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

KK Tan; V Tiong; JY Tan; JE Wong; BT Teoh; J Abd-jamil; J Johari; Nor&rsquo; SS e; CS Khor; CN Yaacob; Mms Zulkifli; A Chematseri; NH Mahfodz; NS Azizan; S Abubakar

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

Author: Tan, K.K. ¹ ; Tiong, V. ¹ ; Tan, J.Y. ¹ ; Wong, J.E. ¹ ; Teoh, B.T. ¹ ; Abd-Jamil, J. ¹ ; Johari, J. ¹ ; Nor’e, S.S. ¹ ; Khor, C.S. ¹ ; Yaacob, C.N. ¹ ; Zulkifli, M.M.S. ¹ ; CheMatSeri, A. ¹ ; Mahfodz, N.H. ¹ ; Azizan, N.S. ¹ ; AbuBakar, S. ^{1
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Author Information

1. Tropical Infectious Diseases Research and Education Centre (TIDREC), Higher Institution Centre of Excellence (HICoE), University of Malaya, 50603 Kuala Lumpur, Malaysia&
2. Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
Publication Type:Journal Article
Keywords: SARS-CoV-2; COVID-19; infectious diseases; next generation sequencing; Malaysia
From:Tropical Biomedicine 2021;38(No.3):283-288
CountryMalaysia
Language:English
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 4Apr20-64Hu. 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™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.
Full text:8.2021my1268.pdf