Targeting ultrashort fragments: Development and validation of a 44-InDel panel for highly degraded DNA

Degraded samples are common yet challenging evidence types in forensic casework, and obtaining complete profiles from highly degraded samples has long been a major difficulty in forensic DNA analysis. However, there is a lack of effective methods for obtaining complete profiles from single-source highly degraded samples. Insertion/deletion (InDel) genetic markers, which combine the advantages of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), are widely distributed throughout the human genome, exhibit low mutation rates, possess short amplicon sizes, and are compatible with capillary electrophoresis (CE) platforms. These features make InDels ideal genetic markers for the analysis of highly degraded forensic samples. To address this issue, we selected 44 InDel genetic markers with amplicon sizes of no more than 125 bp, together with an amelogenin locus, to establish a five-dye multiplex detection system for accurate genotyping of highly degraded DNA samples. Following the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the system was evaluated in terms of sensitivity, inhibitor tolerance, species specificity, and performance on highly degraded samples. Sensitivity analysis demonstrated that complete and reliable profiles could be obtained with as little as 62.5 pg of input DNA. In degradation studies, the 44-InDel panel generated complete profiles from DNA subjected to 60 min of boiling, demonstrating a clear advantage over STR-based methods in the analysis of highly degraded samples. Population analysis was conducted using 180 samples from the Southern Han Chinese population, yielding an average observed heterozygosity (Ho) of 0.4898 for all loci. The combined random match probability (CMP) and cumulative probability of exclusion (CPE) were calculated as 2.9522 × 10⁻¹ ⁹ and 0.99985, respectively. The results demonstrated that the 44-InDel panel is highly suitable for individual identification of highly degraded forensic samples and serves as a valuable complementary tool for paternity testing.