MICROBIOMES WHITE PAPER
Advantages of nanopore sequencing nanoporetech.com/publications in microbiome research
nanoporetech.com OXFORD NANOPORE TECHNOLOGIES | ADVANTAGES OF NANOPORE SEQUENCING IN MICROBIOME RESEARCH
Contents
1Challenges of microbiome research
2From microbiome to metatranscriptome and beyond
3Kits and analysis workflows
4Case studies
5Summary
6About Oxford Nanopore Technologies
7References OXFORD NANOPORE TECHNOLOGIES | ADVANTAGES OF NANOPORE SEQUENCING IN MICROBIOME RESEARCH
Introduction
The study of microbiomes — the genetic Classical microbiome research relies material of all microorganisms in a on culturing, which is associated with given sample — has recently attracted long sample-to-result time and biases considerable attention, mainly due to the related to the different susceptibility of realisation that the microbial composition microorganisms to laboratory handling8. of our bodies and environment can have a The advent of modern sequencing profound effect on our health. technologies has brought significant advantages to the field. Speed and The advent of modern accuracy of microbial analysis (i.e. sequencing technologies has species identification and abundance) have increased substantially, with culturing brought significant advantage to no longer being necessary. Nonetheless, the field of microbial analysis. some challenges remain with traditional sequencing approaches. The composition of the human microbiome, for example, has been This review will explore the challenges connected to obesity1, immunity2 and of microbiome research, providing psychiatric conditions3. Of note, there real-world examples of how they are has also been a surge in research efforts being overcome through the use of focusing on analysing the microbiome nanopore sequencing technology. of extreme environments4, water5, soil6, buildings7, etc.
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Challenges of microbiome research
Accurate microbe identification genomic regions, resulting in improved precision of metagenomic species There are two general approaches for the identification4,11. Furthermore, in 16S-based identification of microbes in a biological studies, nanopore technology allows for the 1 sample. The metagenomic sequencing design of primers covering the whole 16S approach results in the whole genome of gene10 or even whole ribosomal operons12, any microbe present in the sample being often leading to nanopore sequencing sequenced. This unbiased method avoids outperforming traditional sequencing the need for culturing and thus allows platforms in the number of species the identification of currently unculturable accurately distinguished10. organisms. Nonetheless, traditional short-read sequencing technologies Analysis at point of collection struggle to reliably resolve repetitive regions9, making the taxonomic For the most accurate understanding of classification and genome assembly of microbiome composition, samples should closely-related species in such samples be analysed quickly — ideally at the site of challenging. This and other concerns, such collection. However, due to the large size as time to result and cost-effectiveness, and infrastructure requirements of traditional led to the introduction of 16S rRNA gene sequencing machines, researchers often sequencing. collect samples, store them for long periods of time (risking loss of material or change in sample composition while in storage13) and Nanopore technology allows for the transport them to centralised laboratories.
