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

Abstract

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13751-13965 and 23941-24106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64-Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13730 and 23929, whereas the other isolates possess T at these positions. The genetic variations observed suggest that the naturally occurring genome variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel. The possible impact of other genetic sequence variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel, hence, should be considered.

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

“…In our dataset, however, those amplicons were not failing (mean percentage of reads within all reads of 0.307% and 0.310%, respectively), even though the 13730T>C and 23929T>C variants did not occur in any of our samples. Moreover, the amplicon that failed consistently throughout our samples did not show underperformance in the Tan et al study [42]. The possible explanation for this may be that even though there are no indications that the high underperformance of r1_1.15.1421280 is caused by a specific SNP, the collection time of samples studied by Tan et al pinpoints them to the period when B.6 was the main lineage that prevailed in Malaysia [43].…”

“…Lineage-dependent amplicon failure was one of the aspects studied by Tan et al [42] in a sample set that contained six isolates obtained from swabs collected between April and May of 2020 in Malaysia. The amplicons that highly underperformed in that study (r1.14.786182 and r1.25388943) were failing for all but one sample, which contained some changes in comparison to the reference Wuhan-Hu-1 sequence nearby the amplicons' range.…”

Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2

“…The comprehensive detection facilitates precision medicine and epidemiological surveillance. In addition, ccAMP-Seq has the power to detect the genetic variation and trace the origin of infectious diseases pathogens based on the sequence of multiple markers ( 1 , 3 , 5 , 24 ). In conclusion, ccAMP-Seq greatly improved the detection efficiency, sensitivity, specificity, accuracy, whereas it reduced the sample size and detection cost; thus ccAMP-Seq was widely applicable in pathogen-related detection scenarios.…”