Bioinformatic Analysis of Natively Paired VH:VL Antibody Repertoires for Antibody Discovery

Ahmed S Fahad; Bharat Madan; Brandon J DeKosky

doi:10.1007/978-1-0716-2609-2_25

Bioinformatic Analysis of Natively Paired VH:VL Antibody Repertoires for Antibody Discovery

Methods Mol Biol. 2023:2552:447-463. doi: 10.1007/978-1-0716-2609-2_25.

Authors

Ahmed S Fahad^#^{1

2}, Bharat Madan^#^{1

2}, Brandon J DeKosky^{3

4

5

6}

Affiliations

¹ The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA.
² Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS, USA.
³ The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA. dekosky@ku.edu.
⁴ Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS, USA. dekosky@ku.edu.
⁵ Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. dekosky@ku.edu.
⁶ Department of Chemical Engineering, The University of Kansas, Lawrence, KS, USA. dekosky@ku.edu.

^# Contributed equally.

PMID: 36346608
DOI: 10.1007/978-1-0716-2609-2_25

Abstract

Next-generation DNA sequencing (NGS) of human antibody repertoires has been extensively implemented to discover novel antibody drugs, to analyze B-cell developmental features, and to investigate antibody responses to infectious diseases and vaccination. Because the antibody repertoire encoded by human B cells is highly diverse, NGS analyses of antibody genes have provided a new window into understanding antibody responses for basic immunology, biopharmaceutical drug discovery, and immunotherapy. However, many antibody discovery protocols analyze the heavy and light chains separately due to the short-read nature of most NGS technologies, whereas paired heavy and light chain data are required for complete antibody characterization. Here, we describe a computational workflow to process millions of paired antibody heavy and light chain DNA sequence reads using the Illumina MiSeq 2x300 NGS platform. In this workflow, we describe raw NGS read processing and initial quality filtering, the annotation and assembly of antibody clonotypes relating to paired heavy and light chain antibody lineages, and the generation of complete heavy+light consensus sequences for the downstream cloning and expression of human antibody proteins.

Keywords: Antibody discovery; B cells; Bioinformatics; Next-generation sequencing.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Antibodies*
Computational Biology* / methods
High-Throughput Nucleotide Sequencing / methods
Humans
Immunoglobulin Light Chains / genetics

Substances

Antibodies
Immunoglobulin Light Chains

Abstract

Publication types

MeSH terms

Substances

Grants and funding