlier renditions of this method used radioisotopes such as 32P for tagging oligonucleotides (oligos). Radioactive labeling is a labor-intensive process, resulting in Unbound probe issues with efficiency, cost and safety [2,5–7]. To address these T : 25˚C T : 25˚C T : 25˚C T : 25˚C challenges, one major improvement has replaced radioisotope 0 0 0 0 T : 30˚C T : 28˚C T : 30˚C T : 28˚C tagging with biotin conjugation. This labeling technique relies F F F F on its affinity against avidin-based proteins for specific isolation of complexes [8–10]. Run time: 7 minRun time: 12 min Biotin end-labeled probes are easier and safer to use than radioisotope-based applications; however, this method still requires additional time-consuming steps after initial electrophoresis. Figure 1 Improved resolution and assay time with lithium boric acid conductive media versus Tris-boric acid-disodium EDTA method. (A) 1× TBE gel and (B) 1× continued online… LB gel ran fast (300 V) for 7 min, as indicated. (C & D) Gels may be run longer without compromising resolution, in which the same gels were returned to the reservoirs, and run for an additional 5 min (total assay time: 12 min).

1Department of Surgery, The Jefferson Pancreas, Biliary & Related Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0111 article here

Christopher R McEvoy*,‡,1, Timothy Semple‡,2, Bhargavi Yellapu1,3, David Y Choong1, Huiling Xu1, Gisela Mir Arnau2, Andrew P Fellowes1 & Stephen B Fox1

CITATION library generation we show that tumor size frequently results in a cation of the standard KAPA Hyper BioTechniques 68: 48–51 (January library yields are dramatically failed analysis. By incorporating a library preparation procedure. 2020) 10.2144/btn-2019-0059 increased, resulting in decreased simple modification to the standard Genomic DNA libraries were sample failure rates. Improved yields sample preparation method, we subjected to a solid-phase ABSTRACT allowed for a reduction in PCR cycles, show that DNA yields can be greatly reversible mobilization bead-based Tumor DNA sequencing results can which translated into improved increased, allowing for more cleanup; however, they were not have important clinical implications. sequencing parameters without samples to be successfully eluted from the beads prior to However, its use is often limited by affecting variant calling. This sequenced. Further, the method- pre-capture PCR. Post-PCR cleanup low DNA input, owing to small tumor methodology should be applicable to ology used also results in a subse- was performed using a 20% PEG, biopsy size. To help overcome this any NGS system in which input DNA quent improvement in sequencing 2.5M NaCl buffer, resulting in a limitation we have developed a simple is a limiting factor. parameters, with no loss in the dramatic yield increase with no loss improvement to a commonly used ability to detect mutations. of sequencing fidelity. next-generation sequencing (NGS) LAY ABSTRACT capture-based library preparation The results of tumor DNA METHOD SUMMARY KEYWORDS method using formalin-fixed paraffin- sequencing can have important To improve the library yield for low DNA library • next-generation embedded-derived tumor DNA. By clinical implications. However, DNA input tumor samples subjected sequencing • solid-phase reversible using on-bead PCR for pre-capture limited DNA input owing to small to NGS, we adopted a simple modifi- immobilization beads

Tumor DNA analysis using next-generation sequencing PCR (NGS) has revolutionized many aspects of cancer Master mix diagnosis, prognosis, and therapeutics [1]. Two major factors, DNA quality and quantity, influence the proportion of successfully sequenced tumor samples. SPRI Ethanol Elution Magnet The quality issue is attributed to the use of formalin- beads wash buffer Separation of Off-bead PCR fixed paraffin-embedded (FFPE) tumor tissue, which beads from DNA results in highly fragmented DNA that is chemically modified, whereas the low quantity results from small PCR tumor biopsy sizes. In our experience, using the KAPA Magnet Magnet master mix Hyper Prep Kit for library preparation (Roche, Basel, End repair & Binding Separation Clean up Elution A-tailed libraries Switzerland), in conjunction with a SureSelectXT targeted capture (Agilent, CA, USA), we found that approximately 15% (61/432) of the tumor samples failed to produce a sufficient yield of pre-capture library. On-bead PCR Therefore, we modified the standard protocol by Figure 1 Overview of the methods used in the study. performing the pre-capture PCR ‘on-bead’. Solid-phase reversible immobilization (SPRI) bead-based DNA purifi- libraries typically undergo SPRI bead-based bead/eluate mixture resulted in improved cation [2] has become the favored method of DNA purifi- purification in which the DNA eluted from pre-capture library yields when using cation in many protocols owing to its ease of use, the beads is used for PCR enrichment. This FFPE-derived tumor samples. cost-effectiveness, ability to size-select, and suitability elution step inevitably results in the loss of continued online… for automation. Following genomic DNA (gDNA) DNA. This study was designed to determine fragmentation and adapter ligation, NGS capture-based whether performing the PCR directly on the

1Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia; 2Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia; 3Department of Molecular Oncology & Cancer Immunology, Epworth Healthcare, Melbourne, VIC 3121, Australia; *Author for correspondence: [email protected]; ‡Joint first authors View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0059 article here

An improved shotgun antisense method for mutagenesis and gene identification

Yu Tang1 & Richard H Gomer*,1

CITATION library to have an antisense duction pathway components BioTechniques 68: 163–165 cDNA for every gene in the in D. discoideum. (March 2020) 10.2144/btn-2019- genome. Second, the unequal

0123 transcription level of genes METHOD SUMMARY Original Ladder Normalized resulted in many antisense We present an improved shotgun ABSTRACT cDNAs in the library for some antisense method for generating Shotgun expression of genes but relatively few gene expression knockdown 3000 bp antisense cDNA, where each antisense cDNAs for other mutants. This method incorpo- transformed cell expresses a genes. Here we report an rates a cDNA-normalization step different antisense cDNA, has improved method for gener- to equalize the transcript 1000 bp been used for mutagenesis and ating a larger antisense cDNA number of each gene in the gene identification in Dictyo- library with a reduced antisense cDNA library. stelium discoideum. However, percentage of cDNA clones the method has two limitations. from highly prevalent mRNAs KEYWORDS First, there were too few clones and demonstrate its utility by cDNA normalization • genetic 250 bp in the shotgun antisense cDNA screening for signal trans- screen • shotgun antisense

Genetic screens are broadly used to generate shotgun antisense has several benefits. First, Discoidin I gene family in Figure 1 Normal- and identify mutants with a desired phenotype. the library is relatively easy to construct and the the model eukaryote Dictyo- ization of cDNA Techniques for genetic screens include mutant pool is easy to generate. It only requires stelium discoideum [10]. removes bands corresponding to chemical and radiation mutagenesis [1,2], RNA isolation and cDNA synthesis, followed by Shotgun antisense highly prevalent insertional mutagenesis [3,4], CRISPR libraries cloning the cDNA into an expression vector in screens have been used mRNAs. [5,6], siRNA libraries [7,8] and shotgun a backward orientation. CRISPR and siRNA for mutagenesis and gene antisense [9]. The basis of shotgun antisense libraries require careful design and CRISPR identification in D. discoideum [9], but the is antisense repression. For antisense mutagenesis requires the expression of original protocol had two disadvantages. First, repression of a single gene, the corresponding CAS9 protein in the target cells [5,6]. Second, there are 12,257 protein-coding genes in D. cDNA is cloned into an expression vector shotgun antisense can be directed to target discoideum [11], but the size of the shotgun plasmid in a backward orientation and then genes expressed in a specific tissue or devel- antisense library from each ligation was only transformed into cells. The expression vector opmental stage by using RNA isolated from a approximately 15,000 individual clones [9], so will generate an antisense RNA that hybridizes specific tissue or developmental stage. Third, that by Poisson statistics many genes will not to, and effectively neutralizes, the selected as a gene knockdown technique, shotgun have a corresponding cDNA in the library [12]. RNA sequence (typically an mRNA). This antisense is able to identify genes where Second, the unequal transcription level of hybridization and neutralization process complete disruption is lethal. Fourth, identi- genes causes the levels of some RNAs and reduces, but does not eliminate, levels of the fication of the gene associated with an inter- corresponding cDNAs in the library to be much selected RNA. Using a transformation vector esting phenotype from shotgun antisense lower than those of other genes. In a eukaryotic or plasmid where cells typically take up only requires only a PCR reaction on whole cells cell, 20–40% of genes only have one to several one copy of the vector and an antisense to amplify and sequence the antisense cDNA dozen transcripts, but as few as five to ten construct with an entire cDNA library rather in the shotgun antisense plasmid. Fifth, genes have several thousand transcripts [13]. than a selected cDNA allows a mutagenesis antisense can repress the expression of continued online… referred to as shotgun antisense [9]. multiple genes whose transcripts share closely Among the mutagenesis techniques, related sequences, such as the three-member

1Department of Biology, Texas A&M University, College Station, TX 77843, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0123 article here

