FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires

Abstract

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5′ RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5′ RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, 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.

IGHV allele similarity clustering improves genotype inference from adaptive immune receptor repertoire sequencing data

“…We validated our ASC-based genotyping method using a paired dataset drawn from six subjects, comprising full length AIRR-seq repertoire sequencing of IGM from RNA isolated from PBMCs, and haplotype-partitioned assemblies of the genomic IGHV locus derived from long-read sequencing ( 29 ). Across the six subjects (Figure 5 ), a total of 304 ASC allele calls were made from the AIRR-seq repertoires, counting the inference of a single allele in a single individual as an allele call.…”

“…Long-read assemblies from six samples generated using Sequel IIe HiFi reads and IGenotyper v1.0.0 ( 4 ) were used to validate the ASC-based genotype approach. The samples are those described previously ( 28 , 29 ). The assemblies were aligned to a custom immunoglobulin heavy chain (IGH) genome reference containing previously discovered IGHV genes using BLASR ( 4 , 30 ).…”

De novo antibody discovery in human blood from full-length single B cell transcriptomics and matching haplotyped-resolved germline assemblies

“…Our focus in this study was to describe the antibody repertoire using a combination of singlecell, full-length transcriptomics and a haplotyped long-read assembly of the IGH and IGL loci. Bulk adaptive immune receptor repertoire (AIRR-seq (Ford et al 2023)) data has been paired with matching IGH genome assemblies before, but those genomes were generated from targeted sequencing approaches, which result in shorter reads and more discontiguous assemblies (mean # of contigs > 100) (Rodriguez et al 2023). In contrast, our donor's IGH locus was assembled into a single contig and phase block, showing that highly contiguous and fully-phased IGH assemblies can be created and reliably annotated from standard long-read whole genome sequencing data.…”

Impact of structural modifications of IgG antibodies on effector functions

“…Recently, 28 novel variant sites in IGHG1-3 alleles were discovered in seven genetically isolated Brazilian population ( 76 ). In addition, 11 novel alleles and 17 extensions of known IGHG and IGHM alleles were observed in a study using a novel sequencing technique ( 80 ). This demonstrates that the diversity of the IGHG genomic region is still far from being fully characterized in whole genome sequencing databases ( 81 ).…”