Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F17%3A00100344" target="_blank" >RIV/00216224:14740/17:00100344 - isvavai.cz</a>

Result on the web

<a href="https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-017-3815-2?site=bmcgenomics.biomedcentral.com" target="_blank" >https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-017-3815-2?site=bmcgenomics.biomedcentral.com</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1186/s12864-017-3815-2" target="_blank" >10.1186/s12864-017-3815-2</a>

Result language

angličtina

Original language name

Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries

Original language description

Background: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps. Results: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification. Conclusion: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10603 - Genetics and heredity (medical genetics to be 3)

Project

<a href="/en/project/LQ1601" target="_blank" >LQ1601: CEITEC 2020</a><br>

Continuities

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

Publication year

2017

Confidentiality

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

Name of the periodical

BMC Genomics

ISSN

1471-2164

e-ISSN

—

Volume of the periodical

18

Issue of the periodical within the volume

JUN

Country of publishing house

GB - UNITED KINGDOM

Number of pages

11

Pages from-to

440

UT code for WoS article

000404077700008

EID of the result in the Scopus database

2-s2.0-85020216758