![]() ![]() We devised a computational pipeline for cell detection, de novo contig assembly, variable domain annotation, and pairing of full-length VH and VL sequences (Fig. This resulted in sequencing reads with constant 5′ sequence and variable 3′ sequence, allowing de novo assembly of full-length VH and VL sequences from short-read data (2 × 150 bp). The library construction method involves 3′ cDNA shearing after amplification to create a set of fragments with variable 3′ end, while retaining the 5′ end for all fragments. The 5′ barcoded VH and VL domain-encoding cDNAs were sheared and converted into sequencing-ready libraries by addition of appropriate adapter oligonucleotides (Supplementary Fig. Briefly, we generated 5′ barcoded cDNA from thousands of individual B cells in parallel, and amplified the VH and VL regions using custom primers while retaining the cell barcode (Fig. We analyzed >250,000 individual IgG pos B cells from three human donors and two mice, and IgM neg B cells from two rats using emulsion-based encapsulation, cDNA generation and sequencing. High-throughput single-cell B-cell receptor sequencing We demonstrate the potential of direct sequencing of individual antigen-reactive B cells to rapidly generate a large and diverse panel of antigen-specific antibody variable regions and thus expand immune repertoire sampling and expedite antibody discovery processes. Further, we show the utility of the technique for antibody discovery by expressing and testing predicted antigen-reactive antibody sequences. We developed a bioinformatics framework to analyze the sequence data and identify accurate VH and VL pairing. Here we describe the application of high-throughput single-cell sequencing to obtain the VH and VL sequences for antibodies from individual human, rat, and mouse B cells. High-throughput approaches that yield full-length variable regions for individual B cells at scale would enable routine application of large-scale immune repertoire sequencing to antibody discovery and detailed repertoire characterization. However, these techniques only infer full-length variable region sequences indirectly, and single-cell information is lost during library construction. Recently, techniques that isolate individual cells in microwell plates or droplets of an emulsion, followed by physical linking of VH and VL regions through overlap extension RT-PCR, have demonstrated the potential for obtaining VH–VL pairing information in a high-throughput manner 5, 6, 7. Until recently, the majority of high-throughput sequencing approaches produced unpaired VH and VL repertoires, as generating paired information requires obtaining data at the individual cell level 4. Next-generation sequencing has been applied to understand the diversity of the variable regions of heavy (VH) and light chains (VL) that determine the antigen specificity of antibodies. The heavy and light chains are encoded in separate gene loci, and each B cell normally expresses a single functional heavy and light chain sequence. A mature antibody consists of two identical heavy chains linked through disulphide bonds and two identical light chains each linked to one of the heavy chains, generating two identical antigen-binding sites formed by the first immunoglobulin domain of each chain pair 2. Somatic hypermutation (SHM) and class switching add to antibody diversity. B cells produce a diverse array of antibodies by rearranging variable, diversity, and joining immunoglobulin germline gene segments 1, 2, 3. Our results establish scBCR-seq as a powerful tool for antibody discovery.Īntibody diversity is an important feature of the adaptive immune system. ![]() We synthesized, expressed and tested 93 clones from the identified lineages and found that 99% ( n = 92/93) of the clones were antigen-reactive. ![]() Importantly, scBCR-seq identified an additional 710 candidate lineages not recovered as hybridomas. 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. In addition, we immunized rats with chicken ovalbumin and profiled antigen-reactive B cells from lymph nodes of immunized animals. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion. 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. Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem. ![]()
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