{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T21:44:31Z","timestamp":1790372671914,"version":"4.1.0"},"reference-count":27,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2020,5,19]],"date-time":"2020-05-19T00:00:00Z","timestamp":1589846400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000274","name":"British Heart Foundation","doi-asserted-by":"publisher","award":["RG\/12\/15\/29935"],"award-info":[{"award-number":["RG\/12\/15\/29935"]}],"id":[{"id":"10.13039\/501100000274","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Anatomy"],"published-print":{"date-parts":[[2020,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin\u2010fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil\u2010N\u2010glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of genomic studies using these collections. We describe a novel method of DNA extraction capable of providing PCR amplicons of at least 400\u00a0bp length from such excessively formalin\u2010fixed human tissues. When compared with a leading commercial formalin\u2010fixed DNA extraction kit, our method produced greater yields of DNA and reduced sequence variations. Analysis of PCR products using bacterial sub\u2010cloning and Sanger sequencing from UNG\u2010treated DNA unexpectedly revealed increased sequence variations, compared with untreated samples. Finally, whole exome NGS was performed on a myocardial sample fixed in formalin for 2 years and compared with lymphocyte\u2010derived DNA (as a gold standard) from the same patient. Despite the reduction in the number and quality of reads in the formalin\u2010fixed DNA, we were able to show that bioinformatic processing by joint calling and variant quality score recalibration (VQSR) increased the sensitivity four\u2010fold to 56% and doubled specificity to 68% when compared with a standard hard\u2010filtering approach. Thus, high\u2010quality DNA can be extracted from excessively formalin\u2010fixed tissues and bioinformatic processing can optimise sensitivity and specificity of results. Sequencing of several sub\u2010cloned amplicons is an important methodological step in assessing DNA quality.<\/jats:p>","DOI":"10.1111\/joa.13209","type":"journal-article","created":{"date-parts":[[2020,5,19]],"date-time":"2020-05-19T10:10:25Z","timestamp":1589883025000},"page":"587-600","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Isolation and next generation sequencing of archival formalin\u2010fixed DNA"],"prefix":"10.1111","volume":"237","author":[{"given":"Ahlam","family":"Alqahtani","sequence":"first","affiliation":[{"name":"Bioscience Institute Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew","family":"Skelton","sequence":"additional","affiliation":[{"name":"Bioinformatic Support Unit Faculty of Medical Sciences Newcastle University Newcastle upon Tyne 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