biostudies-literatureJohnston ADNational Breast Cancer FoundationUniversity of Queensland Early Career Researcher Grant16051https://www.ebi.ac.uk/biostudies/studies/S-EPMC9512909biostudies-literatureUnknown12(1)In fragmented DNA, PCR-based methods quantify the number of intact regions at a specific amplicon length. 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.Scientific reportsModelling clinical DNA fragmentation in the development of universal PCR-based assays for bisulfite-converted, formalin-fixed and cell-free DNA sample analysis.PMC9512909CG-12-07UQECR1720545Trau MJohnston ADLu JKorbie DfalseModelling clinical DNA fragmentation in the development of universal PCR-based assays for bisulfite-converted, formalin-fixed and cell-free DNA sample analysis.In fragmented DNA, PCR-based methods quantify the number of intact regions at a specific amplicon length. 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.2022-01-01T00:00:00Z2022 Sep2026-05-10T06:39:29.519Z2024-12-04T03:45:52.683ZS-EPMC95129093616337210.1038/s41598-022-18196-7