High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire

Abstract

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

Figure 1

Overview of the high-throughput methodology for paired V_H:V_L antibody repertoire analysis. (a) B-cell populations are sorted for desired phenotype (e.g., mBCs, memory B cells, naive BCs, naive B cells). (b) Single cells are isolated by random settling into 125-pl wells (56-μm diameter) printed in polydimethylsiloxane (PDMS) slides the size of a standard microscope slide (1.7 × 10⁵ wells/slide); 2.8-μm poly(dT) microbeads are also added to the wells (average 55 beads/well). (c) Wells are sealed with a dialysis membrane and equilibrated with lysis buffer to lyse cells and anneal V_H and V_L mRNAs to poly(dT) beads (blue figure represents a lysed cell, orange circles depict magnetic beads, black lines depict mRNA strands; see Supplementary Video 1). (d) Beads are recovered and emulsified for cDNA synthesis and linkage PCR to generate an ∼850–base pair V_H:V_L cDNA product (Supplementary Fig. 1). (e) Next-generation sequencing is performed to sequence the linked strands. (f) Bioinformatic processing is used to analyze the paired V_H:V_L repertoire.

Figure 2

V_H:V_L gene family usage of unique CDR-H3:CDR-L3 pairs identified by high-throughput sequencing of cell populations from three different individuals in separate experiments using the workflow in Figure 1. (a) Healthy donor peripheral IgG⁺ B cells (n = 2,716 unique CDR3 pairs). (b) Peripheral TT–specific plasmablasts, isolated 7 d post-TT immunization (CD19⁺CD3⁻CD14⁻CD38⁺⁺CD27⁺⁺CD20⁻TT⁺, n = 86 unique pairs). (c) Peripheral memory B cells isolated 14 d after influenza vaccination (CD19⁺CD3⁻CD27⁺CD38^int, n = 240 unique pairs). Each panel presents data from an independent experiment obtained from 61,000 fresh B cells (a), ∼400 frozen/thawed plasmablasts (b) and 8,000 twice frozen/thawed memory B cells (c). Color indicates percentage of unique pairs from the sample; repertoire statistics are reported in Supplementary Tables 1 and 4.