Abstract
Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.
Highlights
The earliest known outbreak of COVID-19 occurred in Wuhan, China in late 2019
RNA isolation and RT-qPCR: Five de-identified samples that had been assessed as positive by other methods were obtained from the New Zealand Auckland District Health Board. 80 μl of Viral Transport Media that had previously stored a nasopharyngeal swab from a patient infected with SARS-CoV-2 were used for RNA isolation using the QIAamp Viral RNA Mini spin kit (Qiagen, Cat No./ID: 52904) according to manufacturer specifications, with the following modifications: sample volume was brought up from 80 μl to 140 μl using 1x PBS, and RNA was eluted using two elutions with 40 μl Buffer AVE for a final volume of approximately 80 μl
Our first goal was to select an amplicon set resulting in even coverage across the SARS-CoV-2 genome, to ensure that we could obtain high-quality, complete genomes with minimal sequencing depth
Summary
The earliest known outbreak of COVID-19 occurred in Wuhan, China in late 2019. By May of 2020, the disease had spread to more than 200 countries and territories, with more than five million total confirmed cases (Johns Hopkins Coronavirus Resource Center). Several approaches have been used to sequence the SARS-CoV-2 genome, including metagenomics (Manning et al 2020), sequence capture (Gohl et al 2020), SISPA (Moore et al 2020), and multiplex PCR (Gohl et al 2020; Itokawa et al 2020; Resende et al 2020; Moore et al 2020; Eden et al 2020), followed by generation sequencing using either the Illumina or Oxford Nanopore platforms. Due to its simplicity and economy, using multiplexed PCR amplicons is perhaps the most common approach. This technique has been used to successfully sequence thousands of genomes over the first few months of the COVID-19 outbreak (GISAID Initiative). The most common methods of library preparation for next-generation sequencing remain relatively expensive, even when samples are multiplexed
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