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, 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.

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

• Original language: English
• Type of outcome: Article in a journal
• Field of Study: 10603 Genetics and heredity
• Country of publisher: United Kingdom of Great Britain and Northern Ireland
• Confidentiality degree: is not subject to a state or trade secret
• WWW: 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
• Tags: OA, rivok
• Tags: International impact, Reviewed

Abstract

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)