Mitogenomes of Polar Bodies and Corresponding Oocytes

<div><p>The objective of the present study was to develop an approach that could assess the chromosomal status and the mitochondrial DNA (mtDNA) content of oocytes and their corresponding polar bodies (PBs) with the goal of obtaining a comparative picture of the segregation process both for nuclear and mtDNA. After Whole Genome Amplification (WGA), sequencing of the whole mitochondrial genome was attempted to analyze the segregation of mutant and wild-type mtDNA during human meiosis. Three triads, composed of oocyte and corresponding PBs, were analyzed and their chromosome status was successfully assessed. The complete mitochondrial genome (mitogenome) was almost entirely sequenced in the oocytes (95.99% compared to 98.43% in blood), while the percentage of sequences obtained in the corresponding PB1 and PB2 was lower (69.70% and 69.04% respectively). The comparison with the mtDNA sequence in blood revealed no changes in the D-loop region for any of the cells of each triad. In the coding region of blood mtDNA and oocyte mtDNA sequences showed full correspondence, whereas all PBs had at least one change with respect to the blood-oocyte pairs. In all, 9 changes were found, either in PB1 or PB2: 4 in <i>MT-ND5</i>, 2 in <i>MT-RNR2</i>, and 1 each in <i>MT-ATP8</i>, <i>MT-ND4</i>, <i>MT-CYTB</i>. The full concordance between oocyte and blood in the 3 triads, and the relegation of changes to PBs, revealed the unexpected coexistence of different variants, giving a refined estimation of mitochondrial heteroplasmy. Should these findings be confirmed by additional data, an active mechanism could be postulated in the oocyte to preserve a condition of ‘normality’.</p></div

Distribution of the 9 detected mutations in the 3 studied triads.

<p>wt: wild type.</p><p>Nd: not detected.</p

Amplification of the entire mitochondrial genome from polar body obtained by the amplification of 46 overlapping fragments using MitoAll kit.

<p>Amplification of the entire mitochondrial genome from polar body obtained by the amplification of 46 overlapping fragments using MitoAll kit.</p

Massive parallel sequencing of human whole mitochondrial genomes with Ion Torrent technology: an optimized workflow for Anthropological and Population Genetics studies

<p>Investigation of human mitochondrial DNA variation patterns and phylogeny has been extensively used in Anthropological and Population Genetics studies and sequencing the whole mitochondrial genome is progressively becoming the gold standard. Among the currently available massive parallel sequencing technologies, Ion Torrent™ semiconductor sequencing represents a promising approach for such studies. Nevertheless, an experimental protocol conceived to enable the achievement of both as high as possible yield and of the most homogeneous sequence coverage through the whole mitochondrial genome is still not available. The present work was thus aimed at improving the overall performance of whole mitochondrial genomes Ion Torrent™ sequencing, with special focus on the capability to obtain robust coverage and highly reliable variants calling. For this purpose, a series of cost-effective modifications in standard laboratory workflows was fine-tuned to optimize them for medium- and large-scale population studies. A total of 54 human samples were thus subjected to sequencing of the whole mitochondrial genome with the Ion Personal Genome Machine™ System in four distinct experiments and using Ion 314 chips. Seven of the selected samples were also characterized by means of conventional Sanger sequencing for the sake of comparison. Obtained results demonstrated that the implemented optimizations had definitely improved sequencing outputs in terms of both variants calling efficiency and coverage uniformity, enabling to setup an effective and accurate protocol for whole mitochondrial genome sequencing and a considerable reduction in experimental time consumption and sequencing costs.</p

Presence of major HV lineages in Italy.

<p>(A) presence of haplogroup HV*(xH,V); (B) presence of major lineages HV0, HV1, HV-73(HV2), HV4, HV-16311. Gray dots indicate sampled sites. The size of the square is proportional to the number of HV individuals in each site (see legend).</p

Discriminating power between R-M269 sub-lineages.

<p>Three STR panels are considered: Yfiler (top), RM11 (middle) and Yfiler + RM11 (bottom). Results are represented with admixture-like plots, in which vertical bars represent probabilities for each individual to be affiliated to a given R-M269 sub-lineage (membership probabilities). Mean per-group membership probabilities are reported in horizontal bars at the bottom of each plot.</p

Median Joining network of HV4 lineages.

<p>Mutations weighted proportionally to their frequency in the phylogeny.</p

Average mutation rates for the considered Y-STR sets (Yfiler, RM11, RM13) in deep rooted pedigrees (Dataset A) and in close relatives (Dataset B).

<p>Dotted vertical lines represent 95% confidence intervals (when available). For comparison, estimates from literature are included^10,8,11.</p

Bayesian Skyline plot of the whole HV dataset, with the coding region only and with the full molecule (see legend).

<p>Bayesian Skyline plot of the whole HV dataset, with the coding region only and with the full molecule (see legend).</p

HV phylogeny and dates.

<p><b>(A)</b> Schematic tree of the HV phylogeny, with important branches highlighted. Major nodes HV0, HV1, HV-72, HV4 and HV-16311 are marked with different colors. Mutations defining major clades are indicated, as well as mutations recurrent in the dataset, in dark red font. HV* and other lineages with an asterisk indicate positions of the tree for which we find potentially new lineages with our Italian data. The problematic position of HV9c is highlighted. (<b>B)</b> TMRCAs for major nodes and for the whole HV tree calculated by BEAST on the single lineages, on the whole dataset with imposing monophyly on major branches, and by mtPhyl. Confidence Intervals (of HPD intervals from BEAST runs) are visualized by error bars. (<b>C)</b> Probability estimates of the root height (TMRCA) calculated by BEAST on the imposed monophyly dataset.</p