Improved detection of rare HIV-1 variants using 454 pyrosequencing.
454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral population...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3788733?pdf=render |
| _version_ | 1818469497096372224 |
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| author | Brendan B Larsen Lennie Chen Brandon S Maust Moon Kim Hong Zhao Wenjie Deng Dylan Westfall Ingrid Beck Lisa M Frenkel James I Mullins |
| author_facet | Brendan B Larsen Lennie Chen Brandon S Maust Moon Kim Hong Zhao Wenjie Deng Dylan Westfall Ingrid Beck Lisa M Frenkel James I Mullins |
| author_sort | Brendan B Larsen |
| collection | DOAJ |
| description | 454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%. |
| first_indexed | 2024-04-13T21:25:19Z |
| format | Article |
| id | doaj.art-bf89a77947c84604bb24e57a99dfe103 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T21:25:19Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-bf89a77947c84604bb24e57a99dfe1032022-12-22T02:29:21ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-01810e7650210.1371/journal.pone.0076502Improved detection of rare HIV-1 variants using 454 pyrosequencing.Brendan B LarsenLennie ChenBrandon S MaustMoon KimHong ZhaoWenjie DengDylan WestfallIngrid BeckLisa M FrenkelJames I Mullins454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.http://europepmc.org/articles/PMC3788733?pdf=render |
| spellingShingle | Brendan B Larsen Lennie Chen Brandon S Maust Moon Kim Hong Zhao Wenjie Deng Dylan Westfall Ingrid Beck Lisa M Frenkel James I Mullins Improved detection of rare HIV-1 variants using 454 pyrosequencing. PLoS ONE |
| title | Improved detection of rare HIV-1 variants using 454 pyrosequencing. |
| title_full | Improved detection of rare HIV-1 variants using 454 pyrosequencing. |
| title_fullStr | Improved detection of rare HIV-1 variants using 454 pyrosequencing. |
| title_full_unstemmed | Improved detection of rare HIV-1 variants using 454 pyrosequencing. |
| title_short | Improved detection of rare HIV-1 variants using 454 pyrosequencing. |
| title_sort | improved detection of rare hiv 1 variants using 454 pyrosequencing |
| url | http://europepmc.org/articles/PMC3788733?pdf=render |
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