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

J 2017

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

SHAGIN, D.A., M.A. TURCHANINOVA, I.A. SHAGINA, Mikhail SHUGAY, A.R. ZARETSKY et. al.

Basic information

Original name

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

Authors

SHAGIN, D.A. (643 Russian Federation), M.A. TURCHANINOVA (643 Russian Federation), I.A. SHAGINA (643 Russian Federation), Mikhail SHUGAY (643 Russian Federation, belonging to the institution), A.R. ZARETSKY (643 Russian Federation), O.I. ZUEVA (643 Russian Federation), D.A. BOLOTIN (643 Russian Federation), S. LUKYANOV (643 Russian Federation) and Dmitriy CHUDAKOV (643 Russian Federation, guarantor, belonging to the institution)

Edition

BMC Genomics, London, BioMed Central Ltd, 2017, 1471-2164

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10603 Genetics and heredity

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 3.730

RIV identification code

RIV/00216224:14740/17:00100344

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.1186/s12864-017-3815-2

UT WoS

000404077700008

Keywords in English

High-throughput sequencing; Unique molecular identifiers; Targeted resequencing; PCR

Abstract

V originále

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.

Links

LQ1601, research and development project

Name: CEITEC 2020 (Acronym: CEITEC2020)

Investor: Ministry of Education, Youth and Sports of the CR

633592, interní kód MU

Name: APERIM - Advanced bioinformatics platform for PERsonalised cancer IMmunotherapy (Acronym: APERIM)

Investor: European Union, APERIM - Advanced bioinformatics platform for PERsonalised cancer IMmunotherapy, Health, demographic change and wellbeing (Societal Challenges)

Citovat

SHAGIN, D.A., M.A. TURCHANINOVA, I.A. SHAGINA, Mikhail SHUGAY, A.R. ZARETSKY, O.I. ZUEVA, D.A. BOLOTIN, S. LUKYANOV and Dmitriy CHUDAKOV. Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries. BMC Genomics. London: BioMed Central Ltd, 2017, vol. 18, JUN, p. 440-450. ISSN 1471-2164. Available from: https://dx.doi.org/10.1186/s12864-017-3815-2.

@article{1411050,
   author = {Shagin, D.A. and Turchaninova, M.A. and Shagina, I.A. and Shugay, Mikhail and Zaretsky, A.R. and Zueva, O.I. and Bolotin, D.A. and Lukyanov, S. and Chudakov, Dmitriy},
   article_location = {London},
   article_number = {JUN},
   doi = {http://dx.doi.org/10.1186/s12864-017-3815-2},
   keywords = {High-throughput sequencing; Unique molecular identifiers; Targeted resequencing; PCR},
   language = {eng},
   issn = {1471-2164},
   journal = {BMC Genomics},
   title = {Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries},
   url = {https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-017-3815-2?site=bmcgenomics.biomedcentral.com},
   volume = {18},
   year = {2017}
}

TY  - JOUR
ID  - 1411050
AU  - Shagin, D.A. - Turchaninova, M.A. - Shagina, I.A. - Shugay, Mikhail - Zaretsky, A.R. - Zueva, O.I. - Bolotin, D.A. - Lukyanov, S. - Chudakov, Dmitriy
PY  - 2017
TI  - Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries
JF  - BMC Genomics
VL  - 18
IS  - JUN
SP  - 440
EP  - 440
PB  - BioMed Central Ltd
SN  - 14712164
KW  - High-throughput sequencing
KW  - Unique molecular identifiers
KW  - Targeted resequencing
KW  - PCR
UR  - https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-017-3815-2?site=bmcgenomics.biomedcentral.com
L2  - https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-017-3815-2?site=bmcgenomics.biomedcentral.com
N2  - 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.
ER  -

SHAGIN, D.A., M.A. TURCHANINOVA, I.A. SHAGINA, Mikhail SHUGAY, A.R. ZARETSKY, O.I. ZUEVA, D.A. BOLOTIN, S. LUKYANOV and Dmitriy CHUDAKOV. Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries. \textit{BMC Genomics}. London: BioMed Central Ltd, 2017, vol.~18, JUN, p.~440-450. ISSN~1471-2164. Available from: https://dx.doi.org/10.1186/s12864-017-3815-2.

Detailed Information on Publication Record