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Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding

B-cell repertoire analysis using next-generation sequencing has become a valuable tool for interrogating the genetic record of humoral response to infection. However, key obstacles such as low throughput, short read length, high error rate, and undetermined bias of multiplex PCR method have hindered...

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Autores principales: He, Linling, Sok, Devin, Azadnia, Parisa, Hsueh, Jessica, Landais, Elise, Simek, Melissa, Koff, Wayne C., Poignard, Pascal, Burton, Dennis R., Zhu, Jiang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894419/
https://www.ncbi.nlm.nih.gov/pubmed/25345460
http://dx.doi.org/10.1038/srep06778
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author He, Linling
Sok, Devin
Azadnia, Parisa
Hsueh, Jessica
Landais, Elise
Simek, Melissa
Koff, Wayne C.
Poignard, Pascal
Burton, Dennis R.
Zhu, Jiang
author_facet He, Linling
Sok, Devin
Azadnia, Parisa
Hsueh, Jessica
Landais, Elise
Simek, Melissa
Koff, Wayne C.
Poignard, Pascal
Burton, Dennis R.
Zhu, Jiang
author_sort He, Linling
collection PubMed
description B-cell repertoire analysis using next-generation sequencing has become a valuable tool for interrogating the genetic record of humoral response to infection. However, key obstacles such as low throughput, short read length, high error rate, and undetermined bias of multiplex PCR method have hindered broader application of this technology. In this study, we report several technical advances in antibody repertoire sequencing. We first demonstrated the ability to sequence antibody variable domains using the Ion Torrent PGM platform. As a test case, we analyzed the PGT121 class of antibodies from IAVI donor 17, an HIV-1-infected individual. We then obtained “unbiased” antibody repertoires by sequencing the 5′-RACE PCR products of B-cell transcripts from IAVI donor 17 and two HIV-1-uninfected individuals. We also quantified the bias of previously published gene-specific primers by comparing the repertoires generated by 5′-RACE PCR and multiplex PCR. We further developed a single-molecule barcoding strategy to reduce PCR-based amplification noise. Lastly, we evaluated several new PGM technologies in the context of antibody sequencing. We expect that, based upon long-read and high-fidelity next-generation sequencing technologies, the unbiased analysis will provide a more accurate view of the overall antibody repertoire while the barcoding strategy will facilitate high-resolution analysis of individual antibody families.
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spelling pubmed-48944192016-06-10 Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding He, Linling Sok, Devin Azadnia, Parisa Hsueh, Jessica Landais, Elise Simek, Melissa Koff, Wayne C. Poignard, Pascal Burton, Dennis R. Zhu, Jiang Sci Rep Article B-cell repertoire analysis using next-generation sequencing has become a valuable tool for interrogating the genetic record of humoral response to infection. However, key obstacles such as low throughput, short read length, high error rate, and undetermined bias of multiplex PCR method have hindered broader application of this technology. In this study, we report several technical advances in antibody repertoire sequencing. We first demonstrated the ability to sequence antibody variable domains using the Ion Torrent PGM platform. As a test case, we analyzed the PGT121 class of antibodies from IAVI donor 17, an HIV-1-infected individual. We then obtained “unbiased” antibody repertoires by sequencing the 5′-RACE PCR products of B-cell transcripts from IAVI donor 17 and two HIV-1-uninfected individuals. We also quantified the bias of previously published gene-specific primers by comparing the repertoires generated by 5′-RACE PCR and multiplex PCR. We further developed a single-molecule barcoding strategy to reduce PCR-based amplification noise. Lastly, we evaluated several new PGM technologies in the context of antibody sequencing. We expect that, based upon long-read and high-fidelity next-generation sequencing technologies, the unbiased analysis will provide a more accurate view of the overall antibody repertoire while the barcoding strategy will facilitate high-resolution analysis of individual antibody families. Nature Publishing Group 2014-10-27 /pmc/articles/PMC4894419/ /pubmed/25345460 http://dx.doi.org/10.1038/srep06778 Text en Copyright © 2014, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by-nc-sa/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/
spellingShingle Article
He, Linling
Sok, Devin
Azadnia, Parisa
Hsueh, Jessica
Landais, Elise
Simek, Melissa
Koff, Wayne C.
Poignard, Pascal
Burton, Dennis R.
Zhu, Jiang
Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding
title Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding
title_full Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding
title_fullStr Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding
title_full_unstemmed Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding
title_short Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding
title_sort toward a more accurate view of human b-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894419/
https://www.ncbi.nlm.nih.gov/pubmed/25345460
http://dx.doi.org/10.1038/srep06778
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