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High-Throughput DNA Sequencing Analysis of Antibody Repertoires

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  • Authors: Scott D. Boyd1, Shilpa A. Joshi2
  • Editors: James E. Crowe Jr.3, Diana Boraschi4, Rino Rappuoli5
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Pathology, Stanford University, Stanford, CA 94305; 2: Department of Pathology, Stanford University, Stanford, CA 94305; 3: Vanderbilt University School of Medicine, Nashville, TN; 4: National Research Council, Pisa, Italy; 5: Novartis Vaccines, Siena, Italy
  • Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.AID-0017-2014
  • Received 15 April 2014 Accepted 08 May 2014 Published 10 October 2014
  • : Scott D. Boyd, sboyd1@stanford.edu
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  • Abstract:

    New high-throughput DNA sequencing (HTS) technologies developed in the past decade have begun to be applied to the study of the complex gene rearrangements that encode human antibodies. This article first reviews the genetic features of Ig loci and the HTS technologies that have been applied to human repertoire studies, then discusses key choices for experimental design and data analysis in these experiments and the insights gained in immunological and infectious disease studies with the use of these approaches.

  • Citation: Boyd S, Joshi S. 2014. High-Throughput DNA Sequencing Analysis of Antibody Repertoires. Microbiol Spectrum 2(5):AID-0017-2014. doi:10.1128/microbiolspec.AID-0017-2014.

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2014-10-10
2017-09-22

Abstract:

New high-throughput DNA sequencing (HTS) technologies developed in the past decade have begun to be applied to the study of the complex gene rearrangements that encode human antibodies. This article first reviews the genetic features of Ig loci and the HTS technologies that have been applied to human repertoire studies, then discusses key choices for experimental design and data analysis in these experiments and the insights gained in immunological and infectious disease studies with the use of these approaches.

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FIGURE 1

Antibody structure and genetic encoding. The germ line (unrearranged) genomic DNA configuration of the immunoglobulin heavy chain locus is depicted at the top of the figure, showing the tandem arrays of V, D, and J gene segments (not to scale). A germ line kappa or lambda light chain locus is depicted on the left-hand side, with unrearranged V and J segments. Stepwise rearrangement of the germ line DNA results in the joining of a heavy chain D and J gene segment, followed by joining of a V segment to the D-J product, to generate the DNA encoding the heavy chain variable region. In the process of rearrangement, the ends of the gene segments are subject to variable amounts of exonuclease digestion, and randomized nontemplated bases are added at the segment ends, to produce additional sequence diversity at the VDJ junctional region that encodes the complementarity-determining region 3 (CDR3) loop, which is often the region of the antibody heavy chain that has the greatest impact on antigen specificity. A similar process of V and J gene rearrangement with diversification of the VJ junction occurs in the light chain locus, to produce the rearranged light chain gene. The constant regions of the heavy and light chains (domains CH1, CH2, and CH3 for the heavy chain, and CL for the light chain) are encoded by downstream exons that are joined to the rearranged V(D)J gene by mRNA splicing. Disulfide bridges joining protein chains in the full antibody structure are shown with black line segments. doi:10.1128/microbiolspec.AID-0017-2014.f1

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.AID-0017-2014
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FIGURE 2

IgH library production from genomic DNA (gDNA) or complementary DNA (cDNA). The top diagram shows the rearranged gDNA encoding an antibody heavy chain. Primer sets designed to hybridize in the framework 1, 2, or 3 (FR1, FR2, FR3) regions (labeled with circled numbers 1, 2, and 3), together with a primer complementary to the J gene segments (labeled 4), can be used for PCR to amplify the VDJ gene rearrangement. Multiple primers are shown for the framework primer sets, indicating the different primers required for amplification of V segments belonging to different families. The leader peptide exon is separated from the V segment by a short intron in the genomic DNA. The lower diagram shows cDNA generated from spliced mRNA encoding a heavy chain. Primers hybridizing to the constant region (labeled 6) can be used as the initial gene-specific primer for 5′ RACE protocols (see main text), or else can be used in PCR with primers in the leader sequences (labeled 5), or framework primers, to amplify the VDJ gene rearrangements. The constant region isotype associated with the VDJ gene rearrangement can be identified in such libraries. doi:10.1128/microbiolspec.AID-0017-2014.f2

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.AID-0017-2014
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