Tatsuki Sugi: Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Nishi10-Kita20, Sapporo, Japan.
Naoko Kawai: Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Nishi10-Kita20, Sapporo, Japan.
Kyoko Hayashida: Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Nishi10-Kita20, Sapporo, Japan.
Junya Yamagishi: Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Nishi10-Kita20, Sapporo, Japan. ORCID
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.
References
PLoS One. 2021 Oct 29;16(10):e0259277
[PMID: 34714886]
