Deep profiling of antibody and T cell-receptor repertoires by means of high-throughput sequencing has become an attractive approach for adaptive immunity studies, but its power is substantially compromised by the accumulation of PCR and sequencing errors. Here we report MIGEC (molecular identifier groups-based error correction), a strategy for high-throughput sequencing data analysis. MIGEC allows for nearly absolute error correction while fully preserving the natural diversity of complex immune repertoires.

High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.

2016) Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucilbased chemotherapy or chemoimmunotherapy. Blood, 128, 395-404.