Characterization of mitochondrial DNA heteroplasmy using a parallel sequencing system

Biotechniques. 2010 Apr;48(4):287-96. doi: 10.2144/000113389.


Characterization of human mitochondrial genome sequences is important for the molecular diagnosis of mitochondrial diseases, especially in samples with a low level of mitochondrial DNA (mtDNA) heteroplasmy (>or=5%). Currently, no single methodology can simultaneously determine complete mtDNA sequences, identify mitochondrial genome-wide heteroplasmies, and quantify mtDNA heteroplasmy levels. The deep sampling inherent in "next-generation" sequencing approaches should enable the efficient detection of low-level DNA heteroplasmies and address this need. Herein, we used the Illumina Genome Analyzer to re-sequence human mtDNA samples from two subjects that were combined at five different ratios (1:99, 5:95, 10:90, 20:80, and 50:50). We assessed the sensitivity, specificity, and accuracy of this system, and our results show that mtDNA heteroplasmies >or=5% were detected 100% of the time with virtually no false positives and that the estimates of mtDNA heteroplasmy levels were remarkably close to the theoretical values (correlation coefficient = 0.96). Therefore, parallel sequencing provides a simple, high-throughput, and cost-effective platform for mitochondrial genome sequencing with sensitivity and specificity for mtDNA heteroplasmy detection.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Computational Biology / methods*
  • DNA Mutational Analysis
  • DNA, Mitochondrial / chemistry*
  • Genome, Human / genetics*
  • Humans
  • Linear Models
  • Polymerase Chain Reaction
  • Polymorphism, Restriction Fragment Length
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods*


  • DNA, Mitochondrial