Jienan Li

Highly degraded samples are common in forensic casework and remain among the most challenging samples for forensic genetic analysis. Conventional STR-based technologies often yield partial or null profiles from such samples, making the acquisition of complete profiles a persistent challenge. To address this limitation, we developed a six-dye Multi-InDels kit based on Multi-InDels markers characterized by short amplicon sizes, high polymorphism, and low mutation and recombination rates, while maintaining full compatibility with capillary electrophoresis platforms. The kit integrates 26 Multi-InDels loci and an amelogenin locus into a multiplex system, with all amplicons not exceeding 125 bp, and is specifically designed for personal identification and paternity testing involving highly degraded DNA. Comprehensive internal validation studies demonstrated robust system performance, with complete profiles consistently obtained from as little as 62.5 pg of input DNA. Comparative analyses with STR kits showed that the Multi-InDels kit markedly improved the recovery of complete profiles from highly degraded samples, including artificially degraded DNA and formalin-fixed paraffin-embedded tissue. Population studies yielded a combined random match probability (CMP) of 2.25 × 10⁻¹⁷ and a cumulative probability of exclusion (CPE) of 0.999925. In paternity testing, 48 confirmed trios produced an average combined paternity index (CPI) of 3.2952 × 10⁶, and all 4,800 non-biological fathers were successfully excluded. In addition, innovative investigations were conducted on Multi-InDels allele nomenclature, evaluation strategies for degraded samples, and rare alleles. Collectively, these findings demonstrate that the Multi-InDels kit provides a convenient, feasible, and robust tool for forensic analysis of highly degraded samples.

DNA mixtures containing semen and vaginal fluid are common biological samples in forensic analysis. However, the analysis of semen–vaginal fluid mixtures remains challenging. In this study, to solve these problems, it is proposed to combine semen-specific CpG sites and closely related microhaplotype sites to form a new composite genetic marker (semen-specific methylation-microhaplotype). Six methylation-microhaplotype loci were selected. To further improve discrimination power, five methylation-SNP loci were also included. The methylation levels and genotypes of these selected loci were obtained using massively parallel sequencing technology. Except for loci MMH04ZHA019 and MMH17ZHA059, the remaining nine loci were successfully sequenced. For the successfully sequenced loci, they performed well in identifying individuals and body fluids. An allele categorization model was developed using K-nearest neighbour algorithm, which was then used to predict allele types in semen–vaginal fluid mixtures. These loci were able to confirm the presence of semen and link semen to a true donor in semen–vaginal fluid mixtures with mixing ratios of 10:1, 9:1, 5:1, 4:1, 1:1, 1:3, 1:4, 1:8 and 1:9 (semen:vaginal fluid). This preliminary study suggests that this new composite genetic marker has great potential as a supplementary tool to commonly used genetic markers (STR, etc.) for analysing semen–vaginal fluid mixtures.

Determining the time since deposition (TsD) of body fluid stains can provide crucial criminal information to forensic researchers. Although there are studies on inferring residual time through DNA and RNA markers, this requires high sample quality, and microorganisms, as a new type of marker with individual and tissue identification capabilities, have the potential for body fluid recognition and TsD inference. Blood and semen are the most common types of bodily fluid stains at crime scenes, but research on the inference of the TsD of these two types of stains through microorganisms still needs to be explored. Thus, this study collected samples of body fluid stains exposed indoors for up to 56 days and selected several microorganisms that were both liquid specific and related to residual time inference in blood (Methylobacterium and Sphingomonas) and semen (Gardnerella) stains via 16 S rRNA high-throughput sequencing. Furthermore, the microorganisms’ ability to infer TsD was verified using qPCR in validation group samples stored under the same conditions, and two multiple logistic regression models were constructed. The average absolute deviation of differences between the predicted and actual retention times of the three types of body fluids in the test set using two estimation methods was 2.15 and 2.06 days, respectively. In conclusion, this study has discovered four novel microorganisms related to the retention time of blood and semen and has preliminarily constructed the TsD prediction models, providing a new direction for future forensic research on the inference of TsD in blood and semen stains.

Background Small amounts of DNA from a perpetrator collected during crime-scene investigations can be masked by large amounts of DNA from the victim. These samples can provide important information for the perpetrator’s conviction. Short tandem repeat (STR) detection system is not sensitive enough to detect trace amounts of minor components in unbalanced mixed DNA. We developed a system using droplet digital polymerase chain reaction (ddPCR) capable of discovering trace components and accurately determining the ratio of mixed DNA in extremely unbalanced mixtures. Methods The non-recombining regions of the X chromosome and Y chromosome were quantified in the DNA of male and female mixtures using duplex ddPCR. Absolute quantification of low-abundance portions of trace samples and unbalanced mixtures was done using different mixing ratios. Results The ddPCR system could be used to detect low-abundance samples with < 5 copies of DNA components in an extremely unbalanced mixture at a mixing ratio of 10000:1. The high sensitivity and specificity of the system could identify the mixing ratio of mixed DNA accurately. Conclusions A ddPCR system was developed for evaluation of mixed samples of male DNA and female DNA. Our system could detect DNA quantities as low as 5 copies in extremely unbalanced mixed samples with good specificity and applicability. This method could assist forensic investigators in avoiding the omission of important physical evidence, and evaluating the ratio of mixed male/female trace samples.

Short tandem repeats (STRs) are the most widely used genetic markers in forensic application, but they are not ideal genetic markers for the analysis of forensic challenging samples such as highly degraded or unbalanced mixed samples because of their relatively large amplicons and stutter peaks. In this study, we developed a set of short microhaplotypes based on non-binary SNPs with molecular extent sizes no longer than 60 bases and genotyped 100 unrelated individuals from northern Han groups. Our results showed this panel has similar discrimination power to STR kits, as the combined random match probability (CMP) reached 1.396 × 10⁻²² and mean effective number of alleles (Ae) was 3.59. The cumulative probability of exclusion for duos (CPE-duos) was 0.999919 and the cumulative probability of exclusion for trios (CPE-trios) was 0.9999999987, suggesting this panel could be applied for forensic personal identification and parentage testing independently. Population differentiation in 26 populations from the 1000 Genomes Project indicated this panel could distinguish populations from Africa, East Asia, South Asia, America, and Europe. These microhaplotypes based on non-binary SNPs have short amplicons, good discrimination power, no stutter artifacts, and have great potential in detection of highly degraded and unbalanced mixtures for personal identification, paternity testing, and ancestry inference.