Metagenomics and microbiome analysis with nanopore technology
Long nanopore sequencing reads deliver enhanced genome assemblies, accurate identification of closely related species, and unambiguous analysis of full-length RNA transcripts from mixed microbial samples. Real-time data streaming enables immediate access to results, such as species identification, abundance, and antimicrobial resistance. Combining long reads with targeted approaches enables sequencing of informative genes (e.g. 16S rRNA) in their entirety, improving resolution of identification.
What is metagenomic sequencing?
The genomic analysis of multiple organisms obtained from a single sample, commonly referred to as ‘metagenomics’, allows insight into the genetic composition of microbial communities. Traditionally, microorganisms have been studied through the culturing of individual species or strains using artificial culture media; however, it has been estimated that less than 2% of bacteria can be cultured in the laboratory.
The advance of DNA sequencing technologies to allow genomic analysis of samples containing many species has made it possible to obtain complete or nearly complete genome sequences from uncultured microorganisms — providing an important means to study their biology, ecology, and evolution. However, a number of challenges remain, particularly for time-critical or remote sampling applications such as outbreak investigation and pathogen surveillance.
Oxford Nanopore sequencing technology provides a number of key benefits for metagenomics research. There is no upper read length with nanopore sequencing — reads of any length can be produced, from short to ultra-long. Long sequencing reads enhance genome assembly, enabling more accurate analysis of known and novel microbes, and precise differentiation of closely-related microbes. Reads exceeding 4 Mb have been generated with nanopore technology, meaning that entire microbial genomes can be obtained in single reads, or with minimal contigs (uninterrupted stretches of overlapping DNA). Long reads also improve the resolution of repeat sequences and structural variants, further enhancing genome assembly and antimicrobial resistance (AMR) gene analysis.
Why nanopore technology for metagenomic sequencing?
Nanopore technology offers a variety of metagenomic approaches — from PCR-free whole-genome sequencing through to amplicon-based 16S sequencing — that can be used in the lab, field, or limited resource settings.
Using nanopore metagenomic sequencing, you can:
- Resolve complete genomes and plasmids using long reads
- Identify species, AMR, and virulence factors in real time
- Sequence at sample source using portable MinION and Flongle devices
- Streamline your workflow with 10-minute library prep
- Detect base modifications and link plasmids to hosts using epigenetic motifs
- Use intuitive EPI2ME data analysis workflows for real-time species ID and AMR profiling
Get started with metagenomic sequencing
Get best practice recommendations to optimise your end-to-end metagenomic sequencing workflow in our Getting started guide.
Experimental approach to metagenomic sequencing
A wide range of library preparation kits are available to suit metagenomic sequencing requirements. Amplification-free kits allow direct sequencing of native DNA, eliminating the potential for PCR bias and enabling the detection of base modifications (e.g. methylation) alongside the nucleotide sequence. Amplification-based kits are also available, for example to enable sequencing of the entire 16S gene or custom regions of interest.
|Amplification-free, native DNA sequencing and retained base modification
|Ligation Sequencing Kit
|Rapid Sequencing Kit
|16S Barcoding Kit 24
|Rapid PCR Barcoding Kit 24
|25 min + PCR
|15 min + PCR
|1,000 ng gDNA; 100–200 fmol amplicons or cDNA
|50–100 ng gDNA
|10 ng gDNA
|1–5 ng gDNA
|Equal to fragment length
|Equal to fragment length
|Full-length 16S gene (~1.5 kb)
|Distribution centred around 2 kb
|24 plex, 96 plex
|24 plex, 96 plex
|Optimised for output; retained base modifications; control over read length
|Simple and rapid; retained base modifications
|Simple and rapid; full-length 16S gene provides higher resolution identification
|Optimised for low DNA input
Which device for metagenomic sequencing?
From portable, yet powerful Flongle and MinION devices through to the flexible GridION device and high-output PromethION devices — scale your sequencing to match your specific metagenomic sequencing requirements.
PromethION 2 Integrated & PromethION 2 Solo
Offering sequencing on up to two independent PromethION Flow Cells for cost-efficient access to high-output sequencing — ideal for obtaining complete circular genomes from complex metagenomics samples.
Combining up to 48 independently addressable, high-capacity flow cells with powerful, integrated compute, PromethION 48 delivers flexible, on-demand access to terabases of sequencing data.
Flexible, large-scale sequencing using up to 24 independent, high-capacity flow cells.
PromethION 2 devices maintain the flexibility associated with the PromethION 24 and PromethION 48 devices, but in a compact, accessible form factor.
A compact benchtop device offering powerful integrated compute. Run multiple projects on a single device using five independent MinION Flow Cells and sample multiplexing.
All the benefits of real-time nanopore sequencing in a pocket-sized, USB-powered device.
Adapting MinION and GridION to run our lowest cost flow cells — ideal for smaller or routine assays.
Analysis techniques for metagenomic sequencing
A range of pipelines and tools are available for the analysis of nanopore metagenomic sequencing data, including workflows that enable bacterial genome assembly, whole-genome and 16S-based taxonomic classification, and AMR profiling. For cloud-based or local analysis, our EPI2ME solutions offer simple, point-and-click workflows via a user-friendly interface for routine metagenomic sample analysis.
Find out more about analysing nanopore metagenomic sequencing data
Featured metagenomic sequencing workflow
For assembly and analysis of microbial genomes from complex metagenomic samples, we recommend the following:
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