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However, the relationship between the population of DNA fragments within a sample and the likelihood they will amplify has not been fully described. To address this, we have derived a mathematical equation that relates the distribution profile of a stochastically fragmented DNA sample to the probability that a DNA fragment within that sample can be amplified by any PCR assay of arbitrary length. Two panels of multiplex PCR assays for quantifying fragmented DNA were then developed: a four-plex panel that can be applied to any human DNA sample and used to estimate the percentage of regions that are intact at any length; and a two-plex panel optimized for quantifying circulating cell-free DNA (cfDNA). For these assays, regions of the human genome least affected by copy number aberration were identified and selected; within these copy-neutral regions, each PCR assay was designed to amplify both genomic and bisulfite-converted DNA; and all assays were validated for use in both conventional qPCR and droplet-digital PCR. Finally, using the cfDNA-optimized assays we find evidence of universally conserved nucleosome positioning among individuals.<\/jats:p>","DOI":"10.1038\/s41598-022-18196-7","type":"journal-article","created":{"date-parts":[[2022,9,26]],"date-time":"2022-09-26T16:03:44Z","timestamp":1664208224000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Modelling clinical DNA fragmentation in the development of universal PCR-based assays for bisulfite-converted, formalin-fixed and cell-free DNA sample analysis"],"prefix":"10.1038","volume":"12","author":[{"given":"Andrew D.","family":"Johnston","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"Lu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Darren","family":"Korbie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matt","family":"Trau","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,9,26]]},"reference":[{"key":"18196_CR1","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1016\/j.ab.2006.07.035","volume":"358","author":"CD Georgiou","year":"2006","unstructured":"Georgiou, C. 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