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Updated: Jun 27, 2025
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
A PCR-independent approach for mtDNA enrichment and next-generation sequencing: comprehensive evaluation and clinical
Dong Liang1, Lin Zhu2, Yuqing Zhu1
1Department of Prenatal Diagnosis, Women's Hospital of Nanjing Medical University, Nanjing Women and Children's Healthcare Hospital, Nanjing, 210004, China.
Background:
Sequencing the mitochondrial genome has been increasingly important for the investigation of primary mitochondrial diseases (PMD) and mitochondrial genetics. To overcome the limitations originating from PCR-based mtDNA enrichment, we set out to develop and evaluate a PCR-independent approach in this study, named Pime-Seq (PCR-independent mtDNA enrichment and next generation Sequencing).
Results:
By using the optimized mtDNA enrichment procedure, the mtDNA reads ratio reached 88.0 ± 7.9% in the sequencing library when applied on human PBMC samples. We found the variants called by Pime-Seq were highly consistent among technical repeats. To evaluate the accuracy and reliability of this method, we compared Pime-Seq with lrPCR based NGS by performing both methods simultaneously on 45 samples, yielding 1677 concordant variants, as well as 146 discordant variants with low-level heteroplasmic fraction, in which Pime-Seq showed higher reliability. Furthermore, we applied Pime-Seq on 4 samples of PMD patients retrospectively, and successfully detected all the pathogenic mtDNA variants. In addition, we performed a prospective study on 192 apparently healthy pregnant women during prenatal screening, in which Pime-Seq identified pathogenic mtDNA variants in 4 samples, providing extra information for better health monitoring in these cases.
Conclusions:
Pime-Seq can obtain highly enriched mtDNA in a PCR-independent manner for high quality and reliable mtDNA deep-sequencing, which provides us an effective and promising tool for detecting mtDNA variants for both clinical and research purposes.
Insights
Pime-Seq, a novel PCR-independent method, enables high-quality mitochondrial DNA sequencing. This approach reliably detects mitochondrial DNA variants, proving effective for both clinical diagnosis and research in mitochondrial genetics.
Area of Science:
- Genomics
- Molecular Biology
- Medical Genetics
Background:
- Mitochondrial genome sequencing is crucial for understanding primary mitochondrial diseases (PMD) and mitochondrial genetics.
- Traditional PCR-based enrichment methods for mitochondrial DNA (mtDNA) have limitations.
- A PCR-independent approach, Pime-Seq, was developed to address these limitations.
Purpose of the Study:
- To develop and evaluate Pime-Seq, a PCR-independent method for mtDNA enrichment and next-generation sequencing.
- To assess the accuracy, reliability, and clinical utility of Pime-Seq for detecting mtDNA variants.
Main Methods:
- Developed and optimized Pime-Seq, a PCR-independent mtDNA enrichment technique.
- Compared Pime-Seq with long-range PCR (lrPCR) based next-generation sequencing (NGS) on 45 samples.
- Applied Pime-Seq retrospectively on PMD patient samples and prospectively in prenatal screening.
Main Results:
- Pime-Seq achieved a high mtDNA reads ratio (88.0 ± 7.9%) in sequencing libraries from human PBMC samples.
- Pime-Seq demonstrated high consistency among technical repeats and superior reliability compared to lrPCR-based NGS for low-level heteroplasmic variants.
- Successfully detected all pathogenic mtDNA variants in PMD patients and identified pathogenic variants in 4 out of 192 pregnant women during prenatal screening.
Conclusions:
- Pime-Seq provides highly enriched mtDNA in a PCR-independent manner for high-quality, reliable deep-sequencing.
- This method is an effective and promising tool for detecting mtDNA variants in both clinical and research settings.
- Pime-Seq enhances diagnostic capabilities for mitochondrial disorders and offers valuable information for health monitoring.
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