Microbiome sequencing

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This is a great example of where nanopore sequencing really opened up a new window into a biological entity that we really didn’t know existed before we applied this tech

Ed DeLong, University of Hawai’i at Mānoa, USA

  • Icon displaying a graphic of any length nanopore reads
    Generate complete, reference-quality metagenome-assembled genomes (MAGs) with long, accurate nanopore reads
  • Blue icon displaying a MinION Mk1D device
    Sequence at source, even in the most extreme environments with the portable MinION
  • Real-time icon blue
    Identify microorganisms immediately during a sequencing run with real-time data streaming
Intro

Simplify genome assembly

The study of microbiomes — the genetic material of all microorganisms in a given sample — is providing new insights into a diverse range of research areas, such as human health and disease, crop improvement, food safety, and species conservation. Microorganisms and their interactions have a profound effect on their environments, and it is only now, through the advent of nanopore sequencing technologies, that we are able to fully characterise microbiome samples — not only identifying each individual microbe but also generating complete, closed genome assemblies and elucidating gene expression within microbial communities.

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Technology comparison

Oxford Nanopore sequencing

Legacy short-read sequencing

Any read length (20 bp to >4 Mb)

Short read length (<300 bp)

  • Generate complete, high-quality genomes with fewer contigs and simplify de novo assembly
  • Resolve genomic regions inaccessible to short reads, including complex structural variants (SVs) and repeats
  • Analyse long-range haplotypes, accurately phase single nucleotide variants (SNVs) and base modifications, and identify parent-of-origin effects
  • Sequence short DNA fragments, such as amplicons
  • Resolve mobile genetic elements — including plasmids and transposons — to generate critical genomic insights
  • Enhance taxonomic resolution using full-length reads of informative loci, such as the entire 16S gene
  • Assembly contiguity is reduced and complex computational analyses are required to infer results
  • Complex genomic regions such as SVs and repeat elements typically cannot be sequenced in single reads (e.g. transposons, gene duplications, and prophage sequences)
  • Important genetic information is missed

Direct sequencing of native DNA/RNA

Amplification required

  • Eliminate amplification- and GC-bias, along with read length limitations, and access genomic regions that are difficult to amplify
  • Detect epigenetic modifications, such as methylation, as standard — no additional, time-consuming sample prep required
  • Create cost-effective, amplification-free, targeted panels with adaptive sampling to detect SVs, repeats, SNVs, and methylation in a single assay
  • Amplification is often required and can introduce bias
  • Base modifications are removed, necessitating additional sample prep, sequencing runs, and expense
  • Uniformity of coverage is reduced, resulting in assembly gaps

Real-time data streaming

Fixed run time with bulk data delivery

  • Analyse data as it is generated for immediate access to actionable results
  • Stop sequencing when sufficient data is obtained — wash and reuse flow cell
  • Combine real-time data streaming with intuitive, real-time EPI2ME data analysis workflows for deeper insights
  • Time to result is increased
  • Workflow errors cannot be identified until it is too late
  • Additional complexities of handling large volumes of bulk data

Accessible and affordable sequencing

Constrained to centralised labs

  • Sequence on demand with flexible end-to-end workflows that suit your throughput needs
  • Sequence at sample source, even in the most extreme or remote environments, with the portable MinION device — minimise potential sample degradation caused by storage and shipping
  • Scale up with modular GridION and PromethION devices — suitable for high-output, high-throughput sequencing to generate ultra-rich data
  • Perform cost-effective targeted analyses with single-use Flongle Flow Cells
  • Sequence as and when needed using low-cost, independently addressable flow cells — no sample batching needed
  • Use sample barcodes to multiplex samples on a single flow cell
  • Bulky, expensive devices that require substantial site infrastructure — use is restricted to well-resourced, centralised locations, limiting global accessibility
  • High sample batching is required for optimal efficiency, delaying time to results

Streamlined, automatable workflows

Laborious workflows

  • Lengthy sample prep is required
  • Long sequencing run times
  • Workflow efficiency is reduced, and time to result is increased