Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the ion Personal Genome Machine (PGM)
Document Type
Article
Publication Date
9-1-2021
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 13 751-13 965 and 23 941-24 106), 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 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq (TM) SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq (TM) SARS CoV 2 Research Panel, hence, should be considered.
Keywords
SARS-CoV-2, COVID-19, Infectious diseases, Next generation sequencing, Malaysia
Divisions
tidrec
Funders
Ministry of Energy, Science, Technology, Environment and Climate Change (MESTECC), Malaysia[UM.0000345/KWJ.AK],Ministry of Education, Malaysia,Higher Institution Centre of Excellence (HICoE) program[MO002-2019],University Malaya RU grant[RU002-2019],Fundamental Research Grant Scheme[MRSA/1/2018/SKK08/UM/01/1 (MO012-2017)]
Publication Title
Tropical Biomedicine
Volume
38
Issue
3
Publisher
Malaysian Society of Parasitology and Tropical Medicine
Publisher Location
21-5-2, BLOCK, E, DIAMOND SQUARE, JALAN 3-50, OFF JALAN GOMBAK, KUALA LUMPUR, MALAYSIA