Dynamics of mitochondrial heteroplasmy in three families investigated via a repeatable re-sequencing study

Abstract

Background: Originally 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.

Results: Here 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.

Conclusions: Our 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.

Figure 1

Individuals and samples used in the study. Numbers in parenthesis are the age of each individual; the number at the bottom of each table is count of sequencing reads.

Figure 2

False positive and false negative rates computed from simulation assuming 1,170× coverage. A Python script used to generate these results can be found in Additional file 3.

Figure 3

A representation of all differences found between each sequenced individual and the reference human mtDNA from genome build hg19. The colored bars (blue = A, green = C, orange = G, red = T) represent the frequency of a given allele in each sample. For example, at position 8,992 one can clearly see a heteroplasmy with two high frequency alleles C and T. Lines on top of the image represent location and orientation of mitochondrial genes. F1 = Family F4, F2 = Family F7, F3 = Family F11.

Figure 4

Reads aligning around the low complexity region 16,184 to 16,190. Set A: a set of random reads aligning across the region with no quality filtering performed. Set B: bridging reads; these were selected by requiring the low complexity region (positions 16,184 to 16,190) to be in the middle of the read. Set C: high quality reads containing indels; these were required to align across positions 16,184 to 16,190 and contain ten aligning high quality bases (phred value of 30 or higher) on each side of the indel.

Figure 5

Workflow for finding heteroplasmic sites from Illumina data. This workflow can be accessed, used, and edited at [18].