A systematic study of WGA strategies for enhanced STR and SNP genotyping on trace DNA.

The robust analysis of trace DNA (≤ 100 pg) remains a persistent challenge in forensic genetics, primarily due to the high failure rates and stochastic effects associated with conventional STR typing at low template quantities. Although whole genome amplification (WGA) offers a potential solution, its effectiveness and influencing factors in forensic applications still require systematic evaluation. This study comprehensively evaluated two prominent WGA methods-REPLI-g and MALBAC-for STR and SNP genotyping of trace DNA across a wide input range (0.2441-2000 pg) on both capillary electrophoresis (CE) and targeted next-generation sequencing (NGS). REPLI-g consistently produced high yields (average 37.40 μg) with excellent stutter stability (~ 0.1), proving optimal for intact to moderately degraded samples (≥ 31.25 pg). Although MALBAC yielded less, it demonstrated superior sensitivity for ultra-low inputs (< 20 pg), retaining 21.37% SNP accuracy even at 0.2441 pg. In general, on the NGS platform, both methods significantly increased sequencing depth and reduced allele drop-out at critical low inputs. By further exploring the factors influencing the effectiveness of WGA-based DNA genotyping, we identified amplicon length as a key factor, with effective amplification and typing achieved particularly when the amplified fragment length was ≤ 300 bp. These observations established an evidence-based framework for WGA selection in trace evidence typing. Combining a contextually chosen WGA method with targeted NGS and short-amplicon assays provides a promising workflow for recovering interpretable genotypes from single-source trace evidence that would otherwise be unanalyzable.