Internal Validation of Mitochondrial DNA Control Region Using the Precision ID mtDNA Control Region Panel.

Area of Science:

• Forensic genetics
• Mitochondrial DNA analysis
• Next-generation sequencing (NGS)

Background:

• Mitochondrial DNA (mtDNA) sequencing is crucial in forensic genetics for degraded or low-quantity samples.
• Internal validation of an NGS-based mtDNA control region typing method was performed.
• This validation aims to improve the scientific robustness and judicial admissibility of forensic results.

Purpose of the Study:

• To internally validate an NGS-based typing method for the mitochondrial DNA control region.
• To assess the reliability and performance of the Precision ID mtDNA Control Region Panel on the Ion S5 sequencer for forensic applications.

Main Methods:

• Six key parameters were evaluated: read depth, sensitivity, repeatability, concordance with Sanger sequencing, reproducibility, and heteroplasmy detection.
• Validation involved negative controls, reference samples, a bone sample, and experimental mixtures.
• Libraries were prepared using the Ion Chef system, quantified, sequenced on the Ion GeneStudio S5, and analyzed with Converge software.

Main Results:

• A minimum read depth of 100 reads per position, an optimal DNA concentration of 20 pg/µL, and a heteroplasmy detection threshold of 20% were established as appropriate.
• These parameters support the method's application in forensic casework, accommodating variable sample concentrations.
• The NGS method demonstrated suitability for forensic mtDNA analysis, even with low-template DNA, and showed higher sensitivity than Sanger sequencing.

Conclusions:

• The NGS platform is suitable for forensic mtDNA analysis, particularly for low-template samples.
• The method offers enhanced sensitivity compared to traditional Sanger sequencing.
• Limitations include coverage variability in specific amplicons, challenges with polymorphisms in homopolymeric regions, and detection of low-level heteroplasmies.