Why was Nanopore sequencing used in the SARS-CoV-2 pandemic?
How did Nanopore Sequencing start?
The concept of using a protein nanopore as a method to read DNA was first realised in 1989 by Professor David Deamer (Figure 1). Since his original design, the concept of individual nucleic acids passing through a nanopore which was embedded in a bi-lipid membrane was first put into practice, and patented, in 1998.
Figure 1 – Professor David Deamers notebook from 1989 depicts his first ideas for how to sequence nucleic acids. Source Nature Biotechnology
The commercialisation of nanopore sequencing, however, wouldn’t become available until 2012 with the release of the MinION device. Later variations, such as the PromethION in 2018, were developed and allowed the parallel sequencing of multiple libraries to be visualised for the first time. Equipped across 48 flow cells, the PromethION can process 7 Tb of sequence data in a single run.
How does Nanopore sequencing work?
The flow cell contains a synthetic electro-resistant membrane across a small surface and is embedded with up to 2000 individual holes known as nanopores. Each nanopore is a nanometre across and contains a helicase enzyme to unwind the double-stranded DNA into its single-strand components upon entry. The nanopore (Figures 2 and 3) acts as a biological vessel that transports the DNA from one side of the flow cell to the other. The DNA or RNA that passes through the narrow sensor of the reader protein is read and base called. When working at optimum capabilities, the nanopores can produce raw data reads of up to 400 base pairs a second resulting in large quantities of data being produced.
Figure 2 – Diagram of nanopore sequencing. Diagram created using Biorender.com
Figure 3 – Image showing sensor array of flow cell saturated with nanopores. Disposable flow cell is inserted into MinION sequencing device. Source: Surgical Neurology International
Why was Nanopore sequencing used in the SARS-CoV-2 pandemic?
Conventional sequencing technologies have a high cost of entry and require space in a laboratory. These challenges limit tracking the evolution of viral outbreaks in more remote and low-resource areas. As the MinION is so portable, it was a game-changer for in-field sequencing during viral outbreaks.
The minION was used during the Ebola virus outbreak of 2013-2016 in West Africa, and again during the Zika virus outbreak in Brazil in 2016. The protocols developed during the Ebola and Zika outbreaks were able to be quickly adapted by a genomics research group, the Artic Network, for sequencing of SARS-CoV-2.
Typically the advantage of using Nanopore sequencing is that it is able to sequence long fragments of DNA. This makes it easier to identify overlapping fragments and reduces ambiguity in genome assembly. However, as SARS-CoV-2 undergoes PCR prior to sequencing, the advantage in this instance is the availability of both portable and high throughput devices. The functionality during this pandemic was to sequence small numbers of viruses, less than 50 per minion flow cell within 16-24 hours. This had massive implications in supporting infection control teams, both clinically and regionally. Being able to scale up lower throughput approaches by using a GridION enables rapid response for smaller investigations. Larger epidemiological studies with big batches of samples can then be run more efficiently and cost-effectively on platforms such as NextSeq.
The advancements in portable sequencing devices have opened up new possibilities for sequencing organisms in the furthest corners of our planet. The accuracy of genetic sequencing is improving continuously and the future for Nanopore Technology is promising and exciting for all those who are part of the current genetic sequencing revolution.
“Our goal is to enable the analysis of anything, by anyone, anywhere.”(Oxford Nanopore Technologies)
Let us know in the comments if you ever had the opportunity to use Oxford Nanopore Technology (ONT). How easy or difficult is ONT sequencing for you?
A Practical Guide for SARS-CoV-2 Whole Genome Sequencing
A Practical Guide for SARS-CoV-2 Whole Genome Sequencing
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