Bradford W Daigneault*,1, Sandeep K Rajput2 & George W Smith1

CITATION and genome analyses. These reliable, simplistic approach for extraction from low-input samples BioTechniques 68: 155–158 (March techniques have broad implications analyzing both the genomic and of frozen-thawed bovine sperm, with 2020) 10.2144/btn-2019-0121 in human biomedical sciences and epigenomic status of whole sperm a simple workflow for PCR amplifi- agriculture, including clinical ejaculates that can be adapted for cation, bisulfite conversion and ABSTRACT diagnoses of infertility, the identifi- laboratory diagnostics, clinical repro- methylation analyses of individual We developed a simplified workflow cation of single-nucleotide polymor- ductive practice and basic research. amplicons. of gDNA extraction from ejaculated phisms and aberrant methylation bovine sperm using a low total patterns that can impact fertility, METHOD SUMMARY KEYWORDS number of sperm and a short time lower embryo development and We developed a cost-effective, user- bisulfite • DNA • epigenome • fertility frame that yields high-quality DNA contribute to heritable disease. The friendly and reliable protocol (see • forensic • methylation • oligo- suitable for downstream methylation methods described here provide a Supplementary data) for DNA spermia • sequencing • sperm

Genomic and epigenomic analyses of sperm times, proprietary reagents and DNA are increasingly necessary techniques, high numbers of total sperm input 1. Collect sperm sample with broad applications that include fertility that may be incompatible with 2. diagnostics, forensic analyses and basic available material [9]. Our goal 2 h Cell lysis and gDNA isolation research [1,2]. Additionally, male infertility is a was to simplify the extraction 3. C-T Bisulfite conversion well-recognized concern that contributes to process and to shorten the time failed pregnancies in humans and agricultural required for DNA extraction of 4. PCR amplification animals [3]. Alterations to the genome or DNA mammalian sperm, while methylation status of sperm can impact sperm maintaining sufficient yield for 5. Sanger sequencing function and embryo development [4]. Recent downstream genome and methyl- 6. CTCCA TTTTA Genomic and epigenomic analyses evidence suggests that aberrant methylation ation analyses. We developed a TTCCA of spermatozoa inhibits proper sperm function commercially viable column- and results in lower embryo survival [5]. Impor- based protocol to meet these Figure 1 Workflow for gDNA extraction of mammalian sperm for tantly, both sperm number and processing objectives that require minimal genomic and epigenomic analyses of whole sperm ejaculates. time are limiting factors that can influence sperm input conducive to in vitro fertilization conducted by the addition of technical repli- downstream analyses. Simplified methods for techniques and a short, single-day extraction cates. Sperm were thawed in a water bath at gDNA extraction of sperm followed by process that is compatible for downstream 37°C for 30 s and then purified using a 45:90 successful bisulfite conversion, PCR amplifi- genomic and epigenomic analyses (Figure 1). Percoll gradient for experiment [10]. Purified cation and downstream sequencing are Unless otherwise specified, reagents were sperm were pooled from two bulls, extended needed to improve the workflow in clinical purchased from Sigma-Aldrich (MO, USA). to 4 × 106–4 × 102 total motile sperm by serial settings and provide more rigorous analyses Primers for PCR amplification are shown dilution in 400 μl volumes and placed in a with minimal processing times. However, in Table 1. Isolated DNA was subjected to Tyrode albumin lactate pyruvate medium [11]. extraction of DNA from sperm presents unique bisulfite conversion followed by purification Sperm aliquots were added to 3.6 ml of sperm challenges that differ from somatic cells, using the EpiTect Bisulfite Kit (cat. no. 59104) wash (SW) reagent buffer (150 mM NaCl and including an acrosomal barrier and protamine and the QIAquick PCR Purification Kit (cat. no. 10 mM EDTA [pH 8.0]) in 10-ml Eppendorf compaction of chromatin that often results in 28104; both Qiagen, Hilden, Germany). tubes. low DNA yield [6–8]. Some protocols have been Frozen sperm from two different bulls continued online… developed to address these challenges; (CentralStar Cooperative, Inc., MI, USA) however, most involve prolonged incubation were pooled for experiment with analyses

1Department of Animal Science, Michigan State University, East Lansing, MI 48879, USA; 2Colorado Center for Reproductive Medicine, Lone Tree, CO 80124, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0121 article here

An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

Yves-Marie Boudehen1, Maximilian Wallat1, Philippe Rousseau2, Olivier Neyrolles1 & Claude Gutierrez*,1

CITATION Xer-cise in Mycobacteria. Zeocin genetic stability. The herein described deletions. Subsequent rounds of BioTechniques 68: 106–110 (February resistance cassettes flanked by method should be generalizable to mutagenesis using cassettes flanked 2020) 10.2144/btn-2019-0119 variants of the natural Mycobacterium virtually any transformable bacterial by a range of dif site variants allow tuberculosis dif site were constructed species. construction of multiple mutants in ABSTRACT and shown to be effective tools to which the different dif sites cannot Xer-cise is a technique using construct multiple unmarked METHOD SUMMARY recombine with each other, yielding antibiotic resistance cassettes mutations in M. tuberculosis and in dif-ZeoR-dif cassettes are used to stable genetic constructs. flanked bydif sites allowing sponta- the model species Mycobacterium replace non-essential genes in neous and accurate excision from smegmatis. The dif site variants mycobacterial genome through KEYWORDS bacterial chromosomes with a high harbor mutations in the central region recombineering. Spontaneous dif site • excisable cassette frequency through the action of the and can therefore not recombine with excision of the cassette is carried out • mutagenesis • Mycobacterium cellular recombinase XerCD. Here, we the wild-type or other variants, under the action of the recombinase • recombineering • unmarked report a significant improvement of resulting in mutants of increased XerCD, resulting in unmarked deletions • Xer-cise

