Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing

Abstract

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer’s instructions.

Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts

“…The latter contains 237 primer pairs targeting SARS-CoV-2 and 5 primer pairs for cellular targets; however, only two of the forward primers are located within the leader sequence and are likely to amplify subgenomic RNAs [9] . Furthermore, the number of cycles must be carefully calibrated because biases due to differential amplification efficiency of primer pairs can skew amplification [21] . This presents a technical challenge for samples with low viral loads that require >20 amplification cycles [9] , [22] .…”

Multiplex Sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

“…The Ion Torrent sequencing platform, one of the popular platforms that was extensively used for viral genome sequencing [19][20][21], however, was not widely used in SARS-CoV-2 sequencing. Several Ion-Torrent based SARS-CoV-2 sequencing work ows were reported [12,22,23] but not very popular among the researchers. Unlike the ARTIC protocol, the published protocol for some of these in-house Ion-Torrent based assay was not detailed enough for it to be replicated in other laboratory settings [12,23].…”

Report of SARS-CoV-2 B1.1.7 Lineage in Morocco

“…The annotation was carried out using COVID19AnnotateSnpEff v 1.3.0.2, a plugin specifically developed for SARS-CoV-2. Default parameters were used for all software ( 7 ).…”