Mitochondrial Genome and Mitochondrial DNA (mtDNA) Sequencing

Overview of Mitochondrial Genome

The mitochondrion is an indispensable organelle for nearly all eukaryotic cells. However, there are some exceptions, like the mature blood red cells. The mitochondrial genome is a double-stranded, circular molecule of approximately 16,569 bp and encodes 37 genes including 22 tRNAs, 2 rRNAs and 13 proteins that are subunits of enzyme complexes of the oxidative phosphorylation system). Therefore, The mitochondrial genome is very important for cellular energetics and survival. Additionally, due to the maternal inheritance, mtDNA can be used to trace the origin and ancestry of eukaryotes.

The structure of human mitochondrial genome (from Wiki).Figure 1. The structure of human mitochondrial genome (from Wiki).

  • Mitochondrial Genome and Human Disease

Mutations in mitochondrial DNA (mtDNA) are associated with a wide variety of human diseases such as autoimmune, cancer and neurodegenerative disorders like Parkinson's and Alzheimer's. But why such a small genome can affect human health so much? Researchers found that mtDNA is highly subject to mutagenic attacks from reactive oxygen species, leading to the accumulation of somatic mutations. Coupled with the limited DNA damage repair ability in the organelle, alterations in mtDNA occur at a much higher rate than those in the nuclear DNA. What's more, eukaryotic cells may contains up to thousands of mitochondria. The mtDNA heteroplasmy is an indicator of cellular metabolic dysfunction. Therefore, there has been increased attention for mitochondrial genome analysis.

Mitochondrial DNA Sequencing

Mitochondrial DNA sequencing is a powerful approach for the detection of mtDNA mutations that are associated with human diseases, determination of mtDNA heteroplasmy, detection of epigenetic modifications and haplogroup classification. Sanger sequencing makes mitochondrial DNA sequencing easy and affordable. The introduction of next-generation sequencing (NGS) technologies is the biggest game-changer in mitochondrial genomics. NGS can generate orders of magnitude more data in a cheaper and faster way.

  • Workflow of Mitochondrial Sequencing Using NGS

The workflow of mitochondrial DNA sequencing includes these steps, genomic DNA isolation (containing both nuclear and mitochondrial DNA), enrichment of mtDNA, library generation, high-throughput sequencing and bioinformatics analysis. The mtDNA of DNA samples is individually amplified using long-range PCR method, with two overlapping fragments (>8 Kb). There are commercial long-range PCR kits available in the market such as Qiagen LongRange PCR Kit and SequalPrep™ Long PCR Kit with dNTPs. Long-range PCR is a flexible, efficient and cost-effective method for sequencing specific genomic regions, especially when combined with NGS platforms. This method uses long-range DNA polymerases that are able to amplify up to 15 Kb or longer sequence. It allows for the amplification of entire mitochondrial chromosomes. After this PCR, agarose gel electrophoresis (0.7%) is used to confirm the presence of PCR product, and the PCR product is then quantitated. Libraries are then constructed for DNA fragmentation, barcoded adapter ligation and amplification, followed by determination of concentration with qPCR and sequencing with Ion Torrent™ PGM™, Illumina HiSeq/MiSeq, other NGS platforms or long-read sequencing platforms such as PacBio RSII and Oxford nanopore MinION™.

  • Bioinformatics Analysis

After sequencing, coverage depth and other parameters are estimated and the quality of reads are assessed for read filtering and trimming. Alignment of mitochondrial sequences can be carried out using ClustalW, and open reading frames (ORFs) and rRNA gene fragments can be identified and annotated. Sequence mutations such as SNPs are detected and their allele frequency is estimated. The association between mutations and human diseases can be predicted. And animal phylogeny can be inferred using Maximum Likelihood or Bayesian Analysis.


  1. Smith D R. The past, present and future of mitochondrial genomics: have we sequenced enough mtDNAs?. Briefings in functional genomics, 2015, 15(1): 47-54.
  2. Taanman J W. The mitochondrial genome: structure, transcription, translation and replication. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1999, 1410(2): 103-123.
  3. Douglas A P, Vance D R, Kenny E M, et al. Next-generation sequencing of the mitochondrial genome and association with IgA nephropathy in a renal transplant population. Scientific reports, 2014, 4: 7379.
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