Genetic studies of bacterial evolution and recombination between two specific sites chromosome maintenance [3]. Xer-cise has physiology require efficient and reliable tools repeated at both sides of the cassette. The been adapted to Mycobacteria and shown to for the construction of specific gene deletion so-called Xer-cise technique is a convenient be efficient both in M. tuberculosis and in the mutants. A range of tools are available to way to achieve auto-excision, because the fast-growing model species Mycobacterium manipulate Mycobacteria, such as Mycobac- recombination between two deletion-induced smegmatis [2,4,5]. terium tuberculosis, the etiologic agent of filamentation dif( ) sites is catalyzed by the continued online… tuberculosis. In particular, the development XerCD recombinase present and active in of recombineering has been a considerable most bacterial species, as it is required for improvement over previous methods [1]. This technique allows replacement dif4-ZeoR-dif4 of a specific gene by an antibiotic resistance cassette through homol- pGET-ZeoR PCR R ogous recombination between linear dif4-Zeo -dif4 DNA fragments, so-called allelic ~500 bp ~500 bp mc2 155/pJV53H exchange substrates (AES) and chromosomal DNA catalyzed by WT RecET-like mycobacteriophage gene X dif4-ZeoR-dif4 proteins gp60 and gp61 (Figure 1D). 2 R However, only a limited number of mc 155 ∆geneX::dif4-Zeo -dif4 selectable marker genes are available for mycobacteria [2] and, for safety reasons, strains used for medical Three-fragment PCR mc2 155 ∆geneX::dif4 studies must not carry resistance to In-frame AES geneX::dif4-ZeoR-dif4 ∆ replacement multiple antibiotics. (78 bp) One way to overcome these problems is to use excisable Figure 1 Construction of unmarked deletions in Mycobacteria by recombineering using zeocin-resistance cassettes that can be removed by cassettes flanked by dif site variants.

1Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, UPS, 205 route de Narbonne, F-31400 Toulouse, France; 2Centre de Biologie Intégrative de Toulouse (CBI-Toulouse), Laboratoire de Microbiologie et de Génétique Moléculaires (LMGM), Université de Toulouse, CNRS, UPS, F-31400 Toulouse, France; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0119 article here

Kui Lin1, Qin Yan2, Audrey Mitchell2, Natasha Funk2, Catherlin Lu2 & Hao Xiao*,2,3

CITATION biotin-tagged protein purification rate is over 85% without apparent specifically bind the biotinylated BioTechniques 68: 41–44 (January under non-denaturing conditions. protein denaturation. The method proteins, followed by competitive 2020) 10.2144/btn-2019-0088 The biotinylated bovine serum is applicable for both immuno elution with free biotin at near- albumin is specifically bound to precipitation and column neutral conditions. This approach ABSTRACT the anti-biotin antibody agarose chromatography. is applicable for both immuno The use of avidin or streptavidin in beads and competitively eluted precipitation and column the purification of biotinylated with free biotin under near-neutral METHOD SUMMARY chromatography. proteins has been highly restricted conditions. The optimized elution We describe a method for the purifi- due to the harsh and denaturing conditions include using 4 mg/ml cation of biotin-tagged proteins KEYWORDS elution conditions. Here, we use biotin (pH 8.5) as the elution buffer under non-denaturing conditions affinity chromatography • anti-biotin biotinylated bovine serum albumin and allowing the buffer to incubate with no change in protein natural antibody agarose • biotinylated as a working model to demonstrate with agarose beads for 30 min prior structure or function. Anti-biotin protein • competitive elution a simple and rapid method for to elution. The elution recovery antibody agarose is employed to • immunoprecipitation • purification

