Performance of COVIDSeq and Swift Normalase Amplicon SARS-CoV-2 Panels for SARS-CoV-2 Genome Sequencing: Practical Guide and Combining FASTQ Strategy

The whole genomic sequencing (WGS) of SARS-CoV-2 has been performed extensively and is playing a crucial role in fighting against COVID-19 pandemic. Obtaining sufficient WGS data from clinical samples is often challenging especially from the samples with low viral load. We evaluated two SARS-CoV-2 sequencing protocols for their efficiency/accuracy and limitations. Sequence coverage of >95% was obtained by Swift normalase amplicon SARS-CoV-2 panels (SNAP) protocol for all the samples with Ct ≤ 35 and by COVIDSeq protocol for 97% of samples with Ct ≤ 30. Sample RNA quantitation obtained using digital PCR provided more precise cutoff values. The quantitative digital PCR cutoff values for obtaining 95% coverage are 10.5 copies/μL for SNAP protocol and 147 copies/μL for COVIDSeq protocol. Combining FASTQ files obtained from 2 protocols improved the outcome of sequence analysis by compensating for missing amplicon regions. This process resulted in an increase of sequencing coverage and lineage call precision.