According to the National Missing and Unidentified Persons System (NamUs), more than 15,000 unidentified human remains cases remain unresolved in the United States. Identification is often hindered by limited quantities of nuclear DNA and extensive degradation resulting from prolonged environmental exposure. Massively parallel sequencing (MPS) of single nucleotide polymorphisms (SNPs) has expanded the analytical potential of compromised forensic samples and contributed to the growth of forensic investigative genetic genealogy (FIGG). However, reliable nuclear DNA profiling from highly degraded or environmentally challenged samples remains a significant obstacle. This study evaluated the performance of the ForenSeq™ Kintelligence HT kit (Qiagen), a high-throughput targeted MPS assay designed to interrogate over 10,320 SNPs for ancestry inference, individual identification, phenotypic prediction, and kinship analysis, on postmortem samples recovered from aquatic environments. A section of a human femur was subdivided into five equal fragments. Four fragments were submerged for four months in aquatic environments: saltwater collected from the Atlantic Ocean (Maryland, USA) and freshwater from a stream (Virginia, USA), each maintained at either room temperature or 4 °C. A fifth fragment was stored at −80 °C as a control. DNA extraction followed the QIAamp® DNA Investigator protocol for bones and teeth. 

Saltwater-exposed samples consistently yielded higher DNA quantities and exhibited lower degradation compared to freshwater counterparts, irrespective of temperature. Refrigerated conditions further improved DNA preservation across both environments. Notably, DNA recovery from saltwater samples stored at 4 °C was approximately 12-fold higher than that from freshwater samples under the same conditions. Extracted DNA (1 ng input) was processed using the Kintelligence HT workflow and sequenced on the MiSeq FGx platform, with data analyzed using ForenSeq Universal Analysis Software. All samples produced successful SNP profiles. Even the lowest-performing condition (freshwater at room temperature) exceeded recommended thresholds for kinship analysis in GEDmatch, with over 7,000 SNPs typed and mean coverage exceeding 50×. Overall SNP call rates exceeded 82% across all samples. Saltwater samples stored at 4 °C demonstrated the highest performance, with the greatest number of SNPs recovered and an average read depth of 297× per locus. Concordance analysis confirmed all profiles originated from the same individual. 

Ongoing work includes assessment of SNP-level performance, ancestry inference concordance, and application of this approach to challenging forensic sample types. These findings highlight the robustness of targeted MPS for recovering informative nuclear DNA profiles from environmentally compromised skeletal remains, expanding the scope of forensic genetic analysis beyond conventional methods.

Authors

• Alyse Ostojic (George Washington University, United States of America)
• Grace Millis (George Washington University, United States of America)
• Daniele Podini (George Washington University, United States of America)
• Elaine Lewis (George Washington University, United States of America)
• Nicole Jewett (Bode Technology, United States of America)