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Updated: Jan 16, 2026
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Nanopore sequencing and haplotyping of mitochondrial DNA hypervariable regions and its application on mixed stain
Yong Liao1, Junrui Shui2, Wenjie Chai2
1Urban Vocational College of Sichuan, Chengdu 610100, China; Sichuan Genegle Forensic Center, Chengdu 610093, China.
Objective:
To explore the feasibility of mitochondrial DNA (mtDNA) haplotype detection by nanopore sequencing and its potential value in the identification of mixed biological samples in forensic DNA analysis.
Methods:
Firstly, validation of the method for long fragments detection of human mtDNA hypervariable regions (HVRs) was carried out on the G-seq500 nanopore sequencing platform. Then, the amplified products of two DNA standards were mixed at different ratios to be sequenced and analyzed. Finally, the method established in this study was used to separate haplotypes of the mixed biological samples from simulated and real crime scenes.
Results:
The nanopore sequencing protocol of mtDNA HVRs established in this study demonstrated human-specificity, accuracy comparable to Sanger sequencing and Next Generation Sequencing (NGS), high sensitivity, repeatability and broad applicability. Using Sanger sequencing results as the standard, the accuracy of DNA standards sequencing was 100 %. The sequencing results for random individual blood cards were consistent with that of NGS. The detection limit of DNA template could be as low as 0.0625 ng. The method was also applicable to various sample types, including blood, oral swab, hair with follicles, different tissues and exfoliated cells, and can effectively interpret the Poly-C region. The mixtures of amplified products at ratios of 1:1, 1:9, 1:19, 1:49 and 1:99 from two DNA standards, as well as amplified products of mixed blood samples from two unrelated individuals at ratios of 3:2, 17:3, and 24:1, were accurately sequenced and haplotype-separated using the G-seq500 nanopore sequencing platform. Using this technical scheme to detect the mixture of exfoliated cells in a rape case can accurately separate the haplotypes to identify the suspect.
Conclusion:
The long-read nanopore sequencing of human mtDNA HVRs based on the G-seq500 platform meets forensic DNA methodological requirements and is operationally feasible. It holds great potential and application value in the identification of mixed biological samples in forensic DNA analysis.
