Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)
Abstract Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2019-05-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-019-43524-9 |
| _version_ | 1818430246298320896 |
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| author | Anna L. McNaughton Hannah E. Roberts David Bonsall Mariateresa de Cesare Jolynne Mokaya Sheila F. Lumley Tanya Golubchik Paolo Piazza Jacqueline B. Martin Catherine de Lara Anthony Brown M. Azim Ansari Rory Bowden Eleanor Barnes Philippa C. Matthews |
| author_facet | Anna L. McNaughton Hannah E. Roberts David Bonsall Mariateresa de Cesare Jolynne Mokaya Sheila F. Lumley Tanya Golubchik Paolo Piazza Jacqueline B. Martin Catherine de Lara Anthony Brown M. Azim Ansari Rory Bowden Eleanor Barnes Philippa C. Matthews |
| author_sort | Anna L. McNaughton |
| collection | DOAJ |
| description | Abstract Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-length virus genome data. Here we describe advances to a protocol that exploits the circular HBV genome structure, using isothermal rolling-circle amplification to enrich HBV DNA, generating concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by analysing reads generated from plus and minus strands. With this approach, we can achieve an improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. Thus while Illumina sequencing may still be the most accurate way to capture within-sample diversity, Nanopore data can contribute to an understanding of linkage between polymorphisms within individual virions. The combination of isothermal amplification and Nanopore sequencing also offers appealing potential to develop point-of-care tests for HBV, and for other viruses. |
| first_indexed | 2024-12-14T15:30:22Z |
| format | Article |
| id | doaj.art-23ce459625d44afd94bf0f71124bebc7 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-14T15:30:22Z |
| publishDate | 2019-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-23ce459625d44afd94bf0f71124bebc72022-12-21T22:55:54ZengNature PortfolioScientific Reports2045-23222019-05-019111410.1038/s41598-019-43524-9Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)Anna L. McNaughton0Hannah E. Roberts1David Bonsall2Mariateresa de Cesare3Jolynne Mokaya4Sheila F. Lumley5Tanya Golubchik6Paolo Piazza7Jacqueline B. Martin8Catherine de Lara9Anthony Brown10M. Azim Ansari11Rory Bowden12Eleanor Barnes13Philippa C. Matthews14Nuffield Department of Medicine, Medawar Building, University of Oxford, South Parks RoadWellcome Centre for Human Genetics, Roosevelt DriveNuffield Department of Medicine, Medawar Building, University of Oxford, South Parks RoadWellcome Centre for Human Genetics, Roosevelt DriveNuffield Department of Medicine, Medawar Building, University of Oxford, South Parks RoadNuffield Department of Medicine, Medawar Building, University of Oxford, South Parks RoadWellcome Centre for Human Genetics, Roosevelt DriveImperial BRC Genomics Facility, Imperial CollegeGastroenterology and Hepatology Clinical Trials Facility, Oxford University Hospitals NHS Foundation Trust, John Radcliffe HospitalNuffield Department of Medicine, Medawar Building, University of Oxford, South Parks RoadNuffield Department of Medicine, Medawar Building, University of Oxford, South Parks RoadNuffield Department of Medicine, Medawar Building, University of Oxford, South Parks RoadWellcome Centre for Human Genetics, Roosevelt DriveNuffield Department of Medicine, Medawar Building, University of Oxford, South Parks RoadNuffield Department of Medicine, Medawar Building, University of Oxford, South Parks RoadAbstract Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-length virus genome data. Here we describe advances to a protocol that exploits the circular HBV genome structure, using isothermal rolling-circle amplification to enrich HBV DNA, generating concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by analysing reads generated from plus and minus strands. With this approach, we can achieve an improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. Thus while Illumina sequencing may still be the most accurate way to capture within-sample diversity, Nanopore data can contribute to an understanding of linkage between polymorphisms within individual virions. The combination of isothermal amplification and Nanopore sequencing also offers appealing potential to develop point-of-care tests for HBV, and for other viruses.https://doi.org/10.1038/s41598-019-43524-9 |
| spellingShingle | Anna L. McNaughton Hannah E. Roberts David Bonsall Mariateresa de Cesare Jolynne Mokaya Sheila F. Lumley Tanya Golubchik Paolo Piazza Jacqueline B. Martin Catherine de Lara Anthony Brown M. Azim Ansari Rory Bowden Eleanor Barnes Philippa C. Matthews Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV) Scientific Reports |
| title | Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV) |
| title_full | Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV) |
| title_fullStr | Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV) |
| title_full_unstemmed | Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV) |
| title_short | Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV) |
| title_sort | illumina and nanopore methods for whole genome sequencing of hepatitis b virus hbv |
| url | https://doi.org/10.1038/s41598-019-43524-9 |
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