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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by a grant by VA SeqCURE which in turn received funding from the American Rescue Plan Act funds (Grant# N/A) with additional support from Central Texas Veterans Healthcare System (Temple, TX). The views expressed in this article are those of the authors and do not necessarily represent the views of the Department of Veteran Affairs or the funding agency. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of Central Texas Veterans Healthcare System waived ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.