Characterization of challenging forensic DNA traces using advanced molecular technologies

Abstract

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.

Keywords: Alu sequences; DNA trace; Degraded DNA; Femto pulse system; Forensic casework; Microorganism analysis.

Fig. 1

Description of the experimental workflow and methodological approaches used in our study

Fig. 2

Electrophoregrams of DNA from various tissues (blood, semen, saliva, touch DNA, and vaginal swab) using the FPS reveal tissue-specific profiles. The Ultra Sensitivity NGS Kit uses two size markers (1 bp, lower marker (LM) and 6 000 bp, upper marker (UM)), while the Genomic DNA 165 kb Kit uses only one size marker (1 bp, lower marker (LM))

Fig. 3

Femto pulse electrophoresis profiles of DNA from vaginal swabs from two donors (donor 1 in black and donor 2 in blue) revealing donor-specific profiles. The amount of DNA relative to size differs between the two donors

Fig. 4

Femto pulse electrophoretic profiles of saliva DNA under different tested parameters.  Results obtained with (a) different separation times. The black square highlights DNA fragments above 6,000 bp, which were not detected at 50 min but were fully visualized at 70 and 100 min, b different ladder and diluent marker reagent dilutions, and (c) different voltage injections

Fig. 5

Femto pulse electrophoretic profiles of six casework samples representing ‘least degraded’ DNA profiles (samples 1 and 2), ‘moderately degraded’ profiles (samples 3 and 4), and profiles with degraded DNA (samples 5 and 6). The results show the diversity of FPS profiles obtained from the 100 touch DNA casework samples

Fig. 6

Representation of the femto pulse electrophoretic profiles from touch DNA casework samples divided into two groups: sample type 1 (black line), and sample type 2 (blue line).The red dashed line represents the cut-off at 400 bp, which could be used to distinguish between non-degraded samples (> 400 bp) and degraded samples (< 400 bp)

Fig. 7

Results of the amplification of the V4 and V5 regions of 16S ribosomal RNA analyzed by electrophoresis using 2100 Bioanalyzer technology with (a) the Nested method and (b) the Fuhrman method. The band at 430 bp is specific to prokaryotes and the band at 600 bp is specific to eukaryotes. Non-specific amplification bands are present in samples 4, 5, and 6 with the nested method

Fig. 8

Results of the proportions of prokaryotes (blue), eukaryotes (green) and archaebacterial (*) with the Nested and Fuhrman method in six casework samples