Enzymatic biotinylation by the Escherichia coli purification [12,13]. Compared with avidin antibodies are able to detect a much broader biotinylase BirA is highly specific in covalently or streptavidin, the binding of anti-biotin range of biotinylated sites in cells compared attaching biotin to a 15-amino-acid peptide antibodies to biotin molecules is a milder with streptavidin [12]. (GLNDIFEAQKIEWHE), also known as AviTag, affinity interaction, allowing the bound continued online… which can be added genetically at the molecules to be released much more easily. N-terminus or C-terminus or in exposed loops Also, it has been reported that the anti-biotin of a target protein [1]. The biotinylated proteins can then be isolated and purified with avidin Anti-biotin antibody Equilibrate with PBST or streptavidin [2–6]. Avidin/streptavidin– Equilibration: agarose beads (pH 7.2) biotin binding is rapid and specific and is considered to be the strongest noncovalent interaction known in nature (KD = 10 -14–10 -15 M). However, elution of the bound proteins from Incubate at RT for Load sample at neutral Binding: avidin or streptavidin requires harsh and 15 min condition (pH 7.0–7.5) denaturing conditions, which inevitably destroy the natural protein structure and are often incompatible with downstream processing [7,8]. To avoid this denaturing process, on-bead Wash with PBST (pH 7.2) Wash: digestion of bound proteins has been intro- and 0.9% NaCl duced [9,10]; however, it may cause undesirable contamination of avidin/streptavidin peptides and the digesting enzymes [11]. Development of a new method for eluting bound biotin- Incubate with biotin Elute biotin-tag Elution: tagged proteins from the affinity matrix without elution buffer for 30 min proteins protein denaturation is therefore necessary. Anti-biotin antibodies have been intro- Figure 5 Scheme for biotin-tagged protein purification using anti-biotin antibody binding and free duced recently for biotinylated protein biotin elution.

1Guangxi Zhuang Autonomous Region Center for Analysis and Test Research, Nanning, Guangxi 530022, China; 2ImmuneChem Pharmaceuticals Inc., Burnaby, BC, V5J 3J1, Canada; 3Faculty of Animal Science and Technology, Gaungxi University, Nanning, Guangxi 530015, China; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0088 article here

A customized program for the identification of conserved protein sequence motifs

Mohammad Mian1, Jeffrey Talada1, Anthony Klobas1, Stephanie Torres1, Yusuf Rasheed1, Hibah Javed1, Zainab Lughmani1 & Reza Forough*,1

CITATION amino acid sequence alignment effective against alphaviruses. complexity for the alignment is BioTechniques 68: 45–47 (January program and identified the O(k∧2*m), where k is the number of 2020) 10.2144/btn-2019-0039 conserved amino acid motif, METHOD SUMMARY viruses containing a matching VAIVLGG, in alphaviruses. The Viral protein sequences are fed into sequence and m is the length of the ABSTRACT VAIVLGG sequence is located on the a battery of rolling hashes of 6–14 longest matching sequence. We searched for viral protein structural capsid protein of the length, and amino acid subse- sequences that could be important chikungunya virus, a mosquito- quences are performed with a time KEYWORDS for tissue tropism. To achieve this borne arthrogenic member of the complexity of O(n). The hashes are alignment software program • alpha- goal, human pathogenic viruses alphaviruses. Capsid protein trans- the keys in a HashMap with values viruses • chikungunya virus • CHIKV were classified according to the location onto the host cell membrane of the sequence ID and index; the spike glycoprotein • CHIKV struc- tissue they infect (e.g., pulmonary), is a required step for virion budding. space complexity is O(n). A normal tural capsid protein • Global Virome irrespective of whether they were Our identified VAIVLGG consensus alignment is done on 14 length Project • protein sequence alignment enveloped or non-enveloped RNA or sequence might potentially be used matches to discover longer matches. • vaccine development • viral protein DNA viruses. Next, we developed an for developing a pan-vaccine The upper bound on the time homologies

We were originally inspired by the recent launch of the Global Virome Project (GVP), a multina- Rolling hash Hashmap for lengths 6 to 14 tional project aimed at discovering new viruses Key List that can potentially infect people or become AAAAAA ...... Collection ...... pandemic [1]. According to the GVP, it is of protein VYPFMW Seq15, index 5268 Seq34, index 3379 predicted that 1.67 million undiscovered viral sequences GVYPFMWGGA ... species exist in mammals and birds, with ...... YYYYYY ...... roughly half of these unknown viruses possessing zoonotic potential. Our reasoning was that, if the GVP is preparing for the mammoth task of identifying roughly 600,000– Insert all results into 800,000 potentially human pathogenic viruses, For all keys of 14 length compare each sequence a database then there is a need for developing new against every other sequence methods for faster data collection and analysis. to check for longer matches In this context, we focused on the following two strategies: developing a new conceptual Figure 1 The schematic diagram represents our customized program written in Java to significantly decrease our search time. method of viral classification to provide a better representation of viral pathogenicity and viruses was placed into one of the following each subcategory (Figure 1). Our goal was to writing customized software to speed up the disease subcategories: fever and joint pain, identify conserved protein sequences within data retrieval and analysis. gastroenteritis, hepatitis, skin lesions, respi- each subcategory with the hope that the First, we proceeded to prepare a list of all ratory diseases, encephalitis or hemorrhagic identified viral conserved protein motifs might 265 known human pathogenic viruses using the fever, regardless of whether they were RNA or play central roles in determining tissue tropism ExPASy ViralZone database [2]. Next, we chose DNA or enveloped or non-enveloped viruses. and successful human host infection. to classify these human pathogenic viruses We then wrote a customized program continued online… into subcategories based on the diseases they to speed up alignments and comparisons cause. Thus, each of the 265 human pathogenic among protein sequences of viruses within

