- •We compared the ability of 454 and dideoxy methods to detect point heteroplasmy.
- •Minority variants above the 2% level were found in one third of the population.
- •Heteroplasmic substitutions were localized at different positions in mtDNA.
- •Majority of heteroplasmic substitutions hit positions observed in human mtDNA tree.
Mitochondrial DNA (mtDNA) heteroplasmy has been widely described from clinical, evolutionary and analytical points of view. Historically, the majority of studies have been based on Sanger sequencing. However, next-generation sequencing technologies are now being used for heteroplasmy analysis. Ultra-deep sequencing approaches provide increased sensitivity for detecting minority variants. However, a phylogenetic a posteriori analysis revealed that most of the next-generation sequencing data published to date suffers from shortcomings. Because implementation of new technologies in clinical, population, or forensic studies requires proper verification, in this paper we present a direct comparison of ultra-deep 454 and Sanger sequencing for the detection of heteroplasmy in complete mitochondrial genomes of normal colon cells. The spectrum of heteroplasmic mutations is discussed against the background of mitochondrial DNA variability in human populations.
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- Mitochondrial DNA in human hair shafts—existence of intra-individual differences.Int. J. Legal Med. 1999; 112: 172-175
- The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age.Am. J. Hum. Genet. 2000; 66: 1384-1397
- A sensitive denaturing gradient-Gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region.Am. J. Hum. Genet. 2000; 67: 432-443
- Results of the 1999–2000 collaborative exercise and proficiency testing program on mitochondrial DNA of the GEP-ISFG: an inter-laboratory study of the observed variability in the heteroplasmy level of hair from the same donor.Forensic Sci. Int. 2002; 125: 1-7
- Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts.Forensic Sci. Int. 2004; 40: 1-11
- Targeted high-throughput sequencing of tagged nucleic acid samples.Nucleic Acids Res. 2007; 35: e97
- Heteroplasmy in hair: differences among hair and blood from the same individuals are still a matter of debate.Forensic Sci. Int. 2007; 173: 117-121
- From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing.Nucleic Acids Res. 2008; 36: e5
- Investigation of heteroplasmy in the human mitochondrial DNA control region: a synthesis of observations from more than 5000 global population samples.J. Mol. Evol. 2009; 68: 516-527
- Application of full mitochondrial genome sequencing using 454 GS FLX pyrosequencing.Forensic Sci. Int. Genet. 2009; 2: 518-519
- Heteroplasmic mitochondrial DNA mutations in normal and tumour cells.Nature. 2010; 464: 610-614
- Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes.Am. J. Hum. Genet. 2010; 87: 237-249
- Mitochondrial DNA variant discovery and evaluation in human cardiomyopathies through next-generation sequencing.PLOS ONE. 2010; 5: e12295
- Second generation sequencing allows for mtDNA mixture deconvolution and high resolution detection of heteroplasmy.Croat. Med. J. 2011; 52: 299-313
- Next-generation sequencing of human mitochondrial reference genomes uncovers high heteroplasmy frequency.PLoS Comput. Biol. 2012; 8: e1002737
- Very low-level heteroplasmy mtDNA variations are inherited in humans.J. Genet. Genomics. 2013; 40: 607-615
- Universal heteroplasmy of human mitochondrial DNA.Hum. Mol. Genet. 2013; 22: 384-390
- Frequency and pattern of heteroplasmy in the complete human mitochondrial genome.PLOS ONE. 2013; 8: e74636
- Recurrent tissue-specific mtDNA mutations are common in humans.PLoS Genet. 2013; 9: e1003929
- Massively parallel pyrosequencing of the mitochondrial genome with the 454 methodology in forensic genetics.Forensic Sci. Int. Genet. 2014; 12: 30-37
- Extensive pathogenicity of mitochondrial heteroplasmy in healthy human individuals.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10654-10659
- Current Next Generation Sequencing technology may not meet forensic standards.Forensic Sci. Int. Genet. 2012; 6: 143-145
- The landscape of mitochondrial DNA variation in human colorectal cancer on the background of phylogenetic knowledge.Biochim. Biophys. Acta. 2012; 1825: 153-159
- Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 714-719
- Comparison of mitochondrial mutation spectra in ageing human colonic epithelium and disease: absence of evidence for purifying selection in somatic mitochondrial DNA point mutations.PLoS Genet. 2012; 8: e1003082
- Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences.BMC Genomics. 2009; 10: 139
- Do the four clades of the mtDNA haplogroup L2 evolve at different rates.Am. J. Hum. Genet. 2001; 69: 1348-1356
- Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA.Nat. Genet. 1999; 23: 147
- Peak height variations in automated sequencing of PCR products using Taq dye-terminator chemistry.Biotechniques. 1995; 19: 116-121
- Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation.Hum. Mutat. 2009; 30 (E386–394, updated 19.02.2014)
- Effects of purifying and adaptive selection on regional variation in human mtDNA.Science. 2004; 303: 223-226
- Comparing phylogeny and the predicted pathogenicity of protein variations reveals equal purifying selection across the global human mtDNA diversity.Am. J. Hum. Genet. 2011; 88: 433-439
- Correcting for purifying selection: an improved human mitochondrial molecular clock.Am. J. Hum. Genet. 2009; 84: 740-759
- Detecting errors in mtDNA data by phylogenetic analysis.Int. J. Legal Med. 2001; 115: 64-69
- A critical reassessment of the role of mitochondria in tumorigenesis.PLoS Med. 2005; 2: e296
- The diversity present in 5140 human mitochondrial genomes.Am. J. Hum. Genet. 2009; 84: 628-640
- Normal oxidative damage to mitochondrial and nuclear DNA is extensive.Proc. Natl. Acad. Sci. U. S. A. 1988; 17: 6465-6467
- Accuracy and quality of massively parallel DNA pyrosequencing.Genome Biol. 2007; 8: R143
- Distribution of nucleotide substitutions in human mitochondrial DNA genes.Russ. J. Genet. 2005; 41: 93-99
- A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine.Annu. Rev. Genet. 2005; 39: 359-407
Published online: October 31, 2014
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