Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

Hong Cui, Fangyuan Li, David Chen, Guoli Wang, Cavatina K. Truong, Gregory M. Enns, Brett Graham, Margherita Milone, Megan L. Landsverk, Jing Wang, Wei Zhang, Lee Jun C Wong

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Purpose: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.Conclusion: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.

Original languageEnglish (US)
Pages (from-to)388-394
Number of pages7
JournalGenetics in Medicine
Volume15
Issue number5
DOIs
StatePublished - May 2013

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Mitochondrial Diseases
Mitochondrial Genome
Mitochondrial DNA
Sequence Analysis
High-Throughput Nucleotide Sequencing
Polymerase Chain Reaction
Genetic Counseling
Sequence Homology
Single Nucleotide Polymorphism
Molecular Biology
Technology
Mutation

Keywords

  • long-range PCR
  • massively parallel sequencing
  • mitochondrial disorders
  • mitochondrial genome
  • mtDNA large deletions
  • nuclear mitochondrial DNA homologs (NUMTs)

ASJC Scopus subject areas

  • Genetics(clinical)

Cite this

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders. / Cui, Hong; Li, Fangyuan; Chen, David; Wang, Guoli; Truong, Cavatina K.; Enns, Gregory M.; Graham, Brett; Milone, Margherita; Landsverk, Megan L.; Wang, Jing; Zhang, Wei; Wong, Lee Jun C.

In: Genetics in Medicine, Vol. 15, No. 5, 05.2013, p. 388-394.

Research output: Contribution to journalArticle

Cui, Hong ; Li, Fangyuan ; Chen, David ; Wang, Guoli ; Truong, Cavatina K. ; Enns, Gregory M. ; Graham, Brett ; Milone, Margherita ; Landsverk, Megan L. ; Wang, Jing ; Zhang, Wei ; Wong, Lee Jun C. / Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders. In: Genetics in Medicine. 2013 ; Vol. 15, No. 5. pp. 388-394.
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abstract = "Purpose: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.Conclusion: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.",
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