1Biology Program, Science Division, Bellevue College, Bellevue, WA 98007, USA; *Author for correspondence: [email protected] View full Please see article online at https://www.future-science.com/doi/10.2144/btn-2019-0039 article here

Bringing the Tumor Microenvironment into Focus: Simplified Development of Seven-Color Multiplex Immunohistochemistry-Immunofluorescence (mIF) Panels Bethyl Laboratories, Inc., P.O. Box 850, Montgomery, TX 77356 USA HIGHLIGHTS • Multiplex immunohistochemistry-immunofluorescence (mIF) panel development enables simultaneous detection of multiple proteins of interest within a single sample, advantageous in analysis of limited tissue such as tumor biopsy. • The applications of mIF span clinical and basic research, but a 7-color mIF can take ≥8 weeks to develop. • Here, a faster, simplified approach to 7-color mIF panel development is described with a focus on the tumor microenvironment.

INTRODUCTION TO mIF Recent advances in multiplex immunohistochemistry (IHC) and multispectral imaging facilitate simultaneous analysis of multiple tissue markers within a given sample using an mIF panel. mIF has several advantages relevant to both clinical and basic research applications including 1) conservation of limited sample by visualizing multiple targets within a single tissue section; 2) preservation of tissue architecture and capture of microenvironment data; 3) provision of data regarding colocalization and spatial orientation of proteins; 4) detection of low-level binding sites and quantitation of target intensity; 5) simplified panel design, wherein any primary antibody validated for IHC, regardless of host species, can be utilized for each target of interest so long as a primary-specific secondary antibody is used; and 6) use of a DAPI (4′,6-diamidino-2-phenylindole) counterstain. mIF is performed on formalin-fixed paraffin-embedded tissue sections, where fluorescent detection dye is covalently attached to the tissue allowing primary/secondary antibodies to be removed for successive rounds of staining (Figure 1). Briefly, the primary and secondary antibodies corresponding to the first target of interest are deposited and incubated with the fluorescent detection substrate. The antibodies

Figure 1. mIF staining cycle. Abbreviations: HRP, horseradish peroxidase TSA, tyramide signal amplification.

Vol. 68 | No. 1–3 | 2020 10.2144/btn-2020-1000AP 87 www.BioTechniques.com are removed from the tissue using heated washes (heat-induced epitope retrieval [HIER]), and the staining process is repeated for subsequent target(s) of interest until all targets have been labeled. Substrate is covalently bonded to the target protein site when tyramide forms bonds with tyrosine residues on or near the antigen and the fluorophore is permanently deposited at the site of the antigen. Multiple rounds of staining are allowed by the process of stripping primary/secondary antibody pairs while preserving antigen- associated fluorescent signal. Prior to imaging, a DAPI counterstain and coverslip is applied.

While a powerful technique, the Target Best Signal real-world application of mIF is (Bethyl Catalog No) st rd th currently limited given the time 1 HIER 3 HIER 6 HIER required to develop an optimized CD68 panel. The following method was X X A500-018A applied to accelerate and simplify the development of 7-color mIF Cytokeran X X X A500-019A panels. CD3E X METHOD FOR DEVELOPMENT A700-016 OF A FASTER, SIMPLIFIED PD-L1 7-COLOR mIF PANEL X A700-020 Formalin-fixed, paraffin-embedded human tissue was stained with FOXP3 X PathPlex™ Panel 4 IHC-validated A700-034 primary antibodies (Bethyl Labora- CD8A X X X tories [A810-004]), mouse or rabbit A700-044 HRP-conjugated secondary antibodies (Bethyl Laboratories [A90-116P, A120-501P]), and detected using Opal™ Polaris 7-color IHC kit fluorophores (Perkin Elmer [NEL861001KT]). Primary antibody order was optimized utilizing tissue microarray serial sections (3 slides per target) by staining after the first, third, or First HIER Third HIER Sixth HIER sixth HIER. Slides were imaged using the same exposure time and 1st HIER 3rd HIER 6th HIER analyzed for target nucleus counts, FOXP3 1684 2419 3281 signal intensity, and background. Primary antibody order in the DAPI+ 35151 32876 33012 7-color mIF was confirmed using Rao 5 7 10 a single stain. Whole slide scans were generated using the Vectra Intensity 77 81 88 Polaris® and analyzed with the ® Background - - - InForm image analysis package. RESULTS Figure 2. Determination of optimal primary antibody order. (A) Using Using optimal primary antibody 3 slides per target, slides were stained after the first, third, and sixth heat- dilution and application order induced epitope retrieval (HIER) to determine the best signal for each primary (Figure 2A; antibody order Option 1 antibody in the panel. Potential optimal primary antibody order was deter- and Option 2), 7-color mIF devel- mined to be either: Option 1 of CD3, cytokeratin, CD8, CD68, PD-L1, and finally opment time was reduced using FOXP3, or Option 2 of CD3, cytokeratin, CD8, CD68, FOXP3, and finally PD-L1. IHC-validated antibodies. The (B) Serial section comparison of FOXP3 staining after the first, third, and number of slides was reduced sixth HIER. (C) InForm® image analysis of FOXP3 staining after the first, from a possible 720 combinations third, and sixth HIER. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole. to 3 per target (18) plus

