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Quantification of rare somatic single nucleotide variants by droplet digital PCR using SuperSelective primers

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dc.contributor.author Pablo Fontecha, Verónica
dc.contributor.author Hernández-Illán, Eva
dc.contributor.author Reparaz, Andrea
dc.contributor.author Asensio Juan, Elena
dc.contributor.author Morata, Jordi
dc.contributor.author Tonda, Raúl
dc.contributor.author Lahoz, Sara
dc.contributor.author Parra, Carolina
dc.contributor.author Lozano, Juan José
dc.contributor.author García-Heredia, Anabel
dc.contributor.author Martínez-Roca, Alejandro
dc.contributor.author Beltran, Sergi
dc.contributor.author Balaguer, Francesc
dc.contributor.author Jover, Rodrigo
dc.contributor.author Castells, Antoni
dc.contributor.author Trullàs, Ramón
dc.contributor.author Podlesniy, Petar
dc.contributor.author Camps-Puchadas, Jordi
dc.date.accessioned 2024-02-12T06:36:43Z
dc.date.available 2024-02-12T06:36:43Z
dc.date.issued 2023
dc.identifier.citation Pablo-Fontecha V, Hernández-Illán E, Reparaz A, Asensio E, Morata J, Tonda R, Lahoz S, Parra C, Lozano JJ, García-Heredia A, Martínez-Roca A, Beltran S, Balaguer F, Jover R, Castells A, Trullàs R, Podlesniy P, Camps J. Quantification of rare somatic single nucleotide variants by droplet digital PCR using SuperSelective primers. Sci Rep. 2023 Nov 3;13(1):18997. DOI: 10.1038/s41598-023-39874-0
dc.identifier.issn 2045-2322
dc.identifier.uri http://hdl.handle.net/10230/59059
dc.description.abstract Somatic single-nucleotide variants (SNVs) occur every time a cell divides, appearing even in healthy tissues at low frequencies. These mutations may accumulate as neutral variants during aging, or eventually, promote the development of neoplasia. Here, we present the SP-ddPCR, a droplet digital PCR (ddPCR) based approach that utilizes customized SuperSelective primers aiming at quantifying the proportion of rare SNVs. For that purpose, we selected five potentially pathogenic variants identified by whole-exome sequencing (WES) occurring at low variant allele frequency (VAF) in at-risk colon healthy mucosa of patients diagnosed with colorectal cancer or advanced adenoma. Additionally, two APC SNVs detected in two cancer lesions were added to the study for WES-VAF validation. SuperSelective primers were designed to quantify SNVs at low VAFs both in silico and in clinical samples. In addition to the two APC SNVs in colonic lesions, SP-ddPCR confirmed the presence of three out of five selected SNVs in the normal colonic mucosa with allelic frequencies ≤ 5%. Moreover, SP-ddPCR showed the presence of two potentially pathogenic variants in the distal normal mucosa of patients with colorectal carcinoma. In summary, SP-ddPCR offers a rapid and feasible methodology to validate next-generation sequencing data and accurately quantify rare SNVs, thus providing a potential tool for diagnosis and stratification of at-risk patients based on their mutational profiling.
dc.description.sponsorship This research was funded by grants from the Instituto de Salud Carlos III and co-funded by the European Regional Development Fund (ERDF) (CPII18/00026, PI17/01304, PI20/00863), the CIBEREHD and CIBERNED programs from Instituto de Salud Carlos III, the Agència de Gestió d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya (2017 SGR 1035), and Fundación Científica de la Asociación Española Contra el Cáncer (GCB13131592CAST) and grant PID2020-115091RB-I00 funded by MCIN/AEI/https://doi.org/10.13039/501100011033. This article is based upon work from COST Action CA17118, supported by COST (European Cooperation in Science and Technology).
dc.format.mimetype application/pdf
dc.language.iso eng
dc.publisher Nature Research
dc.relation.ispartof Sci Rep. 2023 Nov 3;13(1):18997
dc.rights © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
dc.rights.uri http://creativecommons.org/licenses/by/4.0/
dc.title Quantification of rare somatic single nucleotide variants by droplet digital PCR using SuperSelective primers
dc.type info:eu-repo/semantics/article
dc.identifier.doi http://dx.doi.org/10.1038/s41598-023-39874-0
dc.subject.keyword Colorectal cancer
dc.subject.keyword DNA sequencing
dc.subject.keyword Genotyping and haplotyping
dc.subject.keyword Next-generation sequencing
dc.subject.keyword PCR-based techniques
dc.relation.projectID info:eu-repo/grantAgreement/ES/2PE/PID2020-115091RB-I00
dc.rights.accessRights info:eu-repo/semantics/openAccess
dc.type.version info:eu-repo/semantics/publishedVersion

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