{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T03:02:21Z","timestamp":1760151741794,"version":"build-2065373602"},"reference-count":29,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2022,4,21]],"date-time":"2022-04-21T00:00:00Z","timestamp":1650499200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["20-75-10163"],"award-info":[{"award-number":["20-75-10163"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5\u2032\/3\u2032-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 \u00b1 167\/Mb vs. 75 \u00b1 37\/Mb, paired t-test p-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA.<\/jats:p>","DOI":"10.3390\/ijms23094586","type":"journal-article","created":{"date-parts":[[2022,4,22]],"date-time":"2022-04-22T05:07:22Z","timestamp":1650604042000},"page":"4586","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0179-3191","authenticated-orcid":false,"given":"Natalia V.","family":"Mitiushkina","sequence":"first","affiliation":[{"name":"Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9844-4536","authenticated-orcid":false,"given":"Grigory A.","family":"Yanus","sequence":"additional","affiliation":[{"name":"Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, Russia"},{"name":"Department of Medical Genetics, St.-Petersburg Pediatric Medical University, 194100 St.-Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2396-6540","authenticated-orcid":false,"given":"Ekatherina Sh.","family":"Kuligina","sequence":"additional","affiliation":[{"name":"Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tatiana A.","family":"Laidus","sequence":"additional","affiliation":[{"name":"Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandr A.","family":"Romanko","sequence":"additional","affiliation":[{"name":"Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4201-8124","authenticated-orcid":false,"given":"Maksim M.","family":"Kholmatov","sequence":"additional","affiliation":[{"name":"Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandr O.","family":"Ivantsov","sequence":"additional","affiliation":[{"name":"Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, Russia"},{"name":"Department of Medical Genetics, St.-Petersburg Pediatric Medical University, 194100 St.-Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Svetlana N.","family":"Aleksakhina","sequence":"additional","affiliation":[{"name":"Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4529-7891","authenticated-orcid":false,"given":"Evgeny N.","family":"Imyanitov","sequence":"additional","affiliation":[{"name":"Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, Russia"},{"name":"Department of Medical Genetics, St.-Petersburg Pediatric Medical University, 194100 St.-Petersburg, Russia"},{"name":"Department of Oncology, I.I. Mechnikov North-Western Medical University, 191015 St.-Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"14508","DOI":"10.1073\/pnas.1208715109","article-title":"Detection of ultra-rare mutations by next-generation sequencing","volume":"109","author":"Schmitt","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4810","DOI":"10.1038\/s41598-019-41215-z","article-title":"Targeted Single Primer Enrichment Sequencing with Single End Duplex-UMI","volume":"9","author":"Peng","year":"2019","journal-title":"Sci. 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