Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022:2453:317-343.
doi: 10.1007/978-1-0716-2115-8_18.

Bulk gDNA Sequencing of Antibody Heavy-Chain Gene Rearrangements for Detection and Analysis of B-Cell Clone Distribution: A Method by the AIRR Community

Aaron M Rosenfeld  1 , Wenzhao Meng  1 , Kalisse I Horne  1 , Elaine C Chen  2 , Davide Bagnara  3 , Ulrik Stervbo  4 , Eline T Luning Prak  5 ; AIRR Community
Affiliations

Bulk gDNA Sequencing of Antibody Heavy-Chain Gene Rearrangements for Detection and Analysis of B-Cell Clone Distribution: A Method by the AIRR Community

Aaron M Rosenfeld et al. Methods Mol Biol. 2022.

Abstract

In this method we illustrate how to amplify, sequence, and analyze antibody/immunoglobulin (IG) heavy-chain gene rearrangements from genomic DNA that is derived from bulk populations of cells by next-generation sequencing (NGS). We focus on human source material and illustrate how bulk gDNA-based sequencing can be used to examine clonal architecture and networks in different samples that are sequenced from the same individual. Although bulk gDNA-based sequencing can be performed on both IG heavy (IGH) or kappa/lambda light (IGK/IGL) chains, we focus here on IGH gene rearrangements because IG heavy chains are more diverse, tend to harbor higher levels of somatic hypermutations (SHM), and are more reliable for clone identification and tracking. We also provide a procedure, including code, and detailed instructions for processing and annotation of the NGS data. From these data we show how to identify expanded clones, visualize the overall clonal landscape, and track clonal lineages in different samples from the same individual. This method has a broad range of applications, including the identification and monitoring of expanded clones, the analysis of blood and tissue-based clonal networks, and the study of immune responses including clonal evolution.

Keywords: Antibody; Clone; Immune repertoire profiling; Immunoglobulin; Lineage; Next-generation sequencing; V(D)J recombination.

PubMed Disclaimer

References

    1. Tonegawa S (1983) Somatic generation of antibody diversity. Nature 302(5909):575–581. 10.1038/302575a0 doi: 10.1038/302575a0. - DOI - PubMed
    1. Sakano H, Kurosawa Y, Weigert M, Tonegawa S (1981) Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy-chain genes. Nature 290(5807):562–565. 10.1038/290562a0 doi: 10.1038/290562a0. - DOI - PubMed
    1. Weigert MG, Cesari IM, Yonkovich SJ, Cohn M (1970) Variability in the lambda light chain sequences of mouse antibody. Nature 228(5276):1045–1047. 10.1038/2281045a0 doi: 10.1038/2281045a0. - DOI - PubMed
    1. Papavasiliou FN, Schatz DG (2002) Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity. Cell 109(Suppl):S35–S44. 10.1016/s0092-8674(02)00706-7 doi: 10.1016/s0092-8674(02)00706-7. - DOI - PubMed
    1. Georgiou G, Ippolito GC, Beausang J, Busse CE, Wardemann H, Quake SR (2014) The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat Biotechnol 32(2):158–168. 10.1038/nbt.2782 doi: 10.1038/nbt.2782. - DOI - PMC - PubMed

Publication types

LinkOut - more resources