Objective

Body fluid and individual identification of biological trace samples are core tasks in forensic genetics. Transcriptomes offer both gene expression characteristics and transcript sequence variation information, potentially revealing tissue origin and individual genetic background simultaneously. However, forensic samples are often severely degraded or present in trace amounts, posing significant challenges for RNA analysis. This study aims to evaluate the utility of transcriptomic expression profiles and transcript SNPs (tSNPs) in degraded forensic body fluids, exploring their application potential for body fluid identification and auxiliary individual identification.

Methods

Five common forensic body fluids (venous blood, oral swab, semen, menstrual blood, and vaginal secretion) were selected for this study. Transcriptomic libraries were constructed starting with 10 ng of total RNA and sequenced. Samples included fresh body fluids (T0) and body fluid stains degraded at room temperature for 7 days (T7) and 14 days (T14). Differential expression analysis was performed to screen for body fluid-specific genes significantly differentially expressed compared to other fluids. Concurrently, tSNP loci were detected in each sample, and their genotype concordance was compared with paired genomic SNPs (gSNPs).

Results

The sequencing Q30 for all 45 samples exceeded 85%, indicating good data quality. Differential expression analysis revealed that the overall number of specific RNA markers in the five body fluids decreased significantly with prolonged degradation time, yet a certain number of body fluid-specific transcripts remained detectable in T14 samples. Specifically, 4318, 1727, and 1577 mRNAs, and 285, 90, and 67 lncRNAs were detected at T0, T7, and T14, respectively. mRNAs were significantly more abundant than lncRNAs at all time points, suggesting their more prominent application value for identifying the origin of degraded body fluids. tSNP loci demonstrated good detectability across all five body fluid types under degradation, although the number detected varied among fluids. Further analysis showed that the genotype concordance between tSNPs and paired gSNPs exceeded 96% across all body fluid samples and degradation time points, with venous blood samples exhibiting concordance above 99% at all time points.

Conclusion

The transcriptomes of degraded forensic body fluids retain body fluid-specific expression signals that are informative for tissue origin identification, while also yielding tSNP information with high genotyping concordance. These findings indicate that transcriptomic analysis holds promise for the simultaneous inference of tissue origin and auxiliary individual identification in degraded forensic samples, providing a novel research perspective and methodological foundation for the multi-purpose assay of complex forensic body fluid stains.

Authors

• Weiping Zhang (College of Forensic Medicine, Hebei Medical University, China)
• Qian Wang (College of Forensic Medicine, Hebei Medical University, China)
• Guanju Ma (College of Forensic Medicine, Hebei Medical University, China)
• Shujin Li (College of Forensic Medicine, Hebei Medical University, China)