DOCSLIB.ORG

Targeted Bisulfite Sequencing: an Efficient, Streamlined Approach to Maximize Throughput and Minimize Cost

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Targeted Bisulfite Sequencing: an Efficient, Streamlined Approach to Maximize Throughput and Minimize Cost Targeted Bisulfite Sequencing: An Efficient, Streamlined Approach to Maximize Throughput and Minimize Cost Ioanna Andreou1, Brian Dugan2, Jonathan Schaffer2, Mohammad Nezami Ranjbar2, Sascha Strauss1, Quan Peng2, Silke Huebner1, Christin Meerschiff1, Katja Heitz1, Christopher Wach1, Eric Lader2 1 QIAGEN GmbH, QIAGEN Straße 1, 40724 Hilden, Germany 2 QIAGEN Sciences Inc., 6951 Executive Way, Frederick, MD 21703, USA Introduction QIAseq Targeted Methyl Panel Workflow Epigenetic changes have a significant impact on human health and disease susceptibility since they play a crucial role in the DNA regulation of important cellular processes. In recent years, significant progress has been made in this rapidly advancing field. DNA bisulte conversion: EpiTect Fast DNA Bisulte Kit EpiTect Fast Bisulfite Conversion One of the most stable forms of epigenetic regulation is DNA methylation. Bisulfite sequencing (BS) is considered the best (cat. nos. 59802, 59824, 59826) chemistry method for detecting DNA methylation with single nucleotide resolution. However, the high cost of whole genome BS and UUCCU the low throughput of amplicon BS are challenges that must be overcome. DNA end repair Here, we present a streamlined workflow combining optimal bisulfite treatment and targeted bisulfite sequencing. This U CCUU X method utilizes single-primer extension (SPE) and unique molecular identifier (UMI) barcoding technology. This enables Ligation of rst index adapters with UMI highly efficient, flexible and scalable targeted analysis of DNA methylation from genomic DNA (gDNA), formalin-fixed QIAseq Targeted Methyl Panel chemistry for target enrichment UMI U CCUU paraffin-embedded (FFPE) and circulating cell-free DNA (ccfDNA) samples. 7.5 h and generation of NGS library 6 h suitable for Illumina platforms Target enrichment by single primer extension Sample to Insight 5' U CCUU GSP Library FP Sample Bisulfite construction NGS run Data analysis Repeated sampling preparation conversion with target enrichment Library amplication and sample indexing = Methylated C = Non-methylated C ® ® ® ® DNeasy , QIAamp , EpiTect Fast Bisulfite • Primer design tool Illumina platforms • Bisulfite DNA 5' TCTTC UMI: Unique molecular index Gentra®, or GeneRead™ Conversion Kits for: • QIAseq® Targeted analysis pipeline SIP FP: Forward primer technologies for isolation of: Methyl Panel of the GeneGlobe® UP • Purified gDNA GSP Gene-specic primer • QIAseq Methyl DNA Data Analysis Center : • gDNA • FFPE DNA UP Index Kits • CLC Genomics : Universal primer • ccfDNA Library SIP: Sample index primer • FFPE DNA Workbench for Methylation Streamlined workflow for DNA methylation analysis. QIAseq Targeted Methyl Panel workflow. Performance: gDNA Performance: ccfDNA Control gDNA (0% and 100% methylated, with inputs of 1 ng, 10 ng, 50 ng and 100 ng) was bisulfite treated using the ccfDNA was purified using QIAamp chemistry and subsequently bisulfite treated using EpiTect Fast chemistry. 10 ng ccfDNA EpiTect Fast DNA Bisulfite Kit and libraries were generated using the using the Colorectal Cancer Panel (MHS-002Z) and was processed using three different QIAseq Targeted Methyl Panels. Library amplification with 19 cycles resulted in high QIAseq Targeted Methyl Panel chemistry. The libraries were sequenced using a MiSeq® instrument with sequencing depth of yields of the generated library. The expected 350 bp library size represents the combination of 170 bp (typical size of approx. 150x. Data were analyzed using the Bisulfite DNA analysis pipeline of the GeneGlobe Data Analysis Center. The ccfDNA with 180 bp adapters). This shows that no DNA degradation occurred during bisulfite treatment with EpiTect Fast results show high library quality, a high mapping rate – even starting from 10 ng input gDNA and high cytosine (C) coverage. chemistry. The libraries where sequenced on MiSeq 2x150 bp paired end run and a sequencing depth of approx. 