COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance

Abstract

While mass-scale vaccination campaigns are ongoing worldwide, genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to monitor the emergence and global spread of viral variants of concern (VOC). Here, we present a streamlined workflow—COVseq—which can be used to generate highly multiplexed sequencing libraries compatible with Illumina platforms from hundreds of SARS-CoV-2 samples in parallel, in a rapid and cost-effective manner. We benchmark COVseq against a standard library preparation method (NEBNext) on 29 SARS-CoV-2 positive samples, reaching 95.4% of concordance between single-nucleotide variants detected by both methods. Application of COVseq to 245 additional SARS-CoV-2 positive samples demonstrates the ability of the method to reliably detect emergent VOC as well as its compatibility with downstream phylogenetic analyses. A cost analysis shows that COVseq could be used to sequence thousands of samples at less than 15 USD per sample, including library preparation and sequencing costs. We conclude that COVseq is a versatile and scalable method that is immediately applicable for SARS-CoV-2 genomic surveillance and easily adaptable to other pathogens such as influenza viruses.

Development and validation of a high throughput SARS-CoV-2 whole genome sequencing workflow in a clinical laboratory

“…To date, several groups have developed different protocols and workflows for SARS-CoV-2 WGS by NGS. However, most of the published studies are small in study sample size, low in throughput or may require complex enzymatic steps 11,12,[25][26][27][28][29] . In one study, the commercially available COVIDSeq test (Illumina) was scaled up to sequence 752 clinical samples in duplicate for a total of 1536 samples and controls on a NovaSeq 6000 instrument using an S4 flow cell 30 .…”

“…org/).The initial SARS-CoV-2 genome sequence was obtained through a metagenomic approach and confirmed by Sanger sequencing and PCR 2,9,10 . As the pandemic progressed, multiple NGS approaches have been utilized for SARS-CoV-2 sequencing, including shotgun metagenomics, hybrid capture enrichment, and amplicon-based sequencing [11][12][13][14] . Shotgun sequencing requires no prior knowledge of the targeted viral genome 15 but is limited by requirements for a high viral load and a higher sequencing depth.…”

VPipe: an Automated Bioinformatics Platform for Assembly and Management of Viral Next-Generation Sequencing Data

“…The 25× coverage cutoff for VPipe reference recruitment is intermediate between less stringent 10× ( 38 ) and more stringent 75× ( 39 ) cutoffs employed in other SARS-CoV-2 analysis pipelines. Since accurate identification of SNPs is important for classifying SARS-CoV-2 variants and accurate phylogenies ( 38 – 40 ), the >98% SNP concordance between VPipe (25× filter consensus) and Genome Detective supports the results generated by the VPipe reference recruitment module.…”

Fast SARS-CoV-2 Variant Detection Using Snapback Primer High-Resolution Melting

“…Viral whole-genome sequencing has been the primary mechanism to identify variant spread and novel mutations and lineages. While viral whole-genome sequencing is a comprehensive approach for variant detection, it can be both cost-and time-prohibitive for institutions outside of genome centers and reference laboratories [13]. To address this issue, several groups have attempted to develop PCR-(polymerase chain reaction) based methods for clade or variant determination using either allele-specific probes, small amplicon high-resolution melting, fluorescence resonance energy transfer polymerase chain reaction, or CRISPR [14][15][16][17][18].…”