Telogen hair represents a challenging, yet highly relevant, biological trace material in forensic genetics. Unlike anagen hair, telogen hairs lack a robust root sheath and typically contain limited and fragmented DNA, rendering conventional STR-based approaches ineffective or unreliable for human identification (HID). Here, we present a comprehensive evaluation of telogen hair as a novel sample type for forensic genetic analysis using shotgun sequencing, enabling reliable recovery of genomic single-nucleotide polymorphisms (SNPs) for HID and for investigative lead applications such as forensic investigative genetic genealogy (FIGG).

We conducted a systematic evaluation of a complete workflow from telogen hairs to its application as forensic evidence for HID or as an investigative lead. Telogen hair, confirmed by microscopy, demonstrated that genomic recovery was independent of the root end and adjacent hair segments, enabling consistent analysis across serial sections of a hair shaft. Notably, hair fragments as short as 1.5 cm yielded reliable genome-wide SNP profiles. To create the optimal workflow, different DNA extraction protocols were compared and methods optimised for low-template and degraded material resulted in substantial recovery of nuclear DNA. The yield was assessed by human-specific qPCR and total DNA quantification. Library preparation methods were systematically assessed for efficiency and library complexity, with single-stranded library preparation showing superior performance. Downstream bioinformatic processing integrated both hard genotype calling and probabilistic approaches to minimise error rates while maximising data retention. Although inter-individual variation in DNA yield and sequencing performance was observed, all 56 telogen hairs (1.5 - 12 cm in length) produced reliable SNP data suitable for downstream forensic genetic analysis.

To illustrate the use of the SNP profiles, we calculated the weight of the evidence (WoE) for HID using the open-source software, wgsLR, under two competing hypotheses. The SNP profiles were compared with reference samples from both the same and unrelated donors and the minimum WoE>5 for true donors and a minimum WoE<-27 for non-donors. Finally, we showed that it was possible to obtain 224,000 SNPs on average from all 56 hairs, which was sufficient to carry out extended kinship analysis comparable to that used in FIGG analysis.

In conclusion, our results demonstrated that single telogen hairs with lengths down to 1.5 cm can produce informative SNP profiles for HID and investigative applications such as FIGG, when the hair is processed using optimised laboratory and bioinformatic workflows.

Authors

• Marie-Louise Kampmann (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark)
• Stine Bøttcher Jacobsen (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark)
• Alberte Honoré Jepsen (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark)
• Mikkel Meyer Andersen (Department of Mathematical Sciences, Aalborg University, Denmark)
• Claus Børsting (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark)
• Jeppe Dyrberg Andersen (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark)