RT Journal Article
SR Electronic
T1 FLAIRR-seq: A novel method for single molecule resolution of near full-length immunoglobulin heavy chain repertoires
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.09.24.509352
DO 10.1101/2022.09.24.509352
A1 Easton E. Ford
A1 David Tieri
A1 Oscar Rodriguez
A1 Nancy Francoeur
A1 Juan Soto
A1 Justin Kos
A1 Ayelet Peres
A1 William Gibson
A1 Catherine A. Silver
A1 Gintaras Deikus
A1 Elizabeth Hudson
A1 Cassandra R. Woolley
A1 Noam Beckmann
A1 Alexander Charney
A1 Thomas C. Mitchell
A1 Gur Yaari
A1 Robert P. Sebra
A1 Corey T. Watson
A1 Melissa L. Smith
YR 2022
UL http://biorxiv.org/content/early/2022/11/09/2022.09.24.509352.abstract
AB Current Adaptive Immune Receptor Repertoire Sequencing (AIRR-seq) strategies resolve expressed antibody (Ab) transcripts with limited resolution of the constant region. Here we present a novel near full-length AIRR-seq (FLAIRR-Seq) method that utilizes targeted amplification by 5’ rapid amplification of cDNA ends (RACE), combined with single molecule, real-time sequencing to generate highly accurate (>Q40, 99.99%) IG heavy chain transcripts. FLAIRR-seq was benchmarked by comparing IG heavy chain variable (IGHV), diversity (IGHD), and joining (IGHJ) gene usage, complementarity-determining region 3 (CDR3) length, and somatic hypermutation to matched datasets generated with standard 5’ RACE AIRR-seq and full-length isoform sequencing. 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 additionally resolving novel IG heavy chain constant (IGHC) gene features. FLAIRR-seq data provides, for the first time, simultaneous, single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class-switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk expressed Ab repertoires to date.Competing Interest StatementThe authors have declared no competing interest.