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

Tan, Kim-Kee and Tiong, Vunjia and Tan, Jiun Yan and Wong, Jo-Ern and Teoh, Boon-Teong and Abd-Jamil, Juraina and Johari, Mohd Iskandar Jefree and Nor'e, Siti-Sarah and Khor, Chee Sieng and Yaacob, Che-Norainon and Zulkifli, Mulya-Mustika-Sari and CheMatSeri, AsmaAnati and Mahfodz, Nur-Hidayana and Azizan, Noor Syahida and AbuBakar, Sazaly (2021) Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the ion Personal Genome Machine (PGM). Tropical Biomedicine, 38 (3). pp. 283-288. ISSN 2521-9855, DOI

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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.

Item Type: Article
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)]
Uncontrolled Keywords: SARS-CoV-2;COVID-19;Infectious diseases;Next generation sequencing;Malaysia
Subjects: Q Science > QR Microbiology
R Medicine
R Medicine > RB Pathology
Divisions: Deputy Vice Chancellor (Research & Innovation) Office > Tropical Infectious Diseases Research and Education Centre
Depositing User: Ms Zaharah Ramly
Date Deposited: 19 Sep 2022 04:19
Last Modified: 19 Sep 2022 04:19

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