Rapid, High-Throughput, Cost Effective Whole Genome Sequencing of SARS-CoV-2 Using a Condensed One Hour Library Preparation of the Illumina DNA Prep Kit

The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely genomic sequencing of SARS-CoV-2 viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. Additionally, the enormous demand of genomic sequencing on supply chains and the resulting shortages of laboratory supplies necessitate the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method where the same protocol can be used in a STAT scenario, from sample to sequencer in as little as eight hours, and a high-throughput scenario, where one technologist can perform 576 library preparations over the course of one 8-hour shift. This new method uses Freed et al.s 1200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014) and a modified and truncated Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original, application of this new method in hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results suggest that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 whole genome sequencing protocol.

Mutations in emerging variant of concern lineages disrupt genomic sequencing of SARS-CoV-2 clinical specimens

Mutations in emerging SARS-CoV-2 lineages can interfere with the laboratory methods used to generate high-quality genome sequences for COVID-19 surveillance. Here, we identify 46 mutations in current variant of concern lineages affecting the widely used laboratory protocols for SARS-CoV-2 genomic sequencing by Freed et al. and the ARTIC network. We provide laboratory data showing how three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada, and we also demonstrate how we modified the Freed et al. protocol to restore performance.

Optimizing SARS-CoV-2 Variant of Concern Screening: Experience from British Columbia, Canada, Early 2021

Comprehensive and timely testing is required for SARS-CoV-2 variant of concern (VoC) screening. Whole genome sequencing (WGS) provides the broadest means to detect circulating VoCs, but requires longer turnaround time than targeted molecular testing by quantitative polymerase chain reaction (qPCR). We demonstrated the feasibility of a combined testing approach for VoC prevalence assessment in British Columbia, and showed high concordance between qPCR testing and WGS. This directly informed wider VoC screening strategy implementation, and public health efforts.