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

Plitnick, Jonathan; Griesemer, Sara; Lasek-Nesselquist, Erica; Singh, Navjot; Lamson, Daryl; St George, Kirsten

Background: Fast and effective methods are needed for sequencing the SARS-CoV-2 genome to track genetic mutations and identify new and emerging variants during the ongoing pandemic. Objective: Assess the performance of the SARS-CoV-2 AmpliSeq Research Panel and S5 plug-in analysis tools for whole genome sequence analysis of SARS-CoV-2 and compare the results to those obtained with the MiSeq based ARTIC analysis pipeline, using metrics such as depth, coverage, and concordance of single nucleotide variant (SNV) calls. Methods: 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 (Ct 15.58 - 32.54) were also sequenced using an Illumina MiSeq based method with the ARTIC analysis pipeline for a direct comparison. Results: 176 of the 191 clinical specimens sequenced on the S5XL and prepared using the SARS-CoV-2 Research Panel, had near complete coverage (>98%) of the viral genome, with an average depth of 5031x. Similar coverage (>98%) levels were observed for 81/83 primary specimens sequenced with both methods tested. The sample with the lowest viral load (Ct 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 in 81/82 samples, in areas of equal coverage, with a single difference present in one sample. Conclusions: The AmpliSeq approach is as effective as the Illumina based method using ARTIC V3 amplification for sequencing SARS-CoV-2 direct from patient specimens across a range of viral loads (Ct 15.56-32.54, median = 22.18). The AmpliSeq workflow is very easily automated with the Ion Chef and S5 instruments and requires less training and experience with NGS preparation than the Illumina workflow.