Shed hairs are a commonly encountered trace type in forensic casework. These hairs are typically in the telogen phase, with little to no soft tissue attached to the root. As the DNA found in hair shafts is often highly fragmented, standard short tandem repeat (STR) typing by PCR-CE is challenging. 

Recent studies suggest that RNA may be highly preserved within hair shafts. In this study, we explored the potential of shotgun whole transcriptome sequencing of telogen hairs as an alternative approach for obtaining human genetic information to support forensic donor identification and characterisation.

Telogen hairs (confirmed microscopically) of controlled lengths (2, 4, 7, 9 cm) were collected from two donors to assess the influence of hair length on RNA recovery and downstream analysis. Furthermore, telogen hairs of varying lengths (2-15 cm) were obtained from twelve additional donors to evaluate the broader applicability of the approach. Total RNA was extracted from each hair, quantified and assessed for quality, and analysed by shotgun RNA sequencing on the Illumina NovaSeq 6000. 

A single telogen hair yielded up to 74 ng of total RNA. The RNA integrity numbers (RIN) ranged from 1.0 to 3.6, indicating highly degraded fragments. Of the reads mapping to the human reference genome, an average of 83.2% originated from protein-coding transcripts (mRNAs). Of these, 61.3% derived from members of the keratin (KRT) and keratin-associated protein (KRTAP) gene families.

The potential for human identification (HID) using RNA-derived genotypes was evaluated by comparison to high-quality DNA reference profiles. The weight of evidence was calculated using wgsLR. Profiles consisting of 49-92 HID SNPs generated from hair transcriptomes yielded likelihood ratios ranging from 10^8 to 10^27 for true donors and from 10^-15 to 10^-41 for non-donors, demonstrating clear discrimination. Furthermore, the feasibility of inferring donor characteristics (such as biological sex, biogeographical ancestry, and externally visible traits), performing kinship analyses, and conducting hair transcriptome analysis using a desktop sequencing device (e.g., MiSeq) was explored.

Overall, this study demonstrates that shotgun transcriptome sequencing of a single telogen hair shaft can yield genetic data suitable for forensic applications, supporting donor identification and characterisation. These findings highlight the potential of RNA-based approaches as complementary tools in forensic genetics, particularly for challenging trace materials.

Authors

• Alberte Honoré Jepsen (Section of Forensic Genetics, Department of Forensic Medicine, University of Copenhagen, Denmark)
• Vanessa van Ackeren (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland)
• Cordula Haas (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland)
• Stine Bøttcher Jacobsen (Section of Forensic Genetics, Department of Forensic Medicine, University of Copenhagen, Denmark)
• Claus Børsting (Section of Forensic Genetics, Department of Forensic Medicine, University of Copenhagen, Denmark)
• Marie-Louise Kampmann (Section of Forensic Genetics, Department of Forensic Medicine, University of Copenhagen, Denmark)
• Jeppe Dyrberg Andersen (Section of Forensic Genetics, Department of Forensic Medicine, University of Copenhagen, Denmark)