RNA-Based High-Throughput Sequencing of the Human Immunoglobulin Repertoire.

Lymphocytes use somatic diversification processes to express a wide variability of antigen receptors, generating a highly diversified repertoire that is unique to each individual. The study of these repertoires is now possible with the advent of next-generation sequencing (NGS) techniques. Here we describe the "RACE Rep-Seq" methodology for high-throughput sequencing of immunoglobulin (Ig) repertoires using RNA templates. The preparation of libraries is based on the conversion of RNA samples into cDNA using the 5'RACE (Rapid Amplification of cDNA Ends) technique and a set of primers specific for the constant domains of the heavy (Cμ, Cγ, Cα) and light (Cκ, Cλ) chains, thus targeting almost the entire Ig repertoire. During this reverse transcription step, unique molecular identifiers (UMIs) are incorporated at the 5' ends to correct PCR amplification biases and sequencing errors. An initial amplification step is then performed using nested primers specific for the heavy and light constant domains to increase the specificity of the amplification of Ig transcripts. This is followed by two sequential PCRs required for paired-end sequencing using asymmetric paired-end Illumina sequencing to obtain sequences of up to 750 base pairs (bp). This length covers the entire Ig variable domain and part of the constant region required for isotype determination. Finally, the raw sequencing data is processed and analyzed to provide a complete Ig repertoire, enabling significant advances in both basic immunology and various clinical applications.