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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F16%3A00088911" target="_blank" >RIV/00216224:14740/16:00088911 - isvavai.cz</a>

Výsledek na webu

<a href="http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.093.html#access" target="_blank" >http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.093.html#access</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1038/nprot.2016.093" target="_blank" >10.1038/nprot.2016.093</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

CE - Biochemie

OECD FORD obor

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Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2016

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

NATURE PROTOCOLS

ISSN

1754-2189

e-ISSN

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Svazek periodika

11

Číslo periodika v rámci svazku

9

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

18

Strana od-do

1599-1616

Kód UT WoS článku

000382164800004

EID výsledku v databázi Scopus

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