Journal of Translational Medicine (Apr 2024)

A PCR-independent approach for mtDNA enrichment and next-generation sequencing: comprehensive evaluation and clinical application

  • Dong Liang,
  • Lin Zhu,
  • Yuqing Zhu,
  • Mingtao Huang,
  • Ying Lin,
  • Hang Li,
  • Ping Hu,
  • Jun Zhang,
  • Bin Shen,
  • Zhengfeng Xu

DOI
https://doi.org/10.1186/s12967-024-05213-8
Journal volume & issue
Vol. 22, no. 1
pp. 1 – 12

Abstract

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Abstract 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.

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