Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding

Abstract

Rapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, 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 C q values between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 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.

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

“…Other groups have proposed alternate primer schemes using longer amplicons to improve genome coverage [25,26] . Our primer scheme uses 400 bp amplicons which are compatible with both long and short-read sequencing platforms and offer good performance with degraded or high Ct samples.…”

Genomic Evidence of In-Flight Transmission of SARS-CoV-2 Despite Predeparture Testing

“…Independent viral extracts were prepared by the Institute of Environmental Science and Research (Porirua, New Zealand) from the 7 positive respiratory tract samples in which SARS-CoV-2 was initially detected by rRT-PCR. We extracted RNA from SARS-CoV-2–positive samples and subjected it to whole-genome sequencing by following the 1,200-bp amplicon protocol ( 6 ) and Oxford Nanopore Rapid barcoding R9.0 sequencing ( 7 ). Genomic data are available on GISAID ( 5 ) ( Table 1 ).…”

The power and limitations of genomics to track COVID-19 outbreaks: a case study from New Zealand

“…In short, viral extracts were prepared from respiratory tract samples where SARS-CoV-2 was detected by RT-PCR using WHO recommended primers and probes targeting the E and N gene. Extracted RNA from SARS-CoV-2 positive samples were subject to whole genome sequencing following the ARTIC network protocol (V3) (https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye) and the Massey University 1200 bp primer set (https://www.protocols.io/view/ncov-2019-sequencing-protocol-rapid-barcoding-1200-bh7hj9j6) 21 .…”