design of primers covering the whole Oxford Nanopore’s MinION™, weighs length of the 16S gene or even whole less than 100 g, is easily transportable in ribosomal operons. aeroplane luggage11 and is powered to sequence DNA or RNA using the USB port The 16S rRNA gene sequence is commonly on a laptop or the IT accessory, MinIT™, used because of its combination of making it suitable for mobile research conserved and highly variable regions, setups. As a result, samples do not need allowing for accurate species distinction. to be frozen and stored for weeks or even However, the 16S primers typically used in days prior to analysis. For example, the these experiments do not cover the whole time from sample collection to sequencing 16S gene region, which can reduce the data generation with the MinION in an resolution of species identification as some Arctic environment was reported to be 11 taxonomically informative regions fall outside just under 40 hours . Oxford Nanopore of the analysed amplicon10. is also developing Flongle™, a flow cell adapter designed to provide even Nanopore long reads are much more more cost-effective analysis of smaller, suitable for the assembly of repetitive more frequently performed tests and
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experiments. Furthermore, portable library in clinical research and disease surveillance preparation, requiring the bare minimum applications, the potential for accurate and of laboratory equipment, is possible using rapid sample analysis could, in the future, Oxford Nanopore’s VolTRAX™ system. lead to faster implementation of appropriate Together, these devices will deliver the therapeutic intervention or pathogen flexibility to collect and analyse samples containment strategies14. onsite, which not only reduces time to result, but also allows the number of Nanopore sequencing allows sample samples collected to be informed by the analysis both in the lab or field, with the data generated, preventing under or over most recent transposase-based library sampling and further streamlining the preparation kits from Oxford Nanopore project workflow. requiring just 10 minutes of hands-on time (after genomic DNA extraction). Streamlined workflows coupled with In combination with real-time data analysis, these streamlined workflows enable real-time analysis enable metagenomic applications such as species identification species identification in as little as to be achieved in as little as 20 minutes — 20 minutes — from sample to result15. from sample to result15. The facility for real- time sequencing also allows the sequencing run to be stopped as soon as sufficient data Reduced time to result has been generated or a particular species When monitoring changes in the identified, further highlighting the benefits microbiome and handling many samples, of nanopore sequencing for the delivery of being able to process samples quickly and rapid and potentially actionable information. easily has many advantages. For example,
Figure 1 Figure 2 Figure 3 MinION: a pocked-sized, portable device. VolTRAX: designed to perform library Flongle: a flow cell adapter that enables preparation automatically. direct, real-time DNA or RNA sequencing on smaller, single-use flow cells.
Flongle
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From microbiome to metatranscriptome and beyond
The advent of high-throughput Nanopore platforms are capable of sequencing techniques has not only sequencing genomic DNA and cDNA17 enabled researchers to identify species from a metagenomic or target-enriched 2 and construct genomes from sample, generating long reads suitable microbiome samples, but also to for accurate taxonomic classification and investigate gene expression. transcriptomics investigation.
Such metatranscriptomic studies can The technology is also capable of provide more detailed insight into performing direct RNA sequencing the interaction of complex microbial without introducing reverse transcription- communities and their response to or PCR amplification-related biases18. specific environments. Furthermore, the facility for direct DNA and RNA sequencing enables the detection of chemical modifications, Nanopore sequencing technology such as methylation, without the need facilitates more complete for bisulfite treatment or antibody-based characterisation of microbial assays19,20. communities than was previously Accordingly, nanopore sequencing possible on a single platform. technology facilitates more complete characterisation of microbial communities Expanding upon this, the role of DNA than was previously possible on a single and RNA modifications (e.g. methylation) platform, dramatically reducing the time in microbial communities is also of and cost associated with such studies. increasing interest16; however, such modifications are cumbersome to detect with traditional technologies due to the need for bisulfite treatment or antibody pull-down sample pre-processing.
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Kits and analysis workflows
Oxford Nanopore offers easy-to-follow Metagenomic analysis workflows for both metagenomic and 16S-based sequencing approaches Oxford Nanopore provides a range of 3 used in microbiome research. DNA library preparation kits, including low-input and barcoding options to suit 16S analysis all experimental requirements. Library preparation can be undertaken in as Oxford Nanopore has released a 16S little as 10 minutes. Data analysis is library preparation kit with barcoding for achieved using the WIMP bioinformatics cost-effective species identification from workflow, which allows the identification multiple samples (Figure 4). The whole of bacteria, fungi, archaea and viruses in workflow can be performed in just 40 a metagenomic sample in real-time as the minutes following genomic extraction, sequencing progresses (Figure 5). WIMP with less than 20 minutes hands-on uses the Centrifuge software tool22, time21. Additionally, a bioinformatics which is capable of accurately identifying workflow for the analysis of 16S data reads when using databases containing is available, delivering a report that is multiple highly similar reference genomes, updated in real-time as the sequencing such as different strains of a bacterial progresses. The report is based on species. The WIMP workflow also allows the result of comparing basecalled quantification of the microorganisms sequences (via BLAST) against the present in the sample. NCBI 16S bacterial database.
Figure 4 Rapid microorganism identification using 16S sequencing with real-time analysis.