200x. The high-quality libraries resulted in high number of unique mapped reads which led to a high cytosine coverage to allow accurate methylation calls even from low input of the strong fragmented ccfDNA. Colorectal carcinoma panel Colorectal carcinoma panel ccfDNA library characteristics ccfDNA 20 ng input % of total reads Covered cytosines, % Average fragment Total reads, % 100 100 Yields, nM size, bp 35 400 150 355 343 350 80 80 30 350 300 100 25 60 60 250 20 50 13.1 15.4 200 40 40 15 150 9.0 0 10 748 primer 1900 primer 1400 primer 20 20 100 5 50 Methyl panel 0 0 0 0 1 ng 10 ng 50 ng 100 ng 1 ng 10 ng 50 ng 100 ng 1 ng 10 ng 50 ng 100 ng 1 ng 10 ng 50 ng 100 ng Methyl panel (748) Methyl panel (1900) Methyl panel (1400) Reads after trimming, % Reads matched a primer, % Reads unique mapped 0% methylated 100% methylated 0% methylated 100% methylated Input 10 ng ccfDNA On-target, % Off-target, % C covered, % CpG covered >30 UMI, % Molarity, nM Average size After trimming, % Map to primer, % Unique maping, % On-target, % Off-target, % % C covered % CpG covered Yields and mean size of generated libraries using three different panels Quality of generated libraries. Libraries and panels used as a percentage consisting of different primer numbers. of the reads after trimming; reads matched to primer sequence; unique mapped reads on target and off-target reads to total number of reads. Read statistics as percentage of all reads obtained. A high unique mapping Cytosine coverage. Total cytosine coverage and coverage of cytosines in The figure also shows the high coverage of cytosines and cytosines in CpG and on target rate can be achieved even from 10 ng gDNA input in the CpG content as percentage of the theoretical C expected to be covered by content as percentage of the theoretical C expected to be covered by the complete workflow. the corresponding panel. corresponding panels. Performance: FFPE DNA Conclusions A QIAseq Targeted Methyl Panel consisting of 93 primers was used to generated libraries with a starting input of 40 ng We present a workflow and chemistry that enables methylation sequencing in a targeted mode, which in turn, allows FFPE DNA. The DNA was first bisulfite converted using EpiTect Fast chemistry. The generated libraries were sequenced on testing of multiple samples and regions of interest in a cost-efficient manner. a MiSeq instrument with a sequencing depth of approx. 200x, generating a mean of 65 UMI per cytosine. Our method: ® The Methylation degree of 7 CpG sites of the MGMT gene was previously validated with Pyrosequencing . The evaluated • Combines highly efficient EpiTect Fast bisulfite conversion chemistry and the QIAseq Methyl Targeted Panels to reduce methylation degree of the MGMT gene using the QIAseq Targeted Methyl Panel highly correlate with Pyrosequencing data. turnaround time over probe-capture-based enrichment Comparable high C coverage was obtained with all FFPE DNA having different methylation status. • Shows high enrichment specificity with low bias for bisulfite-converted DNA, using an optimized SPE methodology including UMI MGMT methylation degree MGMT methylation degree • Allows accurate detection of CpG methylation at different levels C covered, % Methylation, % • Is compatible with gDNA, FFPE DNA and ccfDNA 100 100 77,8 • Allows lower DNA input 80 80 75 • Increases flexibility of methylation analysis through highly efficient panel design to enrich only the regions of interest 60 60 • Integrates analysis tools to standardize the method of methylation status evaluation 40 40 20 20 13 9,4 For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user 2 2,6 0 0 DNA1 DNA5 DNA9 DNA1 DNA5 DNA9 manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN 250 bp run Technical Services or your local distributor. C covered Pyro QIAseq Targeted Methyl Panel 9 1 0 ® ® ® ® ® ® ® ™ ® ® Cytosine coverage obtained in the region of interest. Comparable high Comparison of evaluated methylation degree with QIAseq Targeted Methyl Trademarks: QIAGEN , Sample to Insight , QIAamp , QIAseq , DNeasy EpiTect , GeneGlobe , GeneRead , Gentra , Pyrosequencing (QIAGEN Group); 0/2 ® 1 C coverage was obtained with all FFPE DNAs having different methylation Panel and Pyrosequencing. The evaluated methylation degree of the MiSeq (Illumina, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered status. MGMT gene highly correlates with the methylation degree of the 7 CpG unprotected by law. sites previously validated with Pyrosequencing. © 2019 QIAGEN, all rights reserved. PROM-15023-001 1119112 Sample to Insight.
