NAb-seq: an accurate, rapid, and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells

Abstract

Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional loss of antibodies with loss of cell lines. Hybridoma cell lines have been the primary means of generating monoclonal antibodies from immunized animals, including mice, rats, rabbits, and alpacas. Excluding therapeutic antibodies, few hybridoma-derived antibody sequences are known. Sanger sequencing has been "the gold standard" for antibody gene sequencing, but this method relies on the availability of species-specific degenerate primer sets for amplification of light and heavy antibody genes and it requires lengthy and expensive cDNA preparation. Here, we leveraged recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing to develop Nanopore Antibody sequencing (NAb-seq): a three-day, species-independent, and cost-effective workflow to characterize paired full-length immunoglobulin light- and heavy-chain genes from hybridoma cell lines. When compared to Sanger sequencing of two hybridoma cell lines, long-read ONT sequencing was highly accurate, reliable, and amenable to high throughput. We further show that the method is applicable to single cells, allowing efficient antibody discovery in rare populations such as memory B cells. In summary, NAb-seq promises to accelerate identification and validation of hybridoma antibodies as well as antibodies from single B cells used in research, diagnostics, and therapeutics.

Keywords: Antibody sequencing; B cell; hybridoma; long-read; nanopore sequencing; rat B cell cloning; single cell; workflow.

Figure 1.

NAb-seq workflow for parallel sequencing of full-length antibody heavy and light chain sequences from hybridoma cell lines and single B cells.

Figure 2.

NAb-seq of two hybridoma cell lines revealed antibody sequences. (a) cDNA library size and amplification. Total RNA from two hybridoma cell lines was extracted and converted to cDNA followed by amplification and barcoding, generating whole transcriptome cDNA libraries A and B. (b) Basecalling in Guppy’s super-high accuracy mode yielded approximately 0.5 million total pass reads. (c) Read length of pass reads varied from ~400 bp to ~3000 bp. (d) Sequence analysis of cDNA libraries A and B reveals V(D)J recombination, C gene usage and complementarity-determining region 3 (CDR3) amino acid sequence. Consensus calling of antibody transcripts revealed IgG isotype.

Figure 3.

Antibody sequences from libraries A and B align with 100% accuracy to the 7D10 and 3C10 sequences. (a) Schematic of alignment of 7D10 and 3C10 antibody chains, as derived from Sanger (top row) and Nanopore (bottom row) sequencing methods. Additional bases present in the Nanopore sequence have been annotated with BLAST as indicated. Short sequences at the beginning of reads (gray) were sometimes unable to be annotated with BLAST, nor did they match the primer sequences used during library preparation. (b) Sequence alignment of the light chain sequence of 3C10 as derived from Sanger (top row) and Nanopore (bottom row) sequencing methods. Additional bases present in the Nanopore sequence have been annotated with BLAST. Regions highlighted as in Figure 3a. See Supplementary Figures 1–3 for other chains of 7D10 and 3C10.

Figure 4.

NAb-seq of four single B cells revealed antibody sequences. (a) cDNA library size and amplification. Total RNA from four sorted single B cells were extracted and converted to whole transcriptome cDNA libraries I–IV. (b) Basecalling in Guppy’s super-high accuracy mode yielded approximately 5 million total pass reads. (c) Read length of pass reads varied from ~400 bp to ~5000 bp. (d) Sequence analysis of cDNA libraries I to IV reveals V(D)J recombination, C gene usage and complementarity-determining region 3 (CDR3) amino acid sequence. Consensus calling of antibody transcripts revealed IgM and IgD isotypes.