Microbiome sequencing
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 modern 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.
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
Oxford Nanopore sequencing
Legacy short-read technologies
Unrestricted read length (>4 Mb achieved)
- Generate complete, reference-quality metagenome-assembled genomes (MAGs) — resolving closely related species and complex genomic regions
- Sequence the full-length 16S ribosomal RNA (rRNA) gene for enhanced taxonomic resolution
- Resolve mobile genetic elements — including plasmids and transposons — to reveal information about antimicrobial resistance and virulence factors
Read length typically 50–300 bp
Short sequencing reads do not typically span complex genomic regions (e.g. repeats, transposons) resulting in fragmented, partial genomes and ambiguous assembly of closely related species. Targeted 16S rRNA sequencing approaches using short reads have also been shown to provide lower taxonomic resolution when compared with long sequencing reads.
In the context of gene expression, short reads from legacy sequencing technology generate fragmented and incomplete assemblies, making metatranscriptomic studies highly challenging.
Real-time data streaming
- Identify microorganisms immediately during the sequencing run
- Stop sequencing when sufficient data is obtained — wash and reuse flow cell
- Combine with intuitive, real-time EPI2ME data analysis workflows, including metagenomic- and 16S rRNA-based identification and quantification
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 what you need with flexible end-to-end workflows that suit your throughput or output needs
- Characterise microbiomes at their source, even in the most extreme environments with the portable MinION devices — minimise potential sample degradation caused by storage and shipping
- Scale up and sequence with high-output, modular GridION and PromethION devices
Limited flexibility
Legacy 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
Eliminate amplification- and GC-bias, along with read length limitations and access genomic regions that are difficult to amplify
Detect base modifications, such as methylation, as standard — no additional library prep required
Use methylation motifs to support genome binning and assembly from metagenome samples — maximising the utility of your data
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
Cost-effectively analyse up to 96 samples in a single run using barcoding kits
Group plasmids with their associated genomes using MetaPore-C
Laborious workflows
Typically lengthy sample preparation requirements and long sequencing run times, reducing workflow efficiency and increasing turnaround times.
Addressing the challenges of metagenomics with nanopore sequencing
Understanding the true diversity and interactions of microorganisms in any given environment has historically been restricted by many factors, including the inability to culture the vast majority of microbes on artificial media. Developments in legacy sequencing technologies removed the need for culture, providing more detailed insights into microbiomes; however, many challenges remained, including the assembly of complete genomes, distinguishing closely related species, lengthy turnaround times, and sequencing infrastructure. This white paper reviews how nanopore sequencing is being used by researchers worldwide to meet these challenges, shedding new light on the composition and function of microbiomes — from the human gut to the most remote environments on Earth and beyond.
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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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Nanopore sequencing improves surveillance of serious respiratory diseases and characterises the causative agents
Respiratory infections can be caused by a variety of infectious agents; however, identifying the causative agent is challenging due to limitations using legacy methods. Luo et al. used nanopore sequencing to analyse fluid research samples and identified pathogens in 59 metagenomic samples with rapid turnaround times.
Read the case study to find out how nanopore sequencing was further used to sequence type Legionella pneumophila and to genotype novel avian influenza strains.
[Oxford Nanopore Technologies] is highly scalable, from the smallest consumable unit, namely "Flongle", to the high-throughput platforms.
Krøvel, et al. Front. Cell. Infect. Microbiol. (2023)
Using nanopore sequencing in a food microbiology laboratory
Microbiome sequencing has multiple potential applications in a food microbiology laboratory, including pathogen characterisation and food spoilage investigation. Long nanopore reads can be used for rapid microbial identification, including for samples that cannot be confirmed with traditional cultural methods. At London Calling 2023, Andrzej Benkowski shared how they used efficient nanopore sequencing workflows for bacterial and metagenomic identification in food samples, with rapid turnaround times.
The use of the [Oxford Nanopore Technologies] long read sequencing is a powerful tool that can be utilised in a high throughput [microbiology] lab due to its ease of use, price competitiveness, reliable data and rapid time to result using basic [Oxford Nanopore Technologies] workflow and EPI2ME bioinformatics.
Andrzej Benkowski, Eurofins, USA
Scalable sequencing for microbiome 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 microbiome analysis requirements.
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PromethION 2 Solo and 2 Integrated
Offering two independent PromethION Flow Cells for cost-efficient access to high-output sequencing ─ ideal for obtaining complete circular genomes from complex metagenomics samples.
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Adapting MinION and GridION to run our lowest cost flow cells — ideal single microbiome samples or routine analysis of microbial swabs.
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All the benefits of real-time nanopore sequencing in a pocket-sized, USB-powered device — available as part of a bundle with sequencing reagents.
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A compact benchtop device offering powerful integrated compute. Run multiple microbiome and other sequencing projects on a single device using five independent MinION Flow Cells and sample multiplexing.
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With up to 24 or 48 individual, high-capacity flow cells and powerful, integrated compute, PromethION 24 and 48 delivers flexible access to terabases of sequencing data — ideal for high-throughput labs and highly multiplexed samples.
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