Sample preparation Library preparation Sequencing & basecalling Data analysis
ample esuspension in buffer ttachment of Gene-specific sequencing or multiplexed adapters PC amplification Cell lysis by
0 10,000 20,000 30,000 bead beating taphylococcus acillus isteria Enterococcus (e g mnilyse actobacillus almonella Escherichia higella lebsiella Enterobacter Total 40 minutes ead washing and elution
E PC 30 minutes dapter attachment 5 minutes nalysis real time
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Figure 5 An example WIMP report.
RNA analysis Direct RNA sequencing, allows simultaneous analysis of epigenetic Nanopore sequencing library preparation modifications and nucleotide sequence kits are available for both direct and from full-length RNA transcripts. indirect (cDNA) analysis of RNA. Full-length Researchers have developed a number cDNA analysis can be achieved from just of analysis pipelines that offer read 50 ng input RNA, with library preparation alignment, isoform identification taking less than 1.5 hours. and quantification.
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4 Case studies
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Case study 1 Baby-Associated MicroBiota of the Intestine (BAMBI) — an example from the clinic
Necrotising enterocolitis (NEC) is one Interestingly, the researchers also of the most devastating gastro-intestinal noted that the nanopore metagenomic diseases affecting pre-term and low- sequencing approach allowed more birth-weight babies23. Microbial dysbiosis detailed taxonomic assignment when (imbalance) has been established as compared with 16S rRNA analysis using the most likely underlying cause of the a short-read sequencing platform, disease24. Previous studies have revealed which failed to differentiate members of that pre-term babies suffering from the Enterobacteriaceae family25. NEC had more potentially pathogenic bacteria and less of the beneficial Utilising real-time analysis of a faecal Bifidobacterium bifidum24. sample from a critically ill infant, the team were able to confidently call the presence Dr. Matthew Clark and Dr. Richard of the pathogen Klebsiella pneumoniae Leggett’s teams at the Earlham Institute along with corresponding AMR profiles in Norwich, UK, are utilising metagenomic within 1 hour of sequencing25. nanopore sequencing to profile pre-term infant gut microbiota, identifying not just Nanopore sequencing data was found species but also quantifying them and to be of similar quality to that provided profiling their antimicrobial resistance by short-read sequencing technology but, (AMR) genes25. Their study is being critically, it could be generated in conducted as part of a clinical trial for a fraction of the time, requiring just 5 probiotic treatment with Dr. Lindsay Hall hours as opposed to 39 hours for the at the Quadram Institute of Biosciences, short-read technology. Norwich, UK. In conclusion, the researchers highlighted how nanopore technology could, in the Nanopore sequencing allowed future, be applied to clinical applications: pathogen identification and AMR ‘Our results demonstrate that MinION profiling within 1 hour of sequencing. sequencers offer the ability to progress from clinical samples to a potential Through a time-course experiment, tailored patient antimicrobial treatment 25 the team was able to observe the in just a few hours’ . impact of probiotic and antibiotic treatments on microbiota composition25. As the probiotic was administered, the amount of B. bifidum (identified to the species level) in the baby’s gut increased, with both long- read nanopore sequencing and short-read platforms giving similar profiles.
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Case study 2 The advantage of long reads in 16S-based microbiome studies
Mouse gut microbiome Sequencing data derived from the two platforms were highly concordant 16S-based species identification is (R2=0.8-0.9) at all taxonomic levels apart the most commonly used method from the species level. It was shown that in microbiome studies26. Shin et al.10 nanopore data allowed the identification of examined the composition of mouse more species (i.e. Bifidobacterium animalis gut microbiota using a 16S approach and and Bifidobacterium pseudolongum, either short-read sequencing technology well-known members of the gut or long-read nanopore sequencing. microbiome27), resulting in the construction of a fuller microbiota The common use of the V3-V4 16S representation. region for species identification is not The study illustrates that the common as sensitive as using the full length use of the V3-V4 16S region for species of the 16S gene, as allowed by identification is not as sensitive as using the full length of the 16S gene, as allowed nanopore sequencing. by nanopore sequencing10.
