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

• Main Authors: Tan, Kim-Kee, Tiong, Vunjia, Tan, Jiun Yan, Wong, Jo-Ern, Teoh, Boon-Teong, Abd-Jamil, Juraina, Johari, Mohd Iskandar Jefree, Nor'e, Siti-Sarah, Khor, Chee Sieng, Yaacob, Che-Norainon, Zulkifli, Mulya-Mustika-Sari, CheMatSeri, AsmaAnati, Mahfodz, Nur-Hidayana, Azizan, Noor Syahida, AbuBakar, Sazaly
• Format: Article
• Published: Malaysian Society of Parasitology and Tropical Medicine 2021
• Subjects: QR Microbiology; R Medicine; RB Pathology
• Online Access: http://eprints.um.edu.my/34664/

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.