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

Abstract

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.

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 single-cell, 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.…”

R2C2+UMI: Combining concatemeric consensus sequencing with unique molecular identifiers enables ultra-accurate sequencing of amplicons on Oxford Nanopore Technologies sequencers

“…For the same full-length rRNA operons, PacBio CCS and UMIs achieved an accuracy of ~Q52 9 . PacBio CCS and UMIs have also been used by FLAIRR-seq which analyzed full-length IGH amplicons but did not empirically determine its method's accuracy 10 . In both cases consensus generation was either done by a multi-step adhoc chain of tools or a likely suboptimal approach designed for Illumina reads.…”

Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling

“…Currently, there are two main high-throughput approaches to sequencing the BCR repertoire, differing by scale and resolution: bulk (bulkBCR-seq) and single-cell (scBCR-seq) sequencing. BulkBCR-seq so far still provides the highest sampling depth for the purpose of covering the diversity of the immune repertoire, with a wide array of established methods [17][18][19][20] . On the contrary, most currently available scBCR-seq methods have 100 to 1000 times lower sampling depth compared to bulkBCR-seq 21 , albeit novel methods are currently being developed to improve throughput.…”