A key difference between the two In conclusion, nanopore sequencing’s approaches was that, for short-read long read advantage was instrumental for experiments, the hypervariable V3-V4 the generation of a more accurate profile 16S region was used for primer design of the mouse gut microbiome compared and amplicon generation (mean read to short-read technology. length – 447 bp), while the full length of the 16S gene was used for the nanopore experiments (mean read length – 1393 bp).
Figure 6 Variability in the 16S rRNA gene sequence. Detected variants between the 16S rRNA gene sequences are represented as vertical lines on the 16S rDNA sequences. The black and red arrows indicate the binding positions of primer sets for the amplification of V3-V4 regions (for short-read sequencing) and nearly full-length regions of 16S rDNA sequences (nanopore sequencing), respectively. Image adapted from Shin et al.10
4 region pseudolongum ariant animalis 4 1 1 14 p
4 region acidifaciens ariant ovatus 4 1 1 14 p
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Case study 3 Using ribosomal operons for species identification
Professor Lee Kerkhof from Rutgers The study concluded that nanopore University employed nanopore seque sequencing of operons allowed better ncing for bacterial microbiome profiling taxonomic resolution than standard 16S using whole rRNA operon sequences sequencing using short-read technology. instead of only 16S12,18. The forward primer The team also reported that their approach was generated in the 16S rRNA gene yielded accurate quantification of OTUs. sequence and the reverse primer was within the 23S rRNA gene, resulting in a In summary, Professor Kerkhof 4.2 kb fragment (Figure 7). commented that: ‘Our analysis demonstrated that the MinION has The team sequenced 6 environmental the ability to provide rRNA operon samples, each comprised of farm sequence data of sufficient quality for soil DNA and bioreactor DNA mixed characterising the microbiota of complex in varying proportions. Twelve hours environmental samples and provided of sequencing generated over 1000 results that are reproducible, quantitative, operational taxonomic units (OTUs)*, with and consistent’28. The team are now good concordance between replicates. extending their research to elucidate The team was able to build consensus the impact of external factors (e.g. toxin operons where each rRNA operon exposure) on the composition of the contained a 16S and a 23S gene. mouse microbiome12.
Figure 7 Amplicon structure for ribosomal operon sequencing (4.2 kb), containing almost full length 16S and 23S sequences. Image adapted from Kerkhof12.
orward pri er ro
1
Anti tRNAs ter Re erse pri er
1
* Organisms are clustered into a single OTU based on similarity of DNA sequences above a pre-determined threshold.
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Case study 4 Portable shotgun metagenomic sequencing of supraglacial microbiota
Glaciers have rich microbiota, which play The team was able to obtain raw an active role in maintaining the glacier’s sequencing data within 36 hours properties and determining melting speed13. of sample collection. The WIMP As such, the study of changing glacial bioinformatics workflow used to resolve microbiomes is important for understanding microbe composition of the sequenced the effects of global warming. Dr. Arwyn metagenomic samples revealed a Edwards from the Aberystwyth University dominant presence of Proteobacteria, (UK) pioneered the establishment of a as expected from previous studies using portable lab setup in Svalbard (between short-read technology. The majority of the Norway and the North Pole) using Oxford remaining microbes identified also followed Nanopore’s MinION to study glacial prior expectations. microbiomes13. Recently, the team have assessed the utility Dr. Edwards’ team set out to study the of 16S nanopore sequencing to understand microbiomes of a cryoconite (windblown the impact of different ecological conditions deposits comprised of rock particles and on microbial communities in the field, with microbes) and a red snow algal bloom initial results showing significant changes in in situ using a metagenomic approach13 microbiome composition29. (Figure 8). All of the equipment necessary for this study was transported in 2 standard Dr. Edwards’ results demonstrate the sized airplane pieces of luggage. utility of nanopore sequencing for accurate microbiome analysis in remote and extreme, resource-limited environments.
Figure 8 Sample collection from a cryoconite (a) and red snow algal bloom (b) in Svalbard. Images courtesy of Dr. Arwyn Edwards, Aberystwyth University, UK.
