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

Abstract

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.

Development and validation of a high throughput SARS-CoV-2 whole genome sequencing workflow in a clinical laboratory

“…To date, several groups have developed different protocols and workflows for SARS-CoV-2 WGS by NGS. However, most of the published studies are small in study sample size, low in throughput or may require complex enzymatic steps 11,12,[25][26][27][28][29] . In one study, the commercially available COVIDSeq test (Illumina) was scaled up to sequence 752 clinical samples in duplicate for a total of 1536 samples and controls on a NovaSeq 6000 instrument using an S4 flow cell 30 .…”

SARS-CoV-2 Whole-Genome Sequencing by Ion S5 Technology—Challenges, Protocol Optimization and Success Rates for Different Strains

“…Consistent with our study are findings by Jacot et al [29] who suggest C T ≥ 30 (78 or less of viral RNA copy number per reaction) can serve as an initial sequencing success predictor. An analysis comparing the Ion AmpliSeq Panel and Illumina-MiSeq ARCTIC Protocol showed both methods are overall equally effective, with the automation of all steps of library preparation within the TFS solution considered a strong advantage [30], which was expressed also by Rachiglio et al [28]. In the beforementioned study [30], one low-quality sample (C T 32,5) achieved 89% coverage with MiSeq-based ARCTIC pipeline, while it failed with the SARS-CoV-2 AmpliSeq Research panel, suggesting the TFS solution may be inferior to the MiSeq-based one in cases of low-template material.…”

Household transmission of SARS-CoV-2 from humans to pets in Washington and Idaho: burden and risk factors

“…Respiratory or fecal swabs: RNA extraction and SARS-Cov-2 reverse transcriptase (RT) real-time PCR was performed as described [18]. Following initial viral detection by PCR, three dog samples and one cat sample were submitted to University of Minnesota Genomics Center (Oakdale, MN 55128) for whole genome sequencing (WGS) [19]. A second cat sample was submitted to the USDA National Veterinary Services Laboratories (NVSL) in Ames Iowa for WGS.…”