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

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. This manuscript details a novel algorithm to evaluate high-throughput DNA sequence data from whole mitochondrial genomes purified from genomic DNA, which also contains multiple fragmented nuclear copies of mtgenomes (numts). 40 samples were selected from 2 distinct reference (HapMap) populations of African (YRI) and European (CEU) origin. While previous technologies did not allow the assessment of individual mitochondrial molecules, next-generation sequencing technology is an excellent tool for obtaining the mtgenome sequence and its heteroplasmic sites rapidly and accurately through deep coverage of the genome. The computational techniques presented optimize reference-based alignments and introduce a new de novo assembly method. An important contribution of our study was obtaining high accuracy of the resulting called bases that we accomplished by quantitative filtering of reads that were error prone. In addition, several sites were experimentally validated and our method has a strong correlation (R2 = 0.96) with the NIST standard reference sample for heteroplasmy. Overall, our findings indicate that one can now confidently genotype mtDNA variants using next-generation sequencing data and reveal low levels of heteroplasmy (>10%). Beyond enriching our understanding and pathology of certain diseases, this development could be considered as a prelude to sequence-based individualized medicine for the mtgenome.