BALDR: a computational pipeline for paired heavy and light chain immunoglobulin reconstruction in single-cell RNA-seq data

Amit A Upadhyay,Colin Havenar-Daughton,Robert C Kauffman,Samantha M Reiss,Reem A Dawoud,Iñaki Sanz,Nirav B Patel,F Eun-Hyung Lee,Shane Crotty,Steven E Bosinger,Bali Pulendran,Amber N Wolabaugh,Gregory K Tharp,Alice Cho,Jens Wrammert

doi:10.1186/s13073-018-0528-3

Abstract

B cells play a critical role in the immune response by producing antibodies, which display remarkable diversity. Here we describe a bioinformatic pipeline, BALDR (BCR Assignment of Lineage using De novo Reconstruction) that accurately reconstructs the paired heavy and light chain immunoglobulin gene sequences from Illumina single-cell RNA-seq data. BALDR was accurate for clonotype identification in human and rhesus macaque influenza vaccine and simian immunodeficiency virus vaccine induced vaccine-induced plasmablasts and naïve and antigen-specific memory B cells. BALDR enables matching of clonotype identity with single-cell transcriptional information in B cell lineages and will have broad application in the fields of vaccines, human immunodeficiency virus broadly neutralizing antibody development, and cancer.BALDR is available at https://github.com/BosingerLab/BALDR.

Highlights

B cells comprise a major component of the immune system, and they function primarily by secreting antibodies that bind and neutralize discrete protein moieties on pathogens
Plasmablasts are a useful population to query emergent B cell responses, as they are highly enriched for antigen-specific cells, and they allow for unbiased interrogation of relevant, vaccine-induced B cells without using fluorescently labeled antigenic probes or other technologies
After complementary DNA (cDNA) amplification of single plasmablasts, prominent peaks representing the Immunoglobulin heavy chain (IgH) and immunoglobulin gene and a “light chain” (IgL) messenger RNA (mRNA) were readily apparent by microcapillary electrophoresis (Fig. 1c)

Summary

Introduction

B cells comprise a major component of the immune system, and they function primarily by secreting antibodies that bind and neutralize discrete protein moieties on pathogens. While the achievable depth of these amplicon-based approaches provided remarkable resolution (105–106 chains in a single experiment) [8], a significant limitation of this technology for functional studies of the immune system is that it only sequences a single chain and cannot provide information on Upadhyay et al Genome Medicine (2018) 10:20 endogenous pairing of IgH/IgL genes to definitively identify a B cell clonotype. A novel, ultra highthroughput method to identify millions of paired IgH + IgL genes was developed by Georgiou, DeKosky, and colleagues [9] This method uses an upfront capture of individual B cells into droplets, after which an elegant in-drop PCR ligation strategy creates a single DNA amplicon containing both IgH and IgL chains for en masse Illumina sequencing [9]. The ability to generate deep sequence data of IgH + IgL pairings constitutes a significant advance over single-chain profiling; it does not provide functional or transcriptional information

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