Identification
The rapid, accurate identification of organisms, across all taxonomic domains, is crucial to numerous applications — from clinical research, to environmental conservation, to forensics. However, identification via traditional sequencing technologies can involve turnaround times of days, weeks or more; furthermore, short sequencing reads can limit taxonomic resolution. With nanopore sequencing, identification can be carried out within hours, enabling rapid responses for time-sensitive experiments, in the lab or at the sample source, whilst long sequencing reads allow for unprecedented taxonomic resolution and characterisation.
Identify and characterise organisms with confidence and precision
Gain rapid sample-to answer times with 10-minute library prep and real-time sequencing
Identify organisms at the sample source with portable nanopore sequencing technology
Identifying organisms with short and long sequencing reads
The use of sequencing technology has revolutionised the identification and differentiation of organisms in biology, enabling unprecedented resolution beyond what is possible with morphology alone. The rapid identification of pathogenic bacteria and their antimicrobial resistance (AMR) genes in clinical research isolates or environmental samples can help inform public health responses, whilst identification of plant pathogens is important in agriculture and conservation. In the monitoring of illegal wildlife trade, animal products can be identified where sufficient morphological clues are not available. However, the use of short-read sequencing technology can lead to limitations in taxonomic resolution. Short reads may map to multiple organisms, making strain- or serotype-level identification of bacteria difficult or impossible. Where PCR is required, regions or whole genomes that are difficult to amplify, such as those with repetitive or high GC content, may not be detected in sequencing.
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Figure 1: Long nanopore sequencing reads can span entire viruses or bacterial genomes, enabling their unambiguous identification from metagenomic samples. Here, complete genomes of marine viruses were sequenced in single reads, negating the need for downstream assembly. View the poster to find out more.
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Figure 2: The EPI2ME 'What’s In My Pot?' (WIMP) analysis workflow enables real-time identification of bacterial, archaeal, fungal, and viral species in a sample.
Rapid identification — in the lab or in the field
For many applications, rapid identification of organisms is critical. Using traditional, centralised methods, samples for sequencing are collected and transported to a laboratory with the necessary equipment for preparation and sequencing, in a process that can take days, weeks or more; samples may also degrade during this process, limiting identification of their contents. Where the cost of sequencing devices is prohibitive, sending samples to a sequencing provider can mean waits of up to months for results.
In contrast, Oxford Nanopore offers simple library preparation solutions and portable sequencing. The pocket-sized MinION can be run from a laptop, enabling sequencing in the lab or at the point of sampling. Sequencing reads are delivered in real time, enabling immediate access to results. For microorganisms, Oxford Nanopore offers the EPI2ME 'What’s In My Pot?' (WIMP) analysis workflow, providing real-time, species-level microbial identification (Fig. 2), while the EPI2ME workflow wf-metagenomics enables real-time classification from metagenomic samples. Combined with rapid library prep options, taking as little as ten minutes, organism identification can be performed within hours of collection or less. With the MinION Starter Pack costing $1,999, sequencing can be accessed at low cost.
Improving surveillance of respiratory diseases and characterising their causative pathogens
The rapid genomic analysis of respiratory pathogens is critical for both their timely identification and for genomic surveillance in outbreak scenarios. However, currently used diagnostic methods frequently on culturing and identifying microorganisms via traditional methods such as PCR, limiting identification to culturable microorganism. In outbreak surveillance, the need to send samples to a laboratory for sequencing can lead to long turnaround times. Researchers around the world are now utilising real-time nanopore sequencing to rapidly sequence pathogens, to provide crucial surveillance information during outbreaks, and showing the potential to sensitively detect pathogens faster than currently used techniques.
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‘This analysis showed that all the isolates that were misassembled or untypeable when using short-read assembly methods were typeable with ONT-only assemblies‘
Krøvel et al., Front Cell Infect Microbiol. (2023)
if we're able to take a deep dive into this data, we can actually identify something which would otherwise not have been possible
Kalinka Sand Knudsen, Aalborg University
Identifying methane-eating microorganisms with nanopore sequencing help mitigate greenhouse gas emissions
Atmospheric methane has doubled in concentration over the last century. In her presentation at London Calling 2023, Kalinka Sand Knudsen (Aalborg University, Denmark) described how she and her colleagues are using nanopore sequencing to identify and characterise methanotrophs — microorganisms that are able to metabolise this potent greenhouse gas. Using shallow metagenomic sequencing with long nanopore reads, they were able to identify numerous novel methanotrophs from complex environmental samples, with the goal of building an encyclopaedia of these critical microbes.
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How do I perform identification using nanopore sequencing?
Nanopore sequencing is uniquely scalable. The portable Flongle and MinION are ideal for performing identification experiments at the point of sampling, while the flexible GridION enables sequencing up to five MinION or Flongle Flow Cells to be run on demand. Scaling up, the ultra-high-throughput PromethION platform outputs terabases of data, for rapid sequencing of larger genomes or highly parallel multiplexed experiments.
For applications where turnaround time is crucial, the Rapid Sequencing Kit enables preparation of sequencing libraries in just ten minutes. For targeted sequencing experiments, there are both PCR-based and PCR-free methods available. For identification of transcripts and RNA viruses, Oxford Nanopore provides streamlined RNA and cDNA sequencing kits for sequencing of full-length transcripts.
The EPI2ME 'What’s In My Pot?' workflow enables real-time identification of bacteria, archaea, fungi, and viruses, without the need for prior bioinformatics experience. For data analysis tutorials, and more information on analysis tools from both the Nanopore Community and Oxford Nanopore, visit the Bioinformatics resource in the Nanopore Community.
Find out more about microbial identification with nanopore sequencing in Metagenomic sequencing with Oxford Nanopore: Getting Started.
Identification of bacteria and antimicrobial resistance genes from an environmental sample
Library preparation with the Ligation Sequencing Kit, followed by sequencing on a MinION Flow Cell on the MinION, delivers up to 50 Gb of data*. Basecalling and subsequent analysis can be performed as soon as sequencing starts, with the EPI2ME workflow wf-metagenomics providing both species identification and AMR profiling in real time.
*Theoretical max output when system is run for 72 hours at 420 bases / second. Outputs may vary according to library type, run conditions, etc.
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