Single‐cell based high‐throughput sequencing of full‐length immunoglobulin heavy and light chain genes

Abstract

Single-cell PCR and sequencing of full-length Ig heavy (Igh) and Igk and Igl light chain genes is a powerful tool to measure the diversity of antibody repertoires and allows the functional assessment of B-cell responses through direct Ig gene cloning and the generation of recombinant mAbs. However, the current methodology is not high-throughput compatible. Here we developed a two-dimensional bar-coded primer matrix to combineKeywords: Antibody repertoire r B cell r Index cell sorting Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionAntibodies are B-cell antigen receptors encoded by two independent Ig heavy (Igh) and light (Igk or Igl) chain genes. Therefore, the accurate assessment of antibody repertoires and clonal relationships between individual B cells requires the combined analysis Correspondence: Hedda Wardemann e-mail: wardemann@mpiib-berlin.mpg.de of Igh and Igk/Igl genes from the same cell. Conventional singlecell (sc) based antibody repertoire studies use Sanger sequencing of Igh and corresponding Igk/Igl PCR products to assess V(D)J gene usage, levels of somatic hypermutations, and B-cell clonality [1][2][3][4][5]. This process is highly efficient, unbiased, and has been widely used to assess antibody repertoires in health and disease in mice and humans. Available single-cell PCR (scPCR) protocols provide full-length gene information and are compatible with the direct cloning of Igh and corresponding Igk/Igl genes [3,4]. This facilitates the production of recombinant mAbs, which have beenwww.eji-journal.eu 598Christian E. Busse et al. Eur. J. Immunol. 2014. 44: 597-603 used to study B-cell repertoires based on Ig reactivity and function. Here we combined the scPCR-based amplification of Igh and Igk/Igl transcripts and NGS into a unified pipeline that allows the high-throughput antibody repertoire analysis of thousands of single isolated B cells. The platform is scalable and provides highquality sequence information by consensus building of multiple reads originating from the same cell. It integrates sequencing and flow cytometry data obtained by indexed cell sorting into a single data set and thereby allows for the retrospective phenotypic characterization of B cells according to their antibody genotype. The strategy provides full-length Ig gene sequences of corresponding Igh and Igk/Igl genes and is therefore ideally suited for phylogenetic B-cell repertoire analyses and fully compatible with established procedures for antibody cloning and expression [3]. Importantly, the tag matrix can be used universally to barcode primer sets for the single-cell-based amplification of Ig genes from any species including humans [4]. Results Primer matrix designThe aim of our experimental strategy was to combine the advantages of scPCR and NGS to facilitate the massive parallel analysis of B-cell antibody repertoires (Fig. 1). To perform high-throughput parallel NGS of pooled PCR products without loss of Igh and Igk/Igl linkage, we desig...

Identifying functional anti-Staphylococcus aureus antibodies by sequencing antibody repertoires of patient plasmablasts

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Clonal Evolution of Autoreactive Germinal Centers

“…Complementary DNA was generated using M-MLV Reverse Transcriptase (ThermoFisher Scientific) at standard conditions. Amplification of V(D)J segments was performed using consensus primers based on Tiller et al, and Busse et al, (see also Table S1) and GoTaq DNA polymerase (Promega) under the conditions described herein (Busse et al, 2013; Tiller et al, 2009). Amplicons were TOPO-TA cloned into the pCR2.1-TOPO vector and used for transformation of DH5α, which were subsequently plated on LB-agar containing kanamycin, X-gal and IPTG.…”

Sequencing and cloning of antigen-specific antibodies from mouse memory B cells

“…The major limitations of these methods are loss of heavy-and light-chain pairing at the cellular level, short read lengths and incompatibility for further cloning of antibodies. To overcome some of these shortcomings, Busse et al 21 used a barcoded primer matrix to combine single mouse B cell heavy-and light-chain gene amplification with high-throughput sequencing. Moreover, DeKosky et al 22 described a microfluidic system and overlap extension PCR to sequence activated human memory B cells.…”