Abstract
BackgroundOriginally believed to be a rare phenomenon, heteroplasmy - the presence of more than one mitochondrial DNA (mtDNA) variant within a cell, tissue, or individual - is emerging as an important component of eukaryotic genetic diversity. Heteroplasmies can be used as genetic markers in applications ranging from forensics to cancer diagnostics. Yet the frequency of heteroplasmic alleles may vary from generation to generation due to the bottleneck occurring during oogenesis. Therefore, to understand the alterations in allele frequencies at heteroplasmic sites, it is of critical importance to investigate the dynamics of maternal mtDNA transmission.ResultsHere we sequenced, at high coverage, mtDNA from blood and buccal tissues of nine individuals from three families with a total of six maternal transmission events. Using simulations and re-sequencing of clonal DNA, we devised a set of criteria for detecting polymorphic sites in heterogeneous genetic samples that is resistant to the noise originating from massively parallel sequencing technologies. Application of these criteria to nine human mtDNA samples revealed four heteroplasmic sites.ConclusionsOur results suggest that the incidence of heteroplasmy may be lower than estimated in some other recent re-sequencing studies, and that mtDNA allelic frequencies differ significantly both between tissues of the same individual and between a mother and her offspring. We designed our study in such a way that the complete analysis described here can be repeated by anyone either at our site or directly on the Amazon Cloud. Our computational pipeline can be easily modified to accommodate other applications, such as viral re-sequencing.
Highlights
Believed to be a rare phenomenon, heteroplasmy - the presence of more than one mitochondrial DNA variant within a cell, tissue, or individual - is emerging as an important component of eukaryotic genetic diversity
The DNA was collected from a cheek swab specimen and from blood by our clinical collaborators at Penn State College of Medicine, and mitochondrial genomes were amplified by polymerase chain reaction (PCR) using two primer pairs
In total we generated (7 individuals × 2 tissues × 2 PCRs) + (2 individuals × 2 tissues × 1 PCR) = 32 single-end 76-bp (100-bp reads were generated for blood of M4, M9, and M4-C3) Illumina datasets (Figure 1)
Summary
Believed to be a rare phenomenon, heteroplasmy - the presence of more than one mitochondrial DNA (mtDNA) variant within a cell, tissue, or individual - is emerging as an important component of eukaryotic genetic diversity. To understand the alterations in allele frequencies at heteroplasmic sites, it is of critical importance to investigate the dynamics of maternal mtDNA transmission. MtDNA has been widely used as a marker in molecular evolution, population genetics and forensics. Knowledge of the size of the bottleneck is critical for modeling mtDNA evolution, assessing its applicability as a genetic marker, and for genetic counseling of patients carrying mtDNA mutations [13]. The size of the mtDNA bottleneck can be estimated more accurately when low frequency heteroplasmic mutations are taken into account [14]
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