Massively parallel single-cell B-cell receptor sequencing enables rapid discovery of diverse antigen-reactive antibodies

Leonard D Goldstein,Aju Antony,Dhaya Seshasayee,Jia Wu,Bijay S Jaiswal,Steve Guerrero,Kam Hon Hoi,Kanika Bajaj Pahuja,Jeremy Stinson,Subhra Chaudhuri,Kellen Schneider,Ying-Jiun J Chen,Zora Modrusan,Somasekar Seshagiri,Zhonghua Lin,Isidro Hötzel,Yi-Chun Hsiao

doi:10.1038/s42003-019-0551-y

Leonard D Goldstein, Aju Antony + Show 15 more

Open Access

https://doi.org/10.1038/s42003-019-0551-y

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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.

Highlights

Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem
Assembled variable regions of heavy (VH) and variable germline gene segment (VL) sequences were parsed for framework regions and complementarity-determining regions (CDR) and selected for open reading frames encoding the entire variable region
Techniques that isolate single cells in microwell plates or droplets of an emulsion, followed by physical linking of VH and VL regions through overlap extension RTPCR, have demonstrated the potential for obtaining VH–VL pairing information in a high-throughput manner[5,6,7]. Such techniques require custom equipment and do not yield fulllength variable region sequences or single-cell information. They allow chain pairing by paired-end sequencing of VH and VL sequences that are physically linked in a single amplicon

Summary

Introduction

Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem. 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 RTPCR, have demonstrated the potential for obtaining VH–VL pairing information in a high-throughput manner[5,6,7]. These techniques only infer full-length variable region sequences indirectly, and single-cell information is lost during library construction. We demonstrate the potential of direct sequencing of individual antigen-reactive B cells to rapidly generate a large and diverse panel of antigenspecific antibody variable regions and expand immune repertoire sampling and expedite antibody discovery processes

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