Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding
View/ Open
Author
He, Linling
Sok, Devin
Azadnia, Parisa
Hsueh, Jessica
Landais, Elise
Simek, Melissa
Koff, Wayne C.
Poignard, Pascal
Burton, Dennis R.
Zhu, Jiang
Published Version
https://doi.org/10.1038/srep06778Metadata
Show full item recordCitation
He, Linling, Devin Sok, Parisa Azadnia, Jessica Hsueh, Elise Landais, Melissa Simek, Wayne C. Koff, Pascal Poignard, Dennis R. Burton, and Jiang Zhu. 2014. “Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding.” Scientific Reports 4 (1): 6778. doi:10.1038/srep06778. http://dx.doi.org/10.1038/srep06778.Abstract
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.Other Sources
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894419/pdf/Terms of Use
This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAACitable link to this page
http://nrs.harvard.edu/urn-3:HUL.InstRepos:27662031
Collections
- HMS Scholarly Articles [17922]
Contact administrator regarding this item (to report mistakes or request changes)