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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

Academic Article
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Overview

related to degree

  • Sok, Devin, Ph.D. in Chemistry, Scripps Research 2008 - 2014

authors

  • He, L.
  • Sok, Devin
  • Azadnia, P.
  • Hsueh, J.
  • Landais, Elise
  • Simek, M.
  • Koff, W. C.
  • Poignard, Pascal
  • Burton, Dennis
  • Zhu, Jiang

publication date

  • October 2014

journal

  • Scientific Reports  Journal

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.

subject areas

  • Antibodies
  • Antibodies, Neutralizing
  • Antibody Formation
  • B-Lymphocytes
  • DNA Barcoding, Taxonomic
  • HIV Infections
  • HIV-1
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Phylogeny
  • Polymerase Chain Reaction
  • Reproducibility of Results
  • Somatic Hypermutation, Immunoglobulin
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Identity

PubMed Central ID

  • PMC4894419

International Standard Serial Number (ISSN)

  • 2045-2322

Digital Object Identifier (DOI)

  • 10.1038/srep06778

PubMed ID

  • 25345460
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Additional Document Info

start page

  • 6778

volume

  • 4

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