title: Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders.
abstract: OBJECTIVE
  The application of massively parallel sequencing technology to the analysis of the mitochondrial genome has demonstrated great improvement in the molecular diagnosis of mitochondrial DNA-related disorders. The objective of this study was to investigate the performance characteristics and to gain new insights into the analysis of the mitochondrial genome.
  
  
  METHODS
  The entire mitochondrial genome was analyzed as a single amplicon using a long-range PCR-based enrichment approach coupled with massively parallel sequencing. The interference of the nuclear mitochondrial DNA homologs was distinguished from the actual mitochondrial DNA sequences by comparison with the results obtained from conventional PCR-based Sanger sequencing using multiple pairs of primers.
  
  
  RESULTS
  Our results demonstrated the uniform coverage of the entire mitochondrial genome. Massively parallel sequencing of the single amplicon revealed the presence of single-nucleotide polymorphisms and nuclear homologs of mtDNA sequences that cause the erroneous and inaccurate variant calls when PCR/Sanger sequencing approach was used. This single amplicon massively parallel sequencing strategy provides an accurate quantification of mutation heteroplasmy as well as the detection and mapping of mitochondrial DNA deletions.
  
  
  CONCLUSIONS
  The ability to quantitatively and qualitatively evaluate every single base of the entire mitochondrial genome is indispensible to the accurate molecular diagnosis and genetic counseling of mitochondrial DNA-related disorders. This new approach may be considered as first-line testing for comprehensive analysis of the mitochondrial genome.Genet Med 2013:15(5):388-394.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Cui
authors.forename: Fangyuan
authors.initials: F
authors.lastname: Li
authors.forename: David
authors.initials: D
authors.lastname: Chen
authors.forename: Guoli
authors.initials: G
authors.lastname: Wang
authors.forename: Cavatina K
authors.initials: CK
authors.lastname: Truong
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Enns
authors.forename: Brett
authors.initials: B
authors.lastname: Graham
authors.forename: Margherita
authors.initials: M
authors.lastname: Milone
authors.forename: Megan L
authors.initials: ML
authors.lastname: Landsverk
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Lee-Jun C
authors.initials: LJ
authors.lastname: Wong
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D005260:Female; D017353:Gene Deletion; D054629:Genome, Mitochondrial; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D028361:Mitochondrial Diseases / Q000175:diagnosis* / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D055815:Young Adult