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High-quality full-length immunoglobulin profiling with unique molecular barcoding
MA. Turchaninova, A. Davydov, OV. Britanova, M. Shugay, V. Bikos, ES. Egorov, VI. Kirgizova, EM. Merzlyak, DB. Staroverov, DA. Bolotin, IZ. Mamedov, M. Izraelson, MD. Logacheva, O. Kladova, K. Plevova, S. Pospisilova, DM. Chudakov,
Language English Country England, Great Britain
Document type Journal Article
Grant support
NV15-30015A
MZ0
CEP Register
Digital library NLK
Full text - Article
Source
NLK
ProQuest Central
from 2006-06-01 to 1 year ago
Health & Medicine (ProQuest)
from 2006-06-01 to 1 year ago
- MeSH
- Immunoglobulin Light Chains genetics MeSH
- Humans MeSH
- Mutation MeSH
- Mice MeSH
- Quality Control MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA methods MeSH
- Immunoglobulin Heavy Chains genetics MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
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.
Center of Molecular Medicine CEITEC Masaryk University Brno Czech Republic
Pirogov Russian National Research Medical University Moscow Russia
Shemiakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Science Moscow Russia
References provided by Crossref.org
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