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COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens
oleh: Tatsuki Sugi, Mizanur Rahman, Rummana Rahim, Abu Hasan, Naoko Kawai, Kyoko Hayashida, Junya Yamagishi
| Format: | Article |
|---|---|
| Diterbitkan: | Wiley 2022-01-01 |
Deskripsi
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.