Load more

Recommended publications
  • Three Optimized Workflows for Cpg Island Methylation Profiling
    APPLICATION NOTE Three Optimized Workflows for CpG Island Methylation Profiling Three Optimized Workflows for CpG Island Methylation Profiling DNA methylation is involved in the Discovery regulation of many cellular processes SOLiD™ Do you know your candidate gene? including X chromosome inactivation, No System chromosome stability, chromatin structure, Yes embryonic development, and transcription. Validation/Screening with Capillary Electrophoresis Do you know your candidate region or In mammals only the 5' carbons of the methylation state of that region? cytosines adjacent to guanines (CpG Yes No dinucleotides: cytosine-phospho-guanine) MSMSA Bisulfite Conversion Bisulfite Conversion are substrates for DNA methylation. (MethylSEQr™ Kit) (MethylSEQr™ Kit) Importance of CpG Islands Are there a high number Bisulfite Specific (BSP) CpG islands are 300−3000 base pair of poly T or A, or large Primer Design fragment size (>300 bps)? (Methyl Primer® Express) stretches of DNA that are CG rich, and are often found within unmethylated Yes No regions of promoters. Profiling of CpG Clone based Sequencing Direct PCR Sequencing island methylation indicates that some Fluorescent BSP PCR genes are more frequently methylated Bisulfite Specific (BSP) Primer Design Bisulfite Specific (BSP) Primer Design Data Analysis (Methyl Primer® Express) (Methyl Primer® Express) than others [1]. Hence, CpG islands play a (GeneMapper® Software v. 4.0) critical role in regulating gene expression. CpG islands found outside promoter BSP PCR BSP PCR Identification of a candidate region or the No regions are commonly methylated, and methylation state of Yes that region? are believed to be responsible for silencing Cloning Yes transcription of repetitive sequences Cycle Sequencing Direct PCR Cycle Sequencing Are there a high number and parasitic sequence elements, such of poly T or A, or large No as viral DNA.
    [Show full text]
  • DNA Methylation Analysis: Choosing the Right Method
    biology Review DNA Methylation Analysis: Choosing the Right Method Sergey Kurdyukov 1,* and Martyn Bullock 2 Received: 8 July 2015; Accepted: 22 December 2015; Published: 6 January 2016 Academic Editor: Melanie Ehrlich 1 Genomics Core facility, Kolling Institute of Medical Research, University of Sydney, Sydney 2065, Australia 2 Cancer Genetics Laboratory, Kolling Institute of Medical Research, University of Sydney, Sydney 2065, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-299-264-756 Abstract: In the burgeoning field of epigenetics, there are several methods available to determine the methylation status of DNA samples. However, choosing the method that is best suited to answering a particular biological question still proves to be a difficult task. This review aims to provide biologists, particularly those new to the field of epigenetics, with a simple algorithm to help guide them in the selection of the most appropriate assay to meet their research needs. First of all, we have separated all methods into two categories: those that are used for: (1) the discovery of unknown epigenetic changes; and (2) the assessment of DNA methylation within particular regulatory regions/genes of interest. The techniques are then scrutinized and ranked according to their robustness, high throughput capabilities and cost. This review includes the majority of methods available to date, but with a particular focus on commercially available kits or other simple and straightforward solutions that have proven to be useful. Keywords: DNA methylation; 5-methylcytosine; CpG islands; epigenetics; next generation sequencing 1. Introduction DNA methylation in vertebrates is characterized by the addition of a methyl or hydroxymethyl group to the C5 position of cytosine, which occurs mainly in the context of CG dinucleotides.
