Feasibility study of a single-primer extension-based microhaplotype NGS system.

Area of Science:

• Forensic Science
• Genetics
• Molecular Biology

Background:

• DNA degradation poses significant challenges in forensic analysis.
• Shortening amplicon length and utilizing microhaplotypes (MH) are effective strategies for degraded DNA.
• Existing methods struggle with the complexities of degraded DNA and novel marker systems.

Purpose of the Study:

• To develop and validate a 146-plex microhaplotype (MH) next-generation sequencing (NGS) system for degraded DNA analysis.
• To implement and assess a single-primer extension (SPE)-based library preparation method for enhanced MH detection.
• To propose and evaluate a flexible allele-calling strategy to overcome challenges in SPE-NGS data.

Main Methods:

• Construction of a 146-plex MH-NGS system.
• Application of a single-primer extension (SPE) library preparation method.
• Development and implementation of a flexible allele-calling strategy for SPE-NGS data.
• Forensic efficacy evaluation using low-template, mock-degraded, and casework DNA samples.

Main Results:

• The SPE method effectively mitigates DNA degradation impacts.
• A flexible allele-calling strategy significantly improves amplicon detection and forensic efficacy.
• The system accurately detected at least 37 loci with low-template DNA (7.8 pg), achieving a random match probability (RMP) of 10^-30.
• With the flexible strategy, 82 loci were detected (RMP 10^-54), and 36 loci remained detectable after severe heat treatment (RMP 10^-5).

Conclusions:

• The developed 146-plex MH-NGS system combined with SPE and a flexible allele-calling strategy is highly effective for degraded DNA analysis.
• This approach significantly enhances the detection of genetic markers in challenging forensic samples.
• The study demonstrates the system's robustness and potential for improving forensic casework investigations.