COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens. (28th September 2022)

• Record Type: Journal Article
• Authors: Sugi, Tatsuki; Rahman, Mizanur; Rahim, Rummana; Hasan, Abu; Kawai, Naoko; Hayashida, Kyoko; Yamagishi, Junya
• Other Names: Ghorbani-Dalini Sadegh Academic Editor.
• Is Part Of: Interdisciplinary perspectives on infectious diseases. Volume 2022(2022)
• Publication Date: 2022-09-28
• Subjects: Communicable diseases -- Periodicals; 571.9805
• DOI: 10.1155/2022/2109641
• Languages: English
• ISSNs: 1687-708X

Abstract

Abstract : With an increasing number of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequences gathered worldwide, we recognize that deletion mutants and nucleotide substitutions that may affect whole-genome sequencing are accumulating. Here, we propose an additional strategy for tiling PCR for whole-genome resequencing, which can make the pipeline robust for mutations at the primer annealing site by a redundant amplicon scheme. We further demonstrated that subtracting overrepresented amplicons from the multiplex PCR products reduced the bias of the next-generation sequencing (NGS) library, resulting in decreasing required sequencing reads per sample. We applied this sequencing strategy to clinical specimens collected in Bangladesh. More than 80% out of the 304 samples were successfully sequenced. Less than 5% were ambiguous nucleotides, and several known variants were detected. With the additional strategies presented here, we believe that whole-genome resequencing of SARS-CoV-2 from clinical samples can be optimized.