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The current standard for analyzing casework samples is the specific amplification of short tandem repeats (STR), which is limited by DNA quality and quantity. Since the goal of forensic science is to identify a suspect or victim regardless of trace quality, we evaluated three technological approaches to better characterize and exploit these traces: (i) ultra-sensitive pulse-field electrophoresis on a Femto Pulse System (FPS) to visualize DNA content, (ii) real-time quantitative PCR based on\n                    <jats:italic>Alu<\/jats:italic>\n                    repeats to quantify human DNA and analyze its integrity, and (iii) 16S ribosomal RNA gene (16S rRNA) amplicon sequencing to identify microbiota. We optimized FPS analysis using DNA from model traces (blood, saliva, semen, touch DNA, and vaginal swabs) and applied the protocol to 100 casework samples. We found differences between the FPS profiles of model and casework samples, showing a variation in fragment size and distribution, suggesting the presence of non-human DNA. Using\n                    <jats:italic>Alu<\/jats:italic>\n                    -qPCR and 16S rRNA amplicon sequencing, we determined the amount and proportion of human and non-human DNA. Human DNA was detected in 84% of traces with an average of 70\u00a0pg per trace, while 16S rRNA revealed microbial DNA as the most abundant DNA in traces. These analyses provide new insights into forensic trace composition, allowing better sorting and profiling of traces.\n                  <\/jats:p>","DOI":"10.1007\/s00414-025-03448-8","type":"journal-article","created":{"date-parts":[[2025,2,28]],"date-time":"2025-02-28T20:27:37Z","timestamp":1740774457000},"page":"1511-1527","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Characterization of challenging forensic DNA traces using advanced molecular technologies"],"prefix":"10.1007","volume":"139","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-3352-3971","authenticated-orcid":false,"given":"Amel","family":"Larnane","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caroline","family":"Lef\u00e8vre-Horgues","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Corinne","family":"Cruaud","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"C\u00e9dric","family":"Fund","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edith","family":"Le Floch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Florian","family":"Sandron","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"B\u00e9atrice","family":"Segurens","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandre","family":"How-Kit","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jean-Fran\u00e7ois","family":"Deleuze","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,3,1]]},"reference":[{"key":"3448_CR1","doi-asserted-by":"publisher","unstructured":"(2016) Buckleton JS, Bright JA, Taylor D, editors. 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The procedures used in this study adhere to the tenets of the French National Charter for research integrity (2015).","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethical approval"}},{"value":"Informed consent was obtained from all individual participants included in the study.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent to participate"}},{"value":"All human participants are in agreement with this study.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Research involving human participants"}},{"value":"The authors have no competing interests to declare and nothing to disclose.","order":5,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}},{"value":"No tool used.","order":6,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declaration of generative AI and AI-assisted technologies in the writing process"}}]}}