In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire

Abstract

High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the >1 × 10(6) B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 10(6) B cells per experiment with demonstrated pairing precision >97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or 'public', VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza.

Immunoglobulin gene analysis as a tool for investigating human immune responses

“…Paired end data can also be limited in ability to distinguish some somatic variants 79. As such, the Pacific Biosciences (PacBio) RSII system which offers reads lengths of 10 000 bp on average has become increasingly attractive for specialized applications80 despite its comparatively poor reads per run and high cost (see Table 1). The use of barcodes, a string of known nucleotides added to individual samples by using multiple specifically produced primers, allows simple multiplexing on higher cost sequencing platforms but is currently still expensive.…”

“…Microfluidic equipment for this “DropSeq” method has been bespoke in a number of labs, although there is now a commercially available system from Dolomite Bio suitable for this application. These single‐cell microfluidic methods rely on a PCR that is simple in concept, joining the heavy and light chain transcripts by over‐lap extension, but difficult in practice given the large number of primers in a single approximately 65 pico‐liter emulsion droplet 79, 80, 91. The joining of both the heavy and the light chain resulting in an amplicon that may be in excess of 1000 bp has made it a prime candidate for long read sequencing technologies.…”

Antibodyomics: bioinformatics technologies for understanding B‐cell immunity to HIV‐1

“…We utilize a technology that links V H and V L sequences prior to high‐throughput sequencing 52. The ability to analyze in high throughput both heavy and light chain sequences permits deconvolution of the ontogeny of HIV broadly neutralizing antibodies and rapid discovery of new antibody variants, both in the context of HIV/SHIV infections and in response to vaccination.…”

“…5A). This method links V H and V L sequences by using overlap extension RT‐PCR prior to NGS, providing the capability to analyze up to 10 million B cells in a single analysis 52, 54, 55…”

“…We have also applied Antibodyomics5 to the quantification of multidonor class antibodies that neutralize both group 1 and group 2 influenza A viruses 16. We delineated antibody sequence signatures and quantified antibodies that satisfy these sequence signatures in NGS datasets from multiple donors with different vaccination histories 52, 68. Notably, one vaccination regimen from the VRC 310 trial involving immunization with a diverse H5 hemagglutinin increased the frequency of two multidonor classes by over 100‐fold,16 despite titers too low to detect in sera.…”