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. 2021 Nov 18;59(12):e0064921.
doi: 10.1128/JCM.00649-21. Epub 2021 Sep 22.

Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol

Jonathan Plitnick  1 , Sara Griesemer  1 , Erica Lasek-Nesselquist  1   2 , Navjot Singh  1 , Daryl M Lamson  1 , Kirsten St George  1   2
Affiliations

Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol

Jonathan Plitnick et al. J Clin Microbiol. .

Abstract

Fast and effective methods are needed for sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome to track genetic mutations and to identify new and emerging variants during the ongoing pandemic. The objectives were to assess the performance of the SARS-CoV-2 AmpliSeq research panel and S5 plug-in analysis tools for whole-genome sequencing analysis of SARS-CoV-2 and to compare the results with those obtained with the MiSeq-based ARTIC analysis pipeline, using metrics such as depth, coverage, and concordance of single-nucleotide variant (SNV) calls. A total of 191 clinical specimens and a single cultured isolate were extracted and sequenced with AmpliSeq technology and analysis tools. Of the 191 clinical specimens, 83 (with threshold cycle [CT] values of 15.58 to 32.54) were also sequenced using an Illumina MiSeq-based method with the ARTIC analysis pipeline, for direct comparison. A total of 176 of the 191 clinical specimens sequenced on the S5XL system and prepared using the SARS-CoV-2 research panel had nearly complete coverage (>98%) of the viral genome, with an average depth of 5,031×. Similar coverage levels (>98%) were observed for 81/83 primary specimens that were sequenced with both methods tested. The sample with the lowest viral load (CT value of 32.54) achieved 89% coverage using the MiSeq method and failed to sequence with the AmpliSeq method. Consensus sequences produced by each method were identical for 81/82 samples in areas of equal coverage, with a single difference present in one sample. The AmpliSeq approach is as effective as the Illumina-based method using ARTIC v3 amplification for sequencing SARS-CoV-2 directly from patient specimens across a range of viral loads (CT values of 15.56 to 32.54 [median, 22.18]). The AmpliSeq workflow is very easily automated with the Ion Chef and S5 instruments and requires less training and experience with next-generation sequencing sample preparation than the Illumina workflow.

Keywords: ARTIC; AmpliSeq; Ion Torrent; MiSeq; SARS-CoV-2; whole-genome sequencing.

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Figures

FIG 1
FIG 1
(A) Genome coverage of the 181 primary samples that returned sequences using the AmpliSeq method, compared to the CT value obtained with the CDC 2019-nCoV real-time RT-PCR diagnostic panel as an indication of viral load. The percentage of bases with at least 20× coverage was reported by the CoverageAnalysis plug-in. (B) Genome coverage of the 83 samples that were processed with both the AmpliSeq Ion Torrent and Illumina MiSeq ARTIC methods.
FIG 2
FIG 2
Workflow for each of the methods described and the associated preparation and incubation times for each step. The ARTIC Illumina workflow is for 94 samples and 2 negative-control samples, and the AmpliSeq workflow is for 64 samples using a single Ion Chef instrument. QC, quality control.
See this image and copyright information in PMC

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