    [Show full text]
  • High-Resolution Bisulfite-Sequencing of Peripheral Blood DNA Methylation in Early-Onset and Familial Risk Breast Cancer Patients
    Author Manuscript Published OnlineFirst on June 7, 2019; DOI: 10.1158/1078-0432.CCR-18-2423 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. High-Resolution Bisulfite-Sequencing of Peripheral Blood DNA Methylation in Early- Onset and Familial Risk Breast Cancer Patients Justin Chen1*, Maria K. Haanpää2*, Joshua J. Gruber1,2, Natalie Jäger1, James M. Ford1,2ǂ, and Michael P. Snyder1ǂ 1Department of Genetics 2Department of Medicine, Oncology Division Stanford University, Stanford, CA 94305, USA. * These authors contributed equally to this work. ǂ Co-corresponding authors Running Title: DNA Methylation and High-Risk Breast Cancer Keywords: DNA Methylation; Breast Cancer; Epigenetics; Cancer Predisposition; Allelic Methylation Additional Information: Financial Support: This work used the Genome Sequencing Service Center by Stanford Center for Genomics and Personalized Medicine Sequencing Center, supported by the grant award NIH S10OD020141. MKH is supported by grants from Sigrid Juselius Foundation, Orion Research Foundation, Päivikki ja Sakari Sohlberg Foundation and Instrumentarium Science Foundation. JJG was supported by fellowships from the Jane Coffin Childs Memorial Fund for Medical Research, Stanford Cancer Institute and Susan G. Komen Foundation, as well as funding from ASCO, the Conquer Cancer Foundation and the Breast Cancer Research Foundation. NJ was supported by an EMBO Long-Term Fellowship (ALTF 325-2014). JMF is supported by the BRCA Foundation and the Breast Cancer Research Foundation. MPS is supported by grants from the NIH including a Centers of Excellence in Genomic Science award (5P50HG00773504). Correspondence: Michael P. Snyder James M. Ford Department of Genetics 269 Campus Dr. Stanford University School of Medicine CCSR Room 1115 300 Pasteur Dr.
    [Show full text]
  • HITTING the MARK in CANCER EPIGENETICS Research in Cancer Epigenetics Is Advancing Rapidly
    HITTING THE MARK IN CANCER EPIGENETICS Research in cancer epigenetics is advancing rapidly. Here’s an overview of the tools and methods scientists use to drive progress in the field. For by CUSTOM MEDIA ADVERTISEMENT FEATURE ADVERTISEMENT FEATURE says. “We usually put these cytosine ring — is the best look and behave. Illumina’s Andrew Feber, a cancer patients on a watch and understood epigenetic marker methylation array covers geneticist at UCL Cancer HITTING THE MARK wait scheme. We could have today. Methylation typically 850,000 sites on the Institute in London, is one spared her all that therapy.” silences gene transcription genome, and analyses with of those scientists. He aims Pfister’s experience in patterns that are it, he says, “are sufficient to find epigenetic traces IN CANCER EPIGENETICS illustrates the growing characteristic to specific cell to capture the cancer cell’s of bladder cancer in urine, potential of genomic types and tissues. But when basic identity”. an endeavour he says is RESEARCH IN CANCER EPIGENETICS IS ADVANCING RAPIDLY. Here’s an overview of the tools technologies in epigenetics to it silences tumour suppressor Esteller points out that particularly well suited to and methods scientists use to drive progress in the field. contribute to understanding, genes, cancer may appear. methylation arrays have sequencing since it can diagnosing and treating The first epigenetic many positive aspects, such read the often fragmented cancer. Epigenetic technologies — methylation as low-cost and amenability arrangements of DNA modifications to DNA and arrays — emerged about 15 to formalin-fixed, paraffin- letters in liquid specimens. n 2013 Stefan Pfister, RNA regulate how and when years ago with the launch embedded tissue samples.
