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

Area of Science:

• Immunology
• Genomics
• Molecular Biology

Background:

• Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) methods using short-read sequencing offer limited resolution of antibody (Ab) C regions.
• Accurate characterization of Ab repertoires is crucial for understanding immune responses and developing targeted therapies.

Purpose of the Study:

• To introduce and validate a novel near-full-length AIRR-seq (FLAIRR-seq) method for highly accurate human Ab H chain transcript sequencing.
• To demonstrate FLAIRR-seq's capability in resolving previously undocumented genetic features and class switch recombination events.

Main Methods:

• FLAIRR-seq employs targeted amplification by 5' RACE combined with single-molecule, real-time sequencing.
• Method was benchmarked against standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing.
• RNA samples from PBMCs, purified B cells, and whole blood were utilized.

Main Results:

• FLAIRR-seq generated highly accurate (99.99%) human Ab H chain transcripts, resolving V (IGHV), D (IGHD), and J (IGHJ) gene usage, CDR3 length, and somatic hypermutation.
• Simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC regions, including allele-resolved subisotype definition and class switch recombination, was achieved.
• Analysis of IgM and IgG repertoires identified 32 unique IGHC alleles, with 28 (87%) being previously uncharacterized.

Conclusions:

• FLAIRR-seq offers a comprehensive view of bulk-expressed Ab repertoires by characterizing IGHV, IGHD, IGHJ, and IGHC gene diversity.
• The method enables high-resolution identification of class switch recombination within clonal lineages.
• FLAIRR-seq advances the study of immune receptor repertoire diversity and has implications for immunological research and therapeutic development.