A rapid one-step NGS-based system integrating microhaplotypes and SNPs for forensic identification

Short tandem repeats (STRs) have limited performance in analysing challenging forensic samples owing to the relatively long amplicon lengths of a subset of STR loci and the presence of stutter artefacts. While single nucleotide polymorphisms (SNPs) offer advantages for degraded DNA analysis through shorter amplicons, microhaplotypes (MHs) provide higher allelic diversity per locus than diallelic SNPs, thereby improving the statistical power of mixture deconvolution. To address these needs, we developed a novel next-generation sequencing (NGS)-based MHs and SNPs system, integrating 24 MHs, 80 SNPs, and a Y-Indel marker. Additionally, we developed a rapid, one-step library preparation method, reverse-probe-guided direct amplification. This method efficiently constructs targeted NGS libraries within 2.5 h by integrating target amplification and adapter ligation in a single tube, eliminating intermediate transfers to minimise contamination risks. We conducted comprehensive forensic validation, including population genetics parameters (total discrimination power = 1-2.0265 × 10^-39), performance on severely degraded DNA (degradation index: 10-15, quantified by qPCR; likelihood ratios supporting suspect contribution in 1:9 mixtures with a known victim > 2.26 × 10¹⁵), performance on formalin-fixed paraffin-embedded tissues (our panel achieved 100% genotyping accuracy, whereas capillary electrophoresis-STR exhibited > 38% allelic dropout), species specificity (six common non-human species: chicken, cattle, pig, mouse, rabbit and sheep, all yielded no interpretable typing data), and inhibitor resistance (evaluated against three common forensic inhibitors: humic acid, indigo and EDTA, at varying concentrations). Taken together, the developed one-step library preparation workflow and integrated MH-SNP panel offer a rapid, reliable approach for individual identification across common challenging forensic samples, including degraded DNA, degraded mixtures, FFPE tissues and inhibitor-contaminated specimens.