COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens
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...
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| Format: | Article |
| Language: | English |
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Wiley
2022-01-01
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| Series: | Interdisciplinary Perspectives on Infectious Diseases |
| Online Access: | http://dx.doi.org/10.1155/2022/2109641 |
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| author | Tatsuki Sugi Mizanur Rahman Rummana Rahim Abu Hasan Naoko Kawai Kyoko Hayashida Junya Yamagishi |
| author_facet | Tatsuki Sugi Mizanur Rahman Rummana Rahim Abu Hasan Naoko Kawai Kyoko Hayashida Junya Yamagishi |
| author_sort | Tatsuki Sugi |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-c2d454c22cab48febb035f9a095f448b |
| institution | Kabale University |
| issn | 1687-7098 |
| language | English |
| publishDate | 2022-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Interdisciplinary Perspectives on Infectious Diseases |
| spelling | doaj-art-c2d454c22cab48febb035f9a095f448b2025-02-03T01:22:54ZengWileyInterdisciplinary Perspectives on Infectious Diseases1687-70982022-01-01202210.1155/2022/2109641COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical SpecimensTatsuki Sugi0Mizanur Rahman1Rummana Rahim2Abu Hasan3Naoko Kawai4Kyoko Hayashida5Junya Yamagishi6Division of Collaboration and EducationEvercare Hospital DhakaEvercare Hospital DhakaEvercare Hospital DhakaDivision of Collaboration and EducationDivision of Collaboration and EducationDivision of Collaboration and EducationWith 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.http://dx.doi.org/10.1155/2022/2109641 |
| spellingShingle | Tatsuki Sugi Mizanur Rahman Rummana Rahim Abu Hasan Naoko Kawai Kyoko Hayashida Junya Yamagishi COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens Interdisciplinary Perspectives on Infectious Diseases |
| title | COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens |
| title_full | COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens |
| title_fullStr | COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens |
| title_full_unstemmed | COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens |
| title_short | COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens |
| title_sort | covid 19 whole genome resequencing with redundant tiling pcr and subtract based amplicon normalization successfully characterized sars cov 2 variants in clinical specimens |
| url | http://dx.doi.org/10.1155/2022/2109641 |
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