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

Kód výsledku v 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>

Výsledek na webu

<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>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

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

Projekt

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

Návaznosti

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

Rok uplatnění

2017

Kód důvěrnosti údajů

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

Název periodika

BMC Genomics

ISSN

1471-2164

e-ISSN

—

Svazek periodika

18

Číslo periodika v rámci svazku

JUN

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

11

Strana od-do

440

Kód UT WoS článku

000404077700008

EID výsledku v databázi Scopus

2-s2.0-85020216758