    [Show full text]
  • BS-Seq Data Processing Lecture (Pdf)
    Bisulfite-Sequencing Theory and Quality Control [email protected] v2021-04 Bisulfite-Seq theory and Quality Control coffee a.m. Mapping and QC practical Visualising and Exploring talk Lunch Visualising and Exploring practical p.m. coffee Differential methylation talk & practical Epigenetics Studies changes in gene expression which are not encoded by the underlying DNA sequence Chromatin • histone modification • non-coding RNAs • higher order structure (accessibility/compaction) DNA cytosine methylation From The Cell Biology of Stem Cells (2010) Types of DNA methylation CG context non-CG context me me T C G T A T C A T A 5’ 5’ 5’ 5’ 3’ A G C A T 3’ 3’ A G T A T 3’ me Mammals Plants CG present present CHG (mostly) absent present H = T/A/C CHH (mostly) absent present DNA methylation is stably maintained from W. Reik & J. Walter, Nat. Rev. Genet. 2001 Regulation by DNA methylation Silencing of gene expression Tissue differentiation and embryonic development Faults in correct DNA methylation may result in - early development failure - epigenetic syndromes Repeat activity - cancer Genomic stability Imprinted Genes: mono-allelic expression Differential allelic DNA methylation CGI (CpG island) X methylated CpG unmethylated CpG Imprinted Genes: Mono-allelic expression with parent-of-origin specificity. Have key roles in energy metabolism, placenta functions. DNA Methylation DNA methyl-transferases DNA-demethylase(s)? TETs? Passive demethylation? Cytosine 5-methyl Cytosine Other cytosine modifications Miguel R. Branco, Gabriella Ficz & Wolf Reik Nature Reviews Genetics 13, 7-13 (January 2012) Measuring DNA methylation by Bisulfite-sequencing Image by Illumina Bisulfite Informatics me me CCAGTCGCTATAGCGCGATATCGTA Convert TTAGTTGCTATAGTGCGATATTGTA Map TTAGTTGCTATAGTGCGATATTGTA ||||||||||||||||||||||||| ...CCAGTCGCTATAGCGCGATATCGTA..
    [Show full text]
  • The Bispcr2 Method for Targeted Bisulfite Sequencing Diana L Bernstein, Vasumathi Kameswaran, John E Le Lay, Karyn L Sheaffer and Klaus H Kaestner*
    Bernstein et al. Epigenetics & Chromatin (2015) 8:27 DOI 10.1186/s13072-015-0020-x METHODOLOGY Open Access The BisPCR2 method for targeted bisulfite sequencing Diana L Bernstein, Vasumathi Kameswaran, John E Le Lay, Karyn L Sheaffer and Klaus H Kaestner* Abstract Background: DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statis- tical significance, but are also high cost if performed genome-wide. Here, we describe a simplified targeted bisulfite sequencing approach in which DNA sequencing libraries are prepared following sodium bisulfite conversion and two rounds of PCR for target enrichment and sample barcoding, termed BisPCR2. Results: We have applied the BisPCR2 technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addi- tion to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR2 compared to prior array-based approaches. Conclusion: This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illus- trates its utility by reanalysis of prior findings from genome-wide studies. Keywords: Targeted bisulfite sequencing, DNA methylation, Next-generation sequencing Background disorders, and cardiovascular disease [9–11]. Many of the DNA methylation refers to the addition of a methyl studies linking DNA methylation to disease have been group to the 5-carbon position of cytosine residues, and prompted by the observation that only a small fraction in mammalian genomes occurs most commonly in the of the inherited risk of these complex disorders can be context of CpG dinucleotides.
    [Show full text]
  • Profiling DNA Methylation Based on Next-Generation Sequencing Approaches
    G C A T T A C G G C A T genes Review Profiling DNA Methylation Based on Next-Generation Sequencing Approaches: New Insights and Clinical Applications Daniela Barros-Silva 1 ID , C. Joana Marques 2,3 ID , Rui Henrique 1,4,5 ID and Carmen Jerónimo 1,5,* ID 1 Cancer Biology and Epigenetics Group, IPO Porto Research Center (CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto), Rua António Bernardino Almeida, 4200-072 Porto, Portugal; [email protected] (D.B.-S.); [email protected] (R.H.) 2 Genetics, Department of Pathology, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal; [email protected] 3 I3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal 4 Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto), 4200-072 Porto, Portugal 5 Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar (ICBAS)—University of Porto, 4050-313 Porto, Portugal * Correspondence: [email protected]; Tel.: +351-225084000; Fax: +351-225084199 Received: 23 May 2018; Accepted: 20 August 2018; Published: 23 August 2018 Abstract: DNA methylation is an epigenetic modification that plays a pivotal role in regulating gene expression and, consequently, influences a wide variety of biological processes and diseases. The advances in next-generation sequencing technologies allow for genome-wide profiling of methyl marks both at a single-nucleotide and at a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, coverage, and bioinformatics analysis. Thus, the selection of the most feasible method according with the project’s purpose requires in-depth knowledge of those techniques.
