Next-Generation Sequencing of Human Mitochondrial Reference Genomes Uncovers High Heteroplasmy Frequency

Abstract

We describe methods for rapid sequencing of the entire human mitochondrial genome (mtgenome), which involve long-range PCR for specific amplification of the mtgenome, pyrosequencing, quantitative mapping of sequence reads to identify sequence variants and heteroplasmy, as well as de novo sequence assembly. These methods have been used to study 40 publicly available HapMap samples of European (CEU) and African (YRI) ancestry to demonstrate a sequencing error rate <5.63×10−4, nucleotide diversity of 1.6×10−3 for CEU and 3.7×10−3 for YRI, patterns of sequence variation consistent with earlier studies, but a higher rate of heteroplasmy varying between 10% and 50%. These results demonstrate that next-generation sequencing technologies allow interrogation of the mitochondrial genome in greater depth than previously possible which may be of value in biology and medicine.

Extensive pathogenicity of mitochondrial heteroplasmy in healthy human individuals

“…Among these 131 individuals, 24.43% were detected to possess heteroplasmy with MAF larger than 10% (13). Moreover, a study used a 454 Genome Sequencer FLX system and sequenced 40 HapMap individuals to a mean coverage of 120×, and 65% individuals were found to have heteroplasmies with MAF higher than 9% (14). With a MAF cutoff of 10%, the prevalence of heteroplasmy is 44.42% in our dataset (SI Appendix, Fig.…”

“…The advent of NGS technologies enables the inquiry of mitochondrial heteroplasmy at the genome-wide scale. Several studies using these approaches allowed detection of medium-and high-frequency heteroplasmy with minor allele frequency (MAF) higher than 9%, and it was found that 25∼65% of the general population have at least one heteroplasmy across the entire mitochondrial genome (12)(13)(14). However, deeper sequencing depth at the order of thousands is required for confident identification of low-frequency heteroplasmy (MAF in the range of 1%∼10%) (15,16).…”

“…However, deeper sequencing depth at the order of thousands is required for confident identification of low-frequency heteroplasmy (MAF in the range of 1%∼10%) (15,16). Without considering these low-frequency heteroplasmy, the population prevalence of mitochondrial heteroplasmy is underestimated (12)(13)(14). Moreover, a preliminary study with ultra-deep sequencing (4,158∼20,803×) of two ∼300-bp mtDNA regions was able to find heteroplasmies with very low frequency (>0.2%) in all tested healthy samples (17).…”

Population-level expression variability of mitochondrial DNA-encoded genes in humans

“…TopHat option -read-mismatches was set to 2 and 3 (ie, allowing up to 2 and 3 mismatches in final read alignments), for CEU and YRI, respectively, because the mtDNA nucleotide diversity in YRI is higher than that in CEU. 15 By setting the TopHat option -g to 1, we deliberately allowed reads to be mapped on a single specific position on the genome. For each population, the expression values of log 10 (FPKM þ 1) for all genes were quantile-normalized across individuals.…”

Discovery of Three Newly Described Single Nucleotide Polymorphisms in Mitochondrial DNA Hypervariable Region I (HVI) and Estimation of Variants and Haplotypes Encompassing Nucleotide Positions 16024- 16365

“…In cases where there is an abundance in the sample, for example mass graves in mass disasters, there are newly discovered forensically validated methods such as ESI-MS [22] Certainly, all such applications should have a strong grasp of the mtDNA variation that is present in the populations concerned. As an example, describing and frequency estimates of common mtDNA types and any population sub-structuring must be at hand [23]. Consequently, this may also increase the pool of samples with degraded and insufficient nuclear DNA for mitochondrial DNA analysis.…”