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 a 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

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 Amplification-based
Ligation Sequencing Kit Rapid Sequencing Kit 16S Barcoding Kit 1-24 Rapid PCR Barcoding Kit
Preparation time 60 min 10 min 10 min + PCR 15 min + PCR
Input requirement 1,000 ng gDNA; 100–200 fmol amplicons or cDNA 100 ng gDNA <10 ng gDNA 1–5 ng gDNA
Fragmentation Optional Not required Not required Not required
Read length Equal to fragment length Equal to fragment length Full length 16S gene (~1.5 kb) Distribution centred around 2 kb
Typical throughput 3/3 2/3 2/3 2/3
Multiplexing options 24 plex, 96 plex 24 plex, 96 plex 24 plex 12 plex
Methylation included Yes Yes - -
Overview 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 Simple and rapid; optimised for low DNA input
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Get started with metagenomic sequencing

Get best practice recommendations to optimise your end-to-end metagenomic sequencing workflow in our Getting started guide.

Sequencing devices

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.

  • Theoretical maximum output. Assumes system is run for 72 hours (or 16 hours for Flongle) at 420 bases / second. Actual output varies according to library type, run conditions, etc. TMO noted might not be available for all applications or all chemistries.
Recommended for metagenomic sequencing

PromethION 2 & 2 Solo

Offering two independent PromethION Flow Cells for cost-efficient access to high-output sequencing — ideal for obtaining complete circular genomes from complex metagenomics samples.

Analysis techniques for metagenomic sequencing

Analysis solutions

A range of pipelines and tools are available for the analysis of nanopore metagenomic sequencing data, including workflows for 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 metagenomics sequencing workflow

For assembly and analysis of microbial genomes from complex metagenomic samples, we recommend the following:

Inspiration for metagenomic sequencing

Discover more about applying metagenomic sequencing to your organism and genomic variants of interest.

Research areas


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