A rapid one-step NGS-based system integrating microhaplotypes and SNPs for forensic identification
- 1School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi 030600 China
- 2Shanxi Key Laboratory of Forensic Medicine, Jinzhong, 030600 Shanxi China
- 3Shanxi Province Engineering Research Center of Forensic Identification, Jinzhong, Shanxi 030600 China
Abstract
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.
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s12864-026-13237-1.
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BibTeX
@article{shi2026rapid,
title = {{A rapid one-step NGS-based system integrating microhaplotypes and SNPs for forensic identification}},
author = {Linyu Shi and Jiaxuan Hao and Jingjing Xu and Mengyang Zhao and Anjie Wei and Xinyi Zhang and Haoliang Fan and Jiangwei Yan},
journal = {BMC Genomics},
year = {2026},
month = aug,
doi = {10.1186/s12864-026-13237-1},
url = {https://doi.org/10.1186/s12864-026-13237-1},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - A rapid one-step NGS-based system integrating microhaplotypes and SNPs for forensic identification
AU - Linyu Shi
AU - Jiaxuan Hao
AU - Jingjing Xu
AU - Mengyang Zhao
AU - Anjie Wei
AU - Xinyi Zhang
AU - Haoliang Fan
AU - Jiangwei Yan
PY - 2026
DA - 2026/08/07
JO - BMC Genomics
DO - 10.1186/s12864-026-13237-1
UR - https://doi.org/10.1186/s12864-026-13237-1
AB - 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. Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s12864-026-13237-1.
SN - 1471-2164
ER -