Vol. 68 | No. 1–3 | 2020 10.2144/btn-2020-1000AP 88 www.BioTechniques.com confirmation 7-color slides. Primary antibody order (confirmed on both tonsil and cancer array cores) was guided by the ratio of target staining/DAPI nuclear counts, average intensity, and overall background of the 3 slides (Figure 2). Certain targets (ie, FOXP3) revealed optimal staining with greater intensity and lower background when stained first, whereas the opposite, or a lack of effect of multiple HIER, may apply to other targets. Optimal primary antibody order was determined to be Option 1, resulting in a panel containing CD3, cytokeratin, CD8, CD68, PD-L1, and FOXP3 (Figure 3).

CONCLUSION AND BROADER APPLICATION TO CLINICAL Option 1 Tonsil Option 2

AND BASIC RESEARCH All channels mIF is a powerful technique that allows for examination of spatial arrangement of proteins of interest as well as protein interactions/co-localization of multiple FO targets within a single tissue XP3 (magenta) PD-L1 (red) specimen. Seven-color mIF panels can take ≥8 weeks to optimize, but use of high-quality IHC-validated antibodies whose order of staining has been pre-determined has immense time- and resource-saving potential. Option 1 Lung Carcinoma Option 2

To create a custom 7-color mIF All channels panel, targets of interest and appropriate high-quality IHC-validated antibodies are selected, and optimal dilution confirmed. Panel order is deter- FO mined by identifying the best XP3 (magenta) PD-L1 (red) signal using serial tissue sections and staining slides after the first, third, and sixth HIER. Resulting images are compared to determine optimal primary antibody order. Antibodies are paired with fluorophores such that antibodies to markers that may have overlapping signal are Figure 3. Representative cores for confirmation of 7-color slides in tonsil separated by ≥1 Opal, and order and lung carcinoma array. Possible optimal primary antibody order is for mIF staining is confirmed. depicted as Option 1 (CD3, cytokeratin, CD8, CD68, PD-L1, and finally FOXP3) and Option 2 (CD3, cytokeratin, CD8, CD68, FOXP3, and finally PD-L1). Option 1 was considered the optimal primary antibody order.

Guidelines for the optimization of mIF, including a step-by-step protocol, can be found at https://www.bethyl.com/content/protocol- multiplexing. For further information regarding validated PathPlex™ antibodies, visit https://promotions.bethyl.com/multiplexing/.

Vol. 68 | No. 1–3 | 2020 10.2144/btn-2020-1000AP 89 www.BioTechniques.com New Products Eppendorf launches innovative, first-of-its-kind 25 ml conical tubes

Eppendorf, a leader in conical tube production and design and inventor of the Eppi™ tube, has launched a brand new 25 ml conical tube that is easier to use, minimizes storage space and is more sustainable. Since many researchers work with sample volumes between 15 ml and 25 ml, but only 15 ml and 50 ml tubes were available, Eppendorf saw the need in the industry for a 25 ml conical tube. The 25 ml conical tube is the same diameter as the conventional 50 ml conical tube and comes with either the new patented SnapTec™ snap cap or screw cap, both of which have high centrifugation stability. The wide opening, combined with the lower height, offers easy sample access, and allows for up to 30% more storage in fridges or freezers. When working with low-volume pipettes and tips, the risk of cross-contamination between pipette and tube by touching the inner tube wall is also minimized. One of the most innovative features is the patented SnapTec cap, which is unique within the conical tube market. This cap is firmly connected to the tube and allows single-handed opening and closing for quick liquid extraction or addition of sample. “In addition to the convincing handling and application benefits, we designed the Eppendorf 25 ml conical tube to work with the lab equipment you already have so there’s straightforward integration into the existing lab environment,” said Nils Gerke, Business Manager of Consumables.

