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Using sequencing to investigate SARS-CoV-2

Article detailing sequencing approaches for SARS-CoV-2

Sequencing of SARS-CoV-2 allowed for the rapid identification of the virus and the development of diagnostic tests and other tools for a rapid response to the pandemic.

The sequences were determined using a range of experimental approaches based on metagenomics, sequence capture or enrichment, amplicon pools by deploying short (e.g., Illumina) or long-read (e.g., Pacific Biosciences, Oxford Nanopore Technologies) sequencing platforms. Many sequencing platforms are currently used to sequence the SARS-CoV-2 virus, including Sanger, Illumina, ION torrent, and Oxford Nanopore Technology, which have their advantages and disadvantages. Short-read sequencing technologies (e.g., Illumina) enable accurate sequence determination. However, long-read sequencing devices from companies such as Oxford Nanopore Technologies (ONT) offer an alternative with several advantages. ONT small throughput devices are portable, cheap, require minimal supporting laboratory infrastructure or technical expertise for sample preparation, and can be used to perform rapid sequencing analysis with flexible scalability. However, ramping up to higher throughput using ONT using PromethIon for example, removes the portability advantages and requires similar laboratory infrastructure to generate the required libraries.

A major challenge with whole-genome sequencing (WGS) is obtaining whole viral genomes from clinical samples promptly. Illumina SARS-CoV-2 sequencing is generally limited by long sequencing times and the high cost and labour associated with library preparation for high-throughput sequencing. Another limitation is their relatively short reads (2 × 300 bp), as genomes generally contain multiple repeated sequences, known as tandem repeats, that may be longer than the NGS reads and may result in gaps and misassemblies. Owing to the large footprint of most sequencers portability can be a challenge, which is unfortunate as there is generally a large distance between sample collection sites and sequencing laboratories.

Nanopore sequencing overcomes these challenges as they sequence in real-time and are long-read sequencing technologies that allow for portability and have a relatively low initial investment on sequencing equipment with the MinION costing $1000. ONT sequencing has, however, traditionally been limited by the high number of single pass false negatives and low sensitivity.

Short-read sequencing technologies are useful for population-level genetic analysis and clinical variant discovery as they provide low-cost, high-accuracy data when done in large batches. Long-read sequencing approaches, however, are well suited for de novo genome assembly, sequencing of genomes with long repetitive regions, copy number alterations, and complex structural variations. However, overwhelmingly during this pandemic, whichever platform has been used, short-read sequencing via an amplicon-based approach has been used to generate viral genome sequences. All ARTIC amplicons are under 500 bases and both ONT and Illumina have kits to deal with this. Longer read options such as the midnight protocol have recently emerged, though, as with all approaches, they are limited by RNA quality, which has been a particular issue due to the variety of lysis buffers deployed globally for diagnostic tests.

Several studies have compared the sequencing of SARS-CoV-2 between Illumina and ONT platforms and have shown that despite the higher single-pass error rates observed with ONT sequencing, highly-accurate SARS-CoV-2 consensus genomes can be achieved. ONT sequencing, however, failed to detect short indels identified by Illumina sequencing. There has also been a lower raw-read accuracy with nanopore sequencing when compared to Illumina sequencing.

A comparison of SARS-CoV-2 WGS genomic coverage and variant detection between Illumina and Nanopore sequencing is necessary as it allows us to determine whether SARS-CoV-2 genomes produced by Nanopore sequencing can be reliably used for genomic surveillance and the development of diagnostic measures.


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Figure 1 – PHRED base call quality score distribution of samples sequenced by Illumina and ONT technologies. Source: PLoS One

Table 1 – Comparison of Illumina and Nanopore-based sequencing approaches for SARS-CoV-2 sequencing

Testing Needs Illumina – Amplicon sequencing – Illumina iSeq100 Nanopore – Arctic and Midnight protocol
Instrument specification Miseq, iSeq, Hiseq; 1536 to 3072 results can be processed on the NovaSeq 6000 system in 12 h using; two SP or S4 reagent kits or 384 results in; 12 h using the NextSeq 2000 or the NextSeq 500/550/550Dx (in RUO mode); HO reagent kit MinION, PromethION, GridION
Average read length 150bp – 400bp Variable up to 900 Kb
Base Error rate 0.2% 8.3%
Per-base error frequency profiles Weaker correlation between replicates (substitution R2 = 0.15; indel R2 = 0.42) – indicating that short-read sequencing errors were less systematic than for ONT libraries Clear correlation between ONT replicates (substitution R2 = 0.67; indel R2 = 0.82) – indicates that ONT sequencing errors are not entirely random but are influenced by local sequence context
Consensus-level sequencing accuracy High across SARS-CoV-2 genome High across SARS-CoV-2 genome
Median read depth 4833X 436X
Genome coverage at reads depth 100X 96.7% 94.9%
Limit of detection <500 copies/mL 10 copies/reaction
Turnaround time ~ 24hr ~ 9h


A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2

Rapid Genomic Characterization of SARS-CoV-2 by Direct Amplicon-Based Sequencing Through Comparison of MinION and Illumina iSeq100TM System

Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols

Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR

Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis

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A Practical Guide for SARS-CoV-2 Whole Genome Sequencing

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