Microbiology and microbial sequencing
Microorganisms are the most abundant and diverse forms of life on Earth, with estimates ranging from millions to trillions of species; however, only a small percentage have been identified, let alone sequenced. Of the ~400,000 microbial strains for which sequencing data is available, the majority of genomes are incomplete, reflecting the inherent challenges associated with legacy short-read sequencing technologies. Combining the ability to sequence any length of DNA or RNA fragment — from short to ultra-long (4.2 Mb achieved) — with affordable portable and benchtop devices, and real-time results, researchers are using scalable nanopore technology to fully characterise microbial diversity for a wide range of applications.
Matching excellence: ONT’s rise to parity with PacBio in genome reconstruction of non-model bacterium with high GC content
... our results demonstrate that [Oxford Nanopore Technologies] can be used as a cost-effective sequencing strategy, without the need for complementing with other sequencing technologies, for the reconstruction of complete genomes of the highest quality
Soto-Serrano et al. bioRxiv (2024)
Oxford Nanopore sequencing
Legacy short-read technologies
Unrestricted read length (>4 Mb achieved)
- Simplify de novo assembly and correct microbial reference genomes using long reads
- Assemble complete genomes and plasmids from metagenomic samples — resolving closely related species and complex genomic regions
- Enhance taxonomic resolution using full-length reads of informative loci (e.g. entire 16S gene)
- Sequence and quantify full-length transcripts for unambiguous gene expression analysis
Read length typically 50–300 bp
Short reads do not typically span complex genomic regions such as repeat elements (e.g. transposons, gene duplications, and prophage sequences), reducing assembly contiguity and potentially missing important genomic information.
Real-time data streaming
- Immediate access to results, including species identification and antimicrobial resistance (AMR) profiling
- Stop sequencing when sufficient data obtained — wash and reuse flow cell
- Combine with intuitive, real-time EPI2ME data analysis workflows
Fixed run time with bulk data delivery
Increased time-to-result and inability to identify workflow errors until it’s too late, plus additional complexities of handling large volumes of bulk data.
Flexible, portable, and scalable
- Sequence on demand with flexible end-to-end workflows that suit your throughput needs
- Sequence at sample source, even in the most extreme environments, with the portable MinION device
- Scale up with high-throughput, modular GridION and PromethION devices
Limited flexbility
Legacy sequencing technologies are typically expensive and require high sample batching for optimal efficiency, delaying time to result. The benchtop devices are often bulky and need substantial site infrastructure, restricting its usage to well-resourced, centralised locations.
Direct sequencing of native DNA/RNA
- Detect base modifications, such as methylation, as standard — no additional sample prep required
- Eliminate amplification- and GC-bias, along with read length limitations, and access genomic regions that are difficult to amplify
Amplification required
Amplification can introduce bias — reducing uniformity of coverage with the potential for coverage gaps — and removes base modifications, necessitating additional sample prep, sequencing runs, and expense.
Streamlined workflows
- Sample prep in as little as 10 minutes, including multiplexing
- Whole genome, metagenomic, targeted (including 16S barcoding), direct RNA, and cDNA sequencing approaches
- Automated and streamlined sequencing
Laborious workflows
Typically, lengthy sample preparation requirements and long sequencing run times, reducing workflow efficiency and increasing turnaround times.
Large insights into microorganisms
This white paper explores how microbiologists are now utilising long reads generated by real-time nanopore sequencing to overcome the challenges associated with legacy short-read sequencing technologies, enabling full characterisation of microbial genomes — shedding new light on microbial evolution, pathogenicity, and AMR. Techniques covered include microbial genome assembly, AMR profiling, complete plasmid assembly, virulence analysis, microbial transcriptomics, and the analysis of modified bases.
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Interested in portable sequencing?
Discover how researchers are using MinION for on-site microbial genomics in a wide range of environments, including entirely off-grid sequencing on Europe’s largest ice cap, the crop fields of Africa, and on board the International Space Station.
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Species-level profiling of environmental microbiota with full-length 16S sequencing
Zhang et al. demonstrated how sequencing of the entire 16S ribosomal RNA gene with long nanopore reads can accurately resolve polymicrobial communities at the species level. In contrast, they found that legacy short-read technologies failed to achieve species-level identification. Read how the team identified more species in environmental samples with nanopore sequencing than with an alternative long-read sequencing platform, highlighting how nanopore sequencing is more likely to detect rare or low-abundance microbes.
Our findings show that the [Oxford Nanopore Technologies] R10.4.1 flowcell for full-length 16S enabled species-level taxonomic identification for environmental samples
Zhang et al. Appl. Environ. Microbiol. (2023)
How do I perform whole-genome sequencing of microbial isolates using nanopore technology?
Follow the NO-MISS workflow — the nanopore-only microbial isolate sequencing solution. Start by selecting the right cell lysis and extraction method for your research requirements and microbial isolate. Libraries can then be prepared for nanopore sequencing using the Rapid Barcoding Kit. We recommend multiplexing 4–24 microbial isolate genomes and sequencing on a MinION Flow Cell. Primary data analysis can be performed via our EPI2ME software using the wf-bacterial-genomes workflow.
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Scalable sequencing for microbial analysis
From portable yet powerful Flongle and MinION devices to the flexible, high-throughput benchtop GridION and PromethION platforms — scale your sequencing to match your specific microbial genomics requirements.
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GridION
A compact benchtop device offering powerful integrated compute. Run multiple microbial sequencing and other projects on a single device — from whole genome assembly and targeted sequencing to transcriptomics — using five independent MinION Flow Cells and sample multiplexing.
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With up to 24 or 48 independently addressable, high-capacity flow cells and powerful, integrated compute, PromethION 28 and 48 devices deliver flexible, on-demand access to terabases of sequencing data.
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PromethION 2 devices maintain the flexibility associated with the PromethION 24 and 48 devices, but in a compact, accessible form factor.
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All the benefits of real-time nanopore sequencing in a pocket-sized, USB-powered device.
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Adapting MinION and GridION to run our lowest cost flow cells — ideal for smaller or routine assays.
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