key: cord-0943038-6c187bck
authors: Choi, Hosoon; Hwang, Munok; Navarathna, Dhammika H.; Xu, Jing; Lukey, Janell; Jinadatha, Chetan
title: Performance of COVIDSeq and Swift Normalase Amplicon SARS-CoV-2 Panels for SARS-CoV-2 Genome Sequencing: Practical Guide and Combining FASTQ Strategy
date: 2022-03-24
journal: J Clin Microbiol
DOI: 10.1128/jcm.00025-22
sha: 365737b6cc0cd775a18af819ce85b42d6277a587
doc_id: 943038
cord_uid: 6c187bck

nan

sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been an irreplaceable tool for epidemiological evaluation, and genomic sequence analysis has provided essential information in the development of antiviral therapeutics and vaccines (1, 2) . Good sequencing coverage needs to be obtained to get information: the default threshold for lineage call at Pangolin is at least 50% of non-N bases (3) . Obtaining sufficient WGS coverage from low-viral-load samples is challenging and often not subject to WGS in many labs. However, WGS of low-viral-load patient samples will enable a more complete picture of viral transmission and viral evolution (4) .

Here, we report the evaluation of two SARS-CoV-2 amplicon-based library prep kits, COVIDSeq with ARTIC v3 primers (Illumina) and Swift Normalase amplicon SARS-CoV-2 panels (SNAP) (Swift Biosciences) (5-7) with digital PCR and WGS using a total of 121 COVID-19-positive samples with threshold cycle (C T ) values between 11 and 45 (BD Max SARS-CoV-2 reverse transcriptase quantitative PCR [RT-qPCR] assay; please refer to supplementary material for library preparation and sequencing). About one-third (42) of samples were low viral load (C T . 30). Out of 121 samples, .95% genome coverage was obtained for 107 samples by SNAP and 89 samples by COVIDSeq. This sequence yield exceeds many previous SARS-CoV-2 sequencing reports, especially for low-viral-load samples.

Through testing of various viral load samples along with digital PCR, we were able to obtain a tentative cutoff value of sample viral load above which one can expect to acquire an informative sequencing outcome. Sequence coverage of .95% was obtained by using SNAP for all of the samples with C T # 35 and by COVIDSeq for 97% of samples with C T # 30. Sample RNA quantitation obtained using digital PCR provided more precise cutoff values. The quantitative digital PCR (TaqPath COVID combo kit [Thermo Fisher] analysis with QIAcuity [Qiagen]) cutoff values for obtaining .95% coverage are 10.5 copies/mL for SNAP and 147 copies/mL for COVIDSeq (Fig. 1) . The median sequence read depth was 2,913Â and 3,578Â with SNAP and COVIDSeq, respectively. All samples .750Â coverage from SNAP achieved .95% sequence coverage. Read depth of .2,800Â is sufficient to have .95% sequence coverage by COVIDSeq.

In addition, combining FASTQ files obtained from two kits improved the sequencing coverage and read depth significantly (please refer to supplementary material for combining FASTQ strategy). Combining FASTQ files was performed for 52 samples in which coverage is ,95% or samples with lineage call discrepancy by either kit. By combining FASTQ files, .95% was obtained from all 21 samples with C T # 30 and from 22 out of 31 samples with

The opinions expressed here are those of the authors and do not represent the views of Department of Veterans Affairs or of Central Texas Veterans Health Care System, Temple, TX.

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FIG 2 Increases of percent coverage by combined FASTQ. (A) Comparison of percent coverage between SNAP protocol and combined FASTQ of low-C T samples. (B) Comparison of percent coverage between SNAP protocol and combined FASTQ of high-C T samples. (C) Comparison of percent coverage between COVIDSeq protocol and combined FASTQ of low-C T samples. (D) Comparison of percent coverage between COVIDSeq protocol and combined FASTQ of high C T samples. (E) Summary of sequencing coverage of SNAP protocol