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Massively Parallel Single-Cell B-cell Receptor Sequencing Enables Rapid Discovery of Diverse Antigen-Reactive Antibodies

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Massively Parallel Single-Cell B-cell Receptor Sequencing Enables Rapid Discovery of Diverse Antigen-Reactive Antibodies

Leonard D Goldstein et al. Commun Biol.

Abstract

Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem. Here we use high-throughput single-cell B-cell receptor sequencing (scBCR-seq) to obtain accurately paired full-length variable regions in a massively parallel fashion. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion. In addition, we immunized rats with chicken ovalbumin and profiled antigen-reactive B cells from lymph nodes of immunized animals. The scBCR-seq data recovered 81% (n = 56/69) of B-cell lineages identified from hybridomas generated from the same set of B cells subjected to scBCR-seq. Importantly, scBCR-seq identified an additional 710 candidate lineages not recovered as hybridomas. We synthesized, expressed and tested 93 clones from the identified lineages and found that 99% (n = 92/93) of the clones were antigen-reactive. Our results establish scBCR-seq as a powerful tool for antibody discovery.

Keywords: B-cell receptor; Next-generation sequencing.

Conflict of interest statement

Competing interestsThe authors as noted in the authors section are employees of Genentech or SciGenom Labs. Genentech authors hold shares in Roche. The authors declare no nonfinancial conflicts of interest.

Figures

Fig. 1
Fig. 1
Schematic of single B cell capture, library construction and sequencing. Chromium controller (10x Genomics, Pleasanton, CA) was used to capture single cells along with gel beads containing the 5′ switch oligo with cell barcode, reverse transcriptase, and poly(dT) primer. The barcoded cDNAs were then converted into a library and sequenced (Illumina, San Diego, CA). Heavy- and light-chain sequences for individual cells were analyzed using a custom computational pipeline
Fig. 2
Fig. 2
Computational framework for VH-VL pairing. a Data analysis workflow. b Cell quality filtering based on the number of reads for VH and VL assemblies and VH and VL certainty. Data shown are from one human sample, Donor 1 (1). c Read coverage for quality-filtered VH and VL assemblies for three different species. Positions within VH and VL are based on Kabat numbers, CDR regions are indicated by shaded regions. Solid lines indicate the median, dashed lines interquartile range. Data shown for rat, mouse, and human are from samples Rat 1, Mouse 1, and Donor 1 (1), respectively. d Overview of B-cell repertoire data generated for this study. Bar graphs show data for independent samples. From top to bottom, number of cells captured, percentage of cells with at least one complete VH and VL assembly, percentage of cells that pass quality filtering, number of cells that pass quality filtering
Fig. 3
Fig. 3
Technology validation. a Comparison of unique VH–VL nucleotide sequences obtained by scBCR-seq (columns) and reference VH–VL pairs obtained by a standard sequencing approach (rows). Blue, gray, and red boxes indicate VH–VL sequences validated with 0, 1–2, and >2 mismatches, respectively. Top panel shows number of cells for a particular VH–VL sequence in scBCR-seq data. b Pairing accuracy based on VL concordance for VH–VL pairings with identical VH and CDR-H3
Fig. 4
Fig. 4
Variable germline gene segment pairing for B-cell repertoires from two rats (a, b) and two mice (c, d). Heatmaps show the percentage of lineages with a particular VH–VL pairing. Row and column histograms indicate marginal VH and VL frequencies, respectively
Fig. 5
Fig. 5
Variable germline gene segment pairing for B-cell repertoires from three human donors. Panels ac and df show data from the same three donors at different time points, respectively. Otherwise as in Fig. 4
Fig. 6
Fig. 6
Discovery and validation of antigen-reactive antibodies. a Lineage expansions among OVA antigen-reactive B cells. Pie charts indicate percentage of cells belonging to expanded lineages. Bar charts indicate the number of cells for the top 50 lineages. b Lineage expansions observed in B-cell repertoires for two nonimmunized rats, otherwise as in a. c Somatic hypermutations (SHM) for heavy- and light-chain variable germline gene segments for B-cell repertoires from nonimmunized Rat 1 (n = 15,338) and Rat 2 (n = 15,042) and OVA antigen-reactive B cells pooled from three immunized rats (n = 3091). Boxes indicate the interquartile range (IQR), center lines the median, whiskers extend to the most extreme data point within 1.5 × IQR from the box. d Overlap in lineages identified from direct sequencing of individual antigen-reactive B cells by scBCR-seq (red) and concomitant hybridoma experiment (blue). e Validation of candidate OVA antigen-reactive clones. Shown are monovalent affinities of expressed antibodies to OVA

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