    [Show full text]
  • DNA Methylation Data by Sequencing: Experimental Approaches and Recommendations for Tools and Pipelines for Data Analysis
    Rauluseviciute et al. Clinical Epigenetics (2019) 11:193 https://doi.org/10.1186/s13148-019-0795-x REVIEW Open Access DNA methylation data by sequencing: experimental approaches and recommendations for tools and pipelines for data analysis Ieva Rauluseviciute1* , Finn Drabløs1 and Morten Beck Rye1,2 Abstract Sequencing technologies have changed not only our approaches to classical genetics, but also the field of epigenetics. Specific methods allow scientists to identify novel genome-wide epigenetic patterns of DNA methylation down to single-nucleotide resolution. DNA methylation is the most researched epigenetic mark involved in various processes in the human cell, including gene regulation and development of diseases, such as cancer. Increasing numbers of DNA methylation sequencing datasets from human genome are produced using various platforms—from methylated DNA precipitation to the whole genome bisulfite sequencing. Many of those datasets are fully accessible for repeated analyses. Sequencing experiments have become routine in laboratories around the world, while analysis of outcoming data is still a challenge among the majority of scientists, since in many cases it requires advanced computational skills. Even though various tools are being created and published, guidelines for their selection are often not clear, especially to non-bioinformaticians with limited experience in computational analyses. Separate tools are often used for individual steps in the analysis, and these can be challenging to manage and integrate. However, in some instances, tools are combined into pipelines that are capable to complete all the essential steps to achieve the result. In the case of DNA methylation sequencing analysis, the goal of such pipeline is to map sequencing reads, calculate methylation levels, and distinguish differentially methylated positions and/or regions.
    [Show full text]
  • Whole Genome Bisulfite Sequencing of Down Syndrome Brain Reveals Regional DNA Hypermethylation and Novel Disease Insights
    bioRxiv preprint doi: https://doi.org/10.1101/428482; this version posted September 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Whole genome bisulfite sequencing of Down syndrome brain reveals regional DNA hypermethylation and novel disease insights Benjamin I. Laufer1, Hyeyeon Hwang1, Annie Vogel Ciernia1, Charles E. Mordaunt1, Janine M. LaSalle1,2,3, * 1 Department of Medical Microbiology and Immunology, School of Medicine 2 Genome Center 3 MIND Institute University of California, Davis, CA 95616, USA *Correspondence: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/428482; this version posted September 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Abstract (250 Words) Down Syndrome (DS) is the most common genetic cause of intellectual disability, where an extra copy of human chromosome 21 (HSA21) produces differential genome-wide DNA methylation profiles. Although DNA methylation has been examined across the genome at select regulatory regions in a variety of DS tissues and cells, the differentially methylated regions (DMRs) have yet to be examined in an unbiased sequencing-based approach. Here, we present the first analysis of DMRs from whole-genome bisulfite sequencing (WGBS) data of human DS and matched control brain, specifically frontal cortex.
    [Show full text]
  • Methylated DNA Immunoprecipitation and High-Throughput Sequencing (Medip-Seq) Using Low Amounts of Genomic DNA
    CELLULAR REPROGRAMMING Volume 16, Number 3, 2014 Original Article ª Mary Ann Liebert, Inc. DOI: 10.1089/cell.2014.0002 Methylated DNA Immunoprecipitation and High-Throughput Sequencing (MeDIP-seq) Using Low Amounts of Genomic DNA Ming-Tao Zhao,1 Jeffrey J. Whyte,1 Garrett M. Hopkins,2 Mark D. Kirk,2 and Randall S. Prather1 Abstract DNA modifications, such as methylation and hydroxymethylation, are pivotal players in modulating gene ex- pression, genomic imprinting, X-chromosome inactivation, and silencing repetitive sequences during embryonic development. Aberrant DNA modifications lead to embryonic and postnatal abnormalities and serious human diseases, such as cancer. Comprehensive genome-wide DNA methylation and hydroxymethylation studies provide a way to thoroughly understand normal development and to identify potential epigenetic mutations in human diseases. Here we established a working protocol for methylated DNA immunoprecipitation combined with next-generation sequencing [methylated DNA immunoprecipitation (MeDIP)-seq] for low starting amounts of genomic DNA. By using spike-in control DNA sets with standard cytosine, 5-methylcytosine (5mC), and 5- hydroxymethylcytosine (5hmC), we demonstrate the preferential binding of antibodies to 5mC and 5hmC, respectively. MeDIP-PCRs successfully targeted highly methylated genomic loci with starting genomic DNA as low as 1 ng. The enrichment efficiency declined for constant spiked-in controls but increased for endogenous methylated regions. A MeDIP-seq library was constructed starting with 1 ng of DNA, with the majority of fragments between 250 bp and 600 bp. The MeDIP-seq reads showed higher quality than the Input control. However, after being preprocessed by Cutadapt, MeDIP (97.53%) and Input (94.98%) reads showed comparable alignment rates.
