Characterization of challenging forensic DNA traces using advanced molecular technologies

The majority of crime scenes contain DNA that is either present in small amounts or degraded, making it difficult to obtain usable DNA profiles using conventional technologies. The current standard for analyzing casework samples is the specific amplification of short tandem repeats (STR), which is limited by DNA quality and quantity. Since the goal of forensic science is to identify a suspect or victim regardless of trace quality, we evaluated three technological approaches to better characterize and exploit these traces: (i) ultra-sensitive pulse-field electrophoresis on a Femto Pulse System (FPS) to visualize DNA content, (ii) real-time quantitative PCR based on Alu repeats to quantify human DNA and analyze its integrity, and (iii) 16S ribosomal RNA gene (16S rRNA) amplicon sequencing to identify microbiota. We optimized FPS analysis using DNA from model traces (blood, saliva, semen, touch DNA, and vaginal swabs) and applied the protocol to 100 casework samples. We found differences between the FPS profiles of model and casework samples, showing a variation in fragment size and distribution, suggesting the presence of non-human DNA. Using Alu-qPCR and 16S rRNA amplicon sequencing, we determined the amount and proportion of human and non-human DNA. Human DNA was detected in 84% of traces with an average of 70 pg per trace, while 16S rRNA revealed microbial DNA as the most abundant DNA in traces. These analyses provide new insights into forensic trace composition, allowing better sorting and profiling of traces.