The recovery of human DNA from burial soils represents a promising, yet under-explored, opportunity for human identification, particularly in scenarios where skeletal remains are highly degraded, absent, or unsuitable for sampling. Although considerable advancement has been made in sedimentary ancient DNA (sedaDNA), a forensic workflow for the reliable detection and authentication of human sedimentary DNA (sediDNA) in modern burial contexts remains elusive. Key challenges include extremely low endogenous DNA content, humic inhibition, molecular fragmentation, and the risk of contamination.

We present a systematic evaluation of laboratory workflows for detecting and authenticating human DNA from modern burial soils collected during controlled human decomposition experiments at two taphonomic research facilities (STAFS and AFTER). The known depositional context, donor identity, and spatial sampling design enable rigorous assessment of workflow performance and authentication criteria under realistic forensic conditions. Drawing from established ancient DNA methodologies, we evaluated silica-based extraction (following Dabney) alongside the DNeasy® PowerSoil® Pro Kit (Qiagen), in combination with library preparation and sequencing strategies.

The primary objective of our investigations were to assess human DNA yield, fragment length distributions, library complexity, and potential cytosine deamination patterns. In our comparison of extraction protocols, the DNeasy® PowerSoil® Pro Kit by Qiagen was identified as the most effective method. We further compared single- and double-stranded library preparation paired with shotgun sequencing and mitochondrial hybridization capture. Single-stranded libraries outperformed double-stranded libraries, particularly under capture conditions, yielding substantially higher mitochondrial coverage and depth. Emphasis was also placed on distinguishing authentic endogenous human DNA from environmental background DNA and potential laboratory contamination.

Preliminary analyses demonstrate reproducible recovery of highly fragmented human DNA across burial contexts, with mitochondrial capture improving sensitivity. Fragment length distributions, assessed before and after mapping, indicate retention of short DNA fragments in burial samples, consistent with degraded DNA recovery. In contrast to typical ancient DNA profiles, cytosine deamination signals were weak and highly variable, which in turn limits their use as a reliable authentication criterion in modern burial contexts. Workflow performance varied by burial condition and sampling location, underscoring the importance of context-driven sampling strategies.

These findings establish a forensic-oriented framework for the recovery and authentication of human DNA from burial sediments. By adapting and systematically evaluating ancient DNA methodologies under controlled forensic conditions, this study advances the integration of sedimentary DNA analysis into forensic genetics and expands investigative options in cases where conventional (skeletal) DNA analysis is not feasible.

Authors

• Nadescha Viviane Hänggi (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark)
• Nadezda Vilgerte Jochumsen (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark)
• Marie-Louise Kampmann (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark)
• Elena I. Zavala (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark)