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Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

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Published on: February 10, 2023

Comparative analysis of ForenSeq mtDNA Control Region Kit, precision ID mtDNA Control Region Panel and Sanger

Hee-Yeon Park¹, Yoonji Noh¹, Kyu-Sik Jeong²

¹Forensic DNA Division, National Forensic Service, 10, Ipchun-ro, Wonju-si, Gangwon-do 26460, Republic of Korea.

Legal Medicine (Tokyo, Japan)

|October 17, 2025

Summary

Next-generation sequencing (NGS) offers a convenient way to analyze mitochondrial DNA (mtDNA) control regions, even detecting heteroplasmy. However, caution is advised when interpreting repetitive regions due to potential sequencing artifacts.

Keywords:

Converge ForenSeq UAS ForenSeq mtDNA Control Region Kit Mitochondrial DNA NGS Precision ID mtDNA Control Region Panel

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Published on: March 22, 2018

Area of Science:

Forensic Science
Genetics
Molecular Biology

Background:

Genomic DNA analysis in forensics is limited by low DNA concentration.
Mitochondrial DNA (mtDNA) serves as a viable alternative for forensic analysis.
The control region of mtDNA is crucial for identification purposes.

Purpose of the Study:

To comparatively analyze two commercial next-generation sequencing (NGS) kits for mtDNA control region analysis.
To evaluate the performance of NGS methods against Sanger sequencing for forensic applications.
To identify potential discrepancies and challenges in mtDNA sequencing technologies.

Main Methods:

Mitochondrial DNA was extracted from 75 Korean samples.
Sequencing was performed using Sanger sequencing, ForenSeq mtDNA Control Region Kit, and Precision ID mtDNA Control Region Panel.
Comparative analysis of sequencing results focused on the mtDNA control region.

Main Results:

Most mtDNA control regions yielded identical results across Sanger and NGS methods.
Discrepancies were observed in 43 out of 75 samples, particularly in repetitive regions (poly-C sites) and InDel/substitution variants.
Next-generation sequencing detected heteroplasmy in 6 samples, which was not observed with Sanger sequencing.

Conclusions:

NGS provides a convenient, high-throughput method for mtDNA control region analysis.
NGS is advantageous for detecting heteroplasmy, offering greater sensitivity.
Careful interpretation is required for repetitive regions in NGS data due to potential sequencing and alignment artifacts.