FLAIRR-seq: A novel method for single molecule resolution of near full-length immunoglobulin heavy chain repertoires

Current Adaptive Immune Receptor Repertoire Sequencing (AIRR-seq) strategies resolve immunoglobulin (IG) variable region transcripts with limited resolution of the constant region. Here we present a novel near full-length AIRR-seq (FLAIRR-Seq) method that utilizes targeted heavy chain amplification by rapid amplification of cDNA ends (RACE), combined with single molecule, real-time (SMRT) sequencing to generate highly accurate (>Q40, 99.99%), immunoglobulin heavy chain transcripts. FLAIRR-seq was benchmarked by comparing V, D, and J gene usage, complementarity-determining region 3 (CDR3) length, and the extent of total variable gene somatic hypermutation to matched datasets generated with standard RACE AIRR-seq and full-length isoform sequencing methods. Together these data demonstrate robust, unbiased FLAIRR-seq performance using RNA samples derived from peripheral blood mononuclear cells, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while resolving novel constant region features. FLAIRR-seq data provides, for the first time, simultaneous, single-molecule characterization of variable and constant region genes and alleles, allele-resolved subisotype definition, and identification of class-switch recombination within a clonal lineage. FLAIRR-seq, in conjunction with IGHC genotyping, of the IgM- and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles; 28/32 (87%) that were not represented by sequences curated in the IMmunoGeneTics Information System (IMGT) database. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, D, J and C gene diversity for the most comprehensive view of B cell repertoires to date. ### Competing Interest Statement The authors have declared no competing interest.