(a) (b)
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Case study 5 Pink microbiome
Lake Hillier in Western Australia is one identification was problematic. of several sites targeted for microbiome In contrast, a whole-shotgun DNA investigation by the eXtreme Microbiome sequencing approach using nanopore Project (XMP - extrememicrobiome.org). technology resulted in species-level The lake’s water is about 10 times saltier taxonomic classification, with a more than sea water and has a bright-pink complete profile starting to emerge after colour4 (Figure 9). just 2 hours of sequencing. The final results were available after 24 hours of sequencing, The unusual colouration was thought to with successful identification of algae, be due to a type of red algae (Dunaliella archaea, bacteria and viruses. salina), but no formal investigations had been performed until recently. Dr. Ken The XMP found that the red bacterium McGrath’s team from the Australian Salinibacter ruber dominated the Genome Research Facility collected water metagenomics profile of the lake, whereas samples from the top and lower levels of D. salina was present at relatively low levels, the lake, as well as sediment samples, and concluded that the pink colour of the for metagenomic analysis. lake is produced by the bacteria, and not the algae as previously thought. Traditional culture-based analysis managed to resolve 13 different microorganisms, The Lake Hillier microbiome study, as well with a culture-dependent bias towards as the rest of the XMP work, has put bacterial species4. A 16S-based microbial nanopore sequencing at the forefront profile using short-read sequencing of microbiome research in extreme technology built a more complete understudied environments. taxonomic representation, but species-level
Figure 9 Lake Hiller. Image courtesy of Dr. Ken McGrath, Australian Genome Research Facility, Australia.
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Case study 6 Direct sequencing of 16S rRNA
The majority of microbiome studies to date nucleotide substitutions, or by gain or loss have focused on the analysis of DNA of base modifications, making their analysis to elucidate microbial composition increasingly important31. and genome structure; however, advances in sequencing technology and The team developed an enrichment strategy workflows, have allowed researchers to that allowed the analysis of just 5 pg of E. investigate RNA, providing insights into coli 16S rRNA in a background of 4.5 µg gene expression in complex bacterial total human RNA. Furthermore, the team communities30. The analysis of epigenetic reported that analysis could be performed modifications adds a further level of in a potentially clinically actionable timeframe characterisation to microbial communities of just 2 hours from sample to result. In fact, and their environmental responses; the real-time analysis provided by nanopore however, such modifications are erased technology enabled 16S rRNA reads to be using traditional sequencing techniques31. generated within 20 seconds of starting the sequencing run. In a proof-of-principle study, Dr. Andrew Smith and colleagues at the University of Examination of the data allowed the California, Santa Cruz, utilised nanopore identification of 7-methyl guanosine (m7G) sequencing to directly analyse full-length at known positions in the 16S rRNA (Figure (1.5 kb) 16S ribosomal RNA (rRNA) of 10) and indicated the presence of additional Escherichia coli to characterise both epigenetic modifications, including nucleotide and epigenetic composition in a pseudouridine. 31 single sequencing run . 16S rRNA is a core In summary, the team suggested constituent of the small ribosomal subunit that: ‘This [nanopore] direct RNA which is expressed in all living cells and sequencing technology has promise for plays an essential role in RNA translation. rapid identification of microbes in the Numerous antibiotics target prokaryotic environment and in patient samples’31.* ribosomes which can acquire resistance via
Figure 10 Nanopore sequencing allowed the accurate detection of the epigenetic modification m7G at a position 527 in E. coli 16S rRNA. Image courtesy of Dr. Andrew Smith, University of California, Santa Cruz, USA.
* Nanopore devices are currently for research use only.
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Summary
The advent of sequencing has introduced Furthermore, the facility for direct significant improvements in the field of DNA and RNA sequencing allows the microbiome research, with metagenomic detection of epigenetic modifications 5 and 16S-based experiments achieving alongside sequence data — adding faster and more accurate microbiota further depth to our understanding of profiling than previous culture-based microbiomes and the complex interactions methods4,8; however, a number of between microorganisms and their challenges remain to fully analysing host environment. Importantly, long microbiome composition. nanopore reads allow complete 16S rRNA genomic regions10 or even whole ribosomal operons12 to be resolved, Nanopore sequencing delivers portable leading to a more detailed, species-level and scalable options for microbiome classification10. research. Long reads also provide a more accurate representation of species diversity in Nanopore sequencing is now being metagenomic samples4,11. used to address these challenges, delivering portable and scalable options for microbiome research, with rapid, streamlined workflows, incorporating automated library preparation and real-time data analysis.