    [Show full text]
  • Techniques to Overcome Bottlenecks in Epigenetics Research
    Scientific article Techniques to overcome bottlenecks in epigenetics research Gerald Schock and Thorsten Traeger QIAGEN GmbH, Qiagen Straße 1, 40724 Hilden, Germany Abstract: This article discusses the obstacles in bisulfite conversion of DNA for epigenetics research applications and uncovers possible solutions to overcome them. Furthermore, various methods for methylation analysis such as high-resolution melting (HRM®), methylation-specific PCR (MSP), MethyLight PCR, bisulfite sequencing, and Pyrosequencing® are described, and their respective uses and limitations are evaluated. Introduction Contents DNA methylation, the process of adding a methyl group to Introduction __________________________________________ 1 cytosines, is one of the key epigenetic mechanisms in mammals. DNA methylation — an important epigenetic mechanism ___ 2 It is closely associated with histone modification, and it alters DNA methylation in disease progression _________________ 2 the chromatin structure to enable control and modulation of Bisulfite conversion — the fundamental step in gene expression. DNA methylation plays a crucial role in DNA methylation analysis ______________________________ 2 mammalian development, genomic imprinting, and in genomic Increasing yield and PCR sensitivity through direct bisulfite stability. Because its deregulation can both cause or result conversion without prior DNA isolation __________________ 3 from abnormal cell regulation in cancer, studies in DNA Stability of bisulfite treatment — a challenge for storing methylation are expected to lead to the development of converted DNA for downstream analysis _________________ 4 effective biomarkers and drugs against cancer or other Overcoming limitations due to small sample amounts ______ 5 malignant diseases. Screening for unknown methylation CpG Assessing DNA methylation typically comprises three major positions — high-resolution melting analysis (HRM) ________ 5 steps — DNA isolation, bisulfite conversion, and analysis of Methylation-specific PCR (MSP) — an easy way to the bisulfite converted DNA.
    [Show full text]
  • Accurate, Customizable, and Cost-Effective Targeted Methylation Sequencing
    PRODUCT TABLE APPLICATION NOTE Cat. No. Description Reactions 30XX16M.v5 Daicel Arbor Biosciences myBaits® Custom Methyl-Seq Kit 16 30XX48M.v5 Daicel Arbor Biosciences myBaits® Custom Methyl-Seq Kit 48 Accurate, Customizable, and Cost-effective 30XX96M.v5 Daicel Arbor Biosciences myBaits® Custom Methyl-Seq Kit 96 30024 Swift Biosciences Accel-NGS® Methyl-Seq Library Prep Kit* 24 Targeted Methylation Sequencing 30096 Swift Biosciences Accel-NGS® Methyl-Seq Library Prep Kit* 96 Coupling the Swift Accel-NGS® Methyl-Seq Library Preparation and * Swift Methyl-Seq Indexing Kits complete the library preparation workflow. Daicel Arbor Biosciences myBaits® Custom Methyl-Seq Systems ABSTRACT Methylation sequencing is an important tool in understanding gene regulation during various processes, such as cell differentiation and disease progression, and is seeing increased use in diagnostic assays including oncology. Whole-genome methylation sequencing is ideal for target discovery with its unbiased and comprehensive single base resolution. Once specific genome regions of interest are identified, targeted methylation sequencing through hybridization capture prior to next generation sequencing (NGS) enables a more cost-effective methylation profiling solution that maximizes both the number of samples analyzed per experiment and the potential depth of sequencing for evaluating low frequency methylation signatures in complex samples (e.g., cell free DNA for liquid biopsy). In this application note, we demonstrate a highly accurate targeted methylation sequencing workflow using Swift Accel-NGS Methyl-Seq and Daicel Arbor Biosciences myBaits Custom Methyl-Seq systems. Through proprietary library preparation and hybridization chemistries, and a unique probe design algorithm, the system overcomes many inherent challenges to the efficient retrieval of specific bisulfite-converted DNA sequences.
    [Show full text]