High-quality full-length immunoglobulin profiling with unique molecular barcoding

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Authors

TURCHANINOVA M. A. DAVYDOV Alexey Nikolayevich BRITANOVA O. V. SHUGAY Mikhail BIKOS Vasileios EGOROV Evgeny KIRGIZOVA V. I. MERZLYAK E. M. STAROVEROV D. B. BOLOTIN D. A. MAMEDOV Ilgar IZRAELSON Mark LOGACHEVA M. D. KLADOVA O. PLEVOVÁ Karla POSPÍŠILOVÁ Šárka CHUDAKOV Dmitriy

Year of publication 2016
Type Article in Periodical
Magazine / Source NATURE PROTOCOLS
MU Faculty or unit

Central European Institute of Technology

Citation
Web http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.093.html#access
Doi http://dx.doi.org/10.1038/nprot.2016.093
Field Biochemistry
Keywords ZEBRAFISH ANTIBODY REPERTOIRE; B-CELL REPERTOIRE; RHEUMATOID-ARTHRITIS; IMMUNE REPERTOIRES; MULTIPLEX PCR; MEMORY; VACCINATION; RESPONSES; REVEALS; DEEP
Description 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.
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