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About Oxford Nanopore Technologies
By introducing the MinION — the first portable, real-time, long-read nanopore sequencing machine — Oxford Nanopore Technologies has expanded the capabilities of microbiome researchers. These specifications make the technology suitable for on-site analysis of 6 microbiomes with streamlined workflows and real-time analysis delivering rapid access to results. Nanopore technology is fully scalable, with the GridION™ X5 and PromethION™ devices providing flexible sequencing capacity for high-throughput applications.
For the latest news, products, application information and developments in nanopore sequencing, visit: www.nanoporetech.com
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References
1. Turnbaugh, P.J. et al. An obesity-associated 10. Shin, J. et al. Analysis of the mouse gut gut microbiome with increased capacity for microbiome using full-length 16S rRNA energy harvest. Nature 444, amplicon sequencing. Scientific Reports 6, 1027-1131 (2006). 29681 (2016).
2. Kumar, P. et al. Intestinal interleukin-17 11. Edwards, A., Debbonaire, A.R., Sattler, B., Mur, receptor signalling mediates reciprocal L.A.J. & Hodson, A.J. Extreme metagenomics control of the gut microbiota and autoimmune using nanopore DNA sequencing: a field report inflammation.Immunity 44, 659-671 (2016). from Svalbard, 78 N. bioRxiv 073965 (2016).
3. Deans, E. Microbiome and mental health in the 12. Kerkhof, L.J. Bacterial microbiome profiling modern environment. Journal of Physiological by MinION sequencing of ribosomal operons. Anthropology 36, 1 (2017). (2017). Available at:
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18. Jenjaroenpun, P et al. Complete genomic 27. Turroni, F. et al. Bifidobacterium bifidum and transcriptional landscape analysis using as an example of a specialized human gut third-generation sequencing: a case study of commensal. Frontiers in Microbiology 5, 437 Saccharomyces cerevisiae CEN.PK113-7D (2014). Nucleic Acids Res (2018). doi: 10.1093/nar/ gky014. [Epub ahead of print] 28. Kerkhof, L.J. et al. Profiling bacterial communities by MinION sequencing of 19. McIntyre, A.B.R. et al. Nanopore detection ribosomal operons. Microbiome 5:16 (2017). of bacterial DNA base modifications. bioRxiv 127100 (2017). 29. Edwards, A. MinION microbiome profiling: going from on-the-go to go-to? Presentation 20. Garalde, D.R et al. Highly parallel direct RNA (2017) Available at: https://vimeo. sequencing on an array of nanopores. Nat com/250451356 [Accessed: 24 January 2018] Methods (2018). doi: 10.1038/nmeth.4577. [Epub ahead of print] 30. Bashiardes, S. Zilberman-Schapira, G. & Elinav, E. Use of metatranscriptomics in 21. Oxford Nanopore Technologies. 16S analysis microbiome research. Bioinform Biol Insights using real-time nanopore sequencing [online] 10 (2016). Available at:
23. Deshpande, G., Rao, S., Patole, S. & Bulsara, M. Updated Meta-analysis of probiotics for preventing necrotizing enterocolitis in preterm neonates. Pediatrics 125, 921 (2010).
24. Clark, M. MinION metagenomics: from mock communities to clinical samples. Presentation (2016). Available at:
25. Leggett, R.M. et al. Rapid MinION metagenomic profiling of the preterm infant gut microbiota to aid in pathogen diagnostics. bioRxiv 180406 (2017).
26. Ranjan, R. et al. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing. Biochem Biophys Res Commun. 469(4) (2016).
17 Oxford Nanopore Technologies phone +44 (0)845 034 7900 email [email protected] twitter @nanopore www.nanoporetech.com
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