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Nat Protoc. 2016 Sep;11(9):1599-616. doi: 10.1038/nprot.2016.093. Epub 2016 Aug 4.

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

Author information

1
Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.
2
Center of Molecular Medicine, CEITEC, Masaryk University, Brno, Czech Republic.
3
Pirogov Russian National Research Medical University, Moscow, Russia.

Abstract

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.

PMID:
27490633
DOI:
10.1038/nprot.2016.093
[Indexed for MEDLINE]

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