For more information, visit www.eppendorf.com/25ml

One for all: the new Pall Touch screen repeating pipette Nanosep® device for nucleic with advanced features from acid binding BrandTech® Scientific

Offering increased NEW! The BRAND® HandyStep® touch S repeating pipette binding capacity has advanced pipetting features for versatility. In addition through its innovative to standard pipetting, dispensing, auto dispensing and a dual-layer silica-based proprietary “learn” function, advanced modes include quartz glass fiber sequential dispensing of different volumes, multi-aspiration membrane, the of different volumes versatile device can and a titration mode. recover from 10,000 Up to ten favorites down to 50 base pairs can be saved for ease – a wide range offering of use. Automatic size recognition of benefits for applica- BRAND® PD-Tip™ II tions requiring efficient capture of small fragments. tips, an easy Overall, the Pall NAB Nanosep offers you flexibility and navigable touch cost savings without sacrificing quantity or quality. Request screen interface and a sample or find out more with Pall’scomprehensive protocols motorized tip ejection and application notes. c o m p l e t e s t h e package. Inductive For more information, visit https://laboratory.pall.com/en/ charging stand forms/pall-nab.html available separately Request a sample: https://laboratory.pall.com/en/forms/ or as part of an attrac- pall-nab/sample_request.html tively priced bundle.

Hear from Pall’s Project Manager for the device, Lori Euler, in this exclusive interview with BioTechniques: www.biotechniques.com/ For more preclinical/two-minutes-with-lori-euler-pall-global-product- information, visit manager-for-the-nanosep-nucleic-acid-binding-device/ www.brandtech.com

Fluidic Analytics Ltd has launched a time-saving and affordable protein labeling kit – fluidiphore rapid amine 503. Absorbing at 503 nm, the new kit can be used with a multitude of techniques including the company’s groundbreaking Fluidity One-W instrument for protein interaction analysis. Fluidic Analytics’ fluidiphore labeling kit is extremely rapid, taking just 30 minutes to bind proteins rather than 4 hours to overnight, as is required by other kits. In addition, the fluidiphore kit uniquely does not require a purification step. This is due to a clever ‘triple-lock’ against background effects. Firstly, the absorbance of the dye shifts from 612 nm (free) to 503 nm when conjugated, meaning that unbound dye will not fluoresce at the same excitation wavelength. Quantum yield (the ratio of fluorescence emission to absorption) also increases upon conjugation meaning that unbound dye will have a much weaker signal than bound. Thirdly, any unused dye hydrolyses during the labeling process becoming inactive and unable to bind, therefore preventing the shift in absorbance and increase in quantum yield. In addition, there is also a useful observable color change from blue to red/yellow which confirms that labeling is taking place, giving researchers additional confidence in the process. The fluidiphore rapid amine 503 labeling kit is ideal for use with Fluidic Analytics’ Fluidity One-W instrument to study proteins such as membrane proteins, multi-protein complexes, and intrinsically disordered proteins. The Fluidity One-W enables researchers to accurately assess on-target protein interactions in solution, even in complex, unpurified backgrounds such as crude lysates or blood plasma. The instrument reports absolute size of bound and unbound species, stoichiometry and the nature of binding can be assessed at the same time as binding affinity—giving a complete picture of binding events.

For more information, visit www.fluidic.com

New OEM LED illumination platform

At ASCB 2019, CoolLED launched a new approach to fast-track the development of high content screening and slide scanning systems. LED illumination systems are the perfect match for fluorescence microscopy. With unbeatable speed, stability and energy-efficiency, what’s not to love? Our new Amora modular platform allows endless opportunities for custom designed LED illumination to suit almost any OEM requirement. Best of all, the option to have up to 16 wavelengths means scientists can now choose fluorophores optimised for the assay rather than the illumination system. The Amora is at the heart of our new streamlined approach to creating bespoke illumination systems and is backed up by our engineering and manufacturing expertise. CoolLED’s renowned service and dedicated project management also ensures the project runs smoothly from start to finish. With over 10 years’ experience in producing quality solutions, in volume, to a worldwide customer base, CoolLED is the ideal OEM illumination partner.

For more information, visit www.oemillumination.com

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Vol. 68 | No. 1–3 | © 2020 Future Science Ltd 91 www.BioTechniques.com In the Next Issue Here’s an exclusive preview of what’s coming up in the next issue of BioTechniques LAURA BOYKIN OPINION TAKING THE HIGHEST TECH GENOMICS TOOLS TO THE FARMERS IN EAST AFRICA

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