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Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping
Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates dat...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411816/ https://www.ncbi.nlm.nih.gov/pubmed/32605062 http://dx.doi.org/10.3390/v12070694 |
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author | Parkin, Neil T. Avila-Rios, Santiago Bibby, David F. Brumme, Chanson J. Eshleman, Susan H. Harrigan, P. Richard Howison, Mark Hunt, Gillian Ji, Hezhao Kantor, Rami Ledwaba, Johanna Lee, Emma R. Matías-Florentino, Margarita Mbisa, Jean L. Noguera-Julian, Marc Paredes, Roger Rivera-Amill, Vanessa Swanstrom, Ronald Zaccaro, Daniel J. Zhang, Yinfeng Zhou, Shuntai Jennings, Cheryl |
author_facet | Parkin, Neil T. Avila-Rios, Santiago Bibby, David F. Brumme, Chanson J. Eshleman, Susan H. Harrigan, P. Richard Howison, Mark Hunt, Gillian Ji, Hezhao Kantor, Rami Ledwaba, Johanna Lee, Emma R. Matías-Florentino, Margarita Mbisa, Jean L. Noguera-Julian, Marc Paredes, Roger Rivera-Amill, Vanessa Swanstrom, Ronald Zaccaro, Daniel J. Zhang, Yinfeng Zhou, Shuntai Jennings, Cheryl |
author_sort | Parkin, Neil T. |
collection | PubMed |
description | Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4–99; reverse transcriptase 38–247). The concordance among laboratories’ sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1–100%), compared to 15% (99.4%, 88.5–100%), 10% (99.2%, 87.4–100%), or 5% (98.5%, 86.4–100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping. |
format | Online Article Text |
id | pubmed-7411816 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-74118162020-08-25 Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping Parkin, Neil T. Avila-Rios, Santiago Bibby, David F. Brumme, Chanson J. Eshleman, Susan H. Harrigan, P. Richard Howison, Mark Hunt, Gillian Ji, Hezhao Kantor, Rami Ledwaba, Johanna Lee, Emma R. Matías-Florentino, Margarita Mbisa, Jean L. Noguera-Julian, Marc Paredes, Roger Rivera-Amill, Vanessa Swanstrom, Ronald Zaccaro, Daniel J. Zhang, Yinfeng Zhou, Shuntai Jennings, Cheryl Viruses Article Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4–99; reverse transcriptase 38–247). The concordance among laboratories’ sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1–100%), compared to 15% (99.4%, 88.5–100%), 10% (99.2%, 87.4–100%), or 5% (98.5%, 86.4–100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping. MDPI 2020-06-27 /pmc/articles/PMC7411816/ /pubmed/32605062 http://dx.doi.org/10.3390/v12070694 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Parkin, Neil T. Avila-Rios, Santiago Bibby, David F. Brumme, Chanson J. Eshleman, Susan H. Harrigan, P. Richard Howison, Mark Hunt, Gillian Ji, Hezhao Kantor, Rami Ledwaba, Johanna Lee, Emma R. Matías-Florentino, Margarita Mbisa, Jean L. Noguera-Julian, Marc Paredes, Roger Rivera-Amill, Vanessa Swanstrom, Ronald Zaccaro, Daniel J. Zhang, Yinfeng Zhou, Shuntai Jennings, Cheryl Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping |
title | Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping |
title_full | Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping |
title_fullStr | Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping |
title_full_unstemmed | Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping |
title_short | Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping |
title_sort | multi-laboratory comparison of next-generation to sanger-based sequencing for hiv-1 drug resistance genotyping |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411816/ https://www.ncbi.nlm.nih.gov/pubmed/32605062 http://dx.doi.org/10.3390/v12070694 |
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