Identification
The timely, sensitive 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, whilst 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.
Introduction
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 it possible with morphology alone. The identification of pathogenic bacteria and their antimicrobial resistance genes in clinical research or environmental samples can help inform public health responses, whilst identifying the cause of a plant disease 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, short-read sequencing 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; furthermore, 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.
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.
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 devices, enabling sequencing in the lab or at the point of sampling. The pocket-sized MinION can be run from a laptop, whilst the new MinION Mk1C is a complete, standalone sequencer, incorporating a screen and onboard compute into one portable device. 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). Combined with library prep kits such as the lyophilised Field Sequencing Kit, with which libraries can be prepared in just ten minutes, organism identification can be performed within hours of collection or less. With the MinION Starter Pack costing $1,000, sequencing can be accessed at low cost.
Case study
Rapid, cost-effective genotyping of human enteroviruses using Flongle
Enteroviruses infect millions of people across the world each year. With over 100 genotypes known to infect humans, associated with disease ranging from mild respiratory symptoms to severe neurological diseases, frequent monitoring of circulating and emerging genotypes is of high importance. Whilst the current gold-standard method of human enterovirus identification is short-read Sanger sequencing, Grädel et al. demonstrated the future potential of Flongle Flow Cells for rapid, low-cost enterovirus genotyping. A nested PCR-based method was used to amplify the VP1 gene from 26 Enterovirus-positive clinical research samples, and barcode these with the PCR Barcoding Expansion Kit 1-96. The barcoded amplicons were then pooled and prepared for sequencing on Flongle using the Ligation Sequencing Kit. Consensus sequences were genotyped via mapping to a custom VP1 enterovirus database. The team found that all 26 samples could be correctly genotyped from a single Flongle Flow Cell run, at a total cost of $10 per sample, with an end-to-end workflow of 22.5 hours. They then demonstrated that each sample could be run in duplicate, enabling 52 samples to be run on a single flow cell, with no reduction in sequencing quality. This reduced genotyping costs to just $7 per sample.
'We conclude that the new Flongle-based assay with its fast turnover time, low cost investment, and low cost per sample represents an accurate, reproducible, and cost-effective platform for enterovirus identification and genotyping.'
Grädel et al.‘The new CZ ID ONT pipeline is successfully able to identify organisms from a range of sample types’
Katrina Kalantar, the Chan Zuckerberg InitiativeCase study
A pipeline for metagenomic pathogen detection and monitoring from nanopore sequencing data
The CZ ID collaboration focuses on the detection, identification, and tracking of infectious diseases and has developed the CZ ID software, an open source, cloud-based metagenomics platform. At the NCM 2022, Katrina Kalantar from the Chan Zuckerberg Initiative explained that the CZ ID software can accept DNA and RNA sequencing data from any sample type or host organism, and has been developed with a metagenomic analysis pipeline that is compatible with Oxford Nanopore Technologies data. The benefits of this platform are simple data upload and cloud-based data processing, data management for samples and projects, single sample reports of microbial abundances and multi-sample analysis and flexible downloads for offline analysis. In her talk, Katrina demonstrated that the new pipeline can accurately identify organisms from a variety of sample types.
Sequencing workflow
How do I perform identification using nanopore sequencing?
Nanopore sequencing is uniquely scalable. The portable Flongle, MinION and MinION Mk1C are ideal for performing identification experiments at point-of-sampling, whilst the flexible GridION enables 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; a fully lyophilised version, the Field Sequencing Kit, is available for sequencing in the field. For targeted sequencing experiments, there are several PCR-based methods available, whilst Cas9 enrichment can be combined with the Ligation Sequencing Kit for PCR-free, long-read targeted sequencing. For identification of transcripts and RNA viruses, Oxford Nanopore provides three streamlined RNA sequencing kits, for long-read RNA sequencing with low GC bias.
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.
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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 ARMA workflow providing both species identification and antimicrobial resistance 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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