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

Median Joining network of HV4 lineages.

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

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

Analysis of Population Substructure in Two Sympatric Populations of Gran Chaco, Argentina

<div><p>Sub-population structure and intricate kinship dynamics might introduce biases in molecular anthropology studies and could invalidate the efforts to understand diseases in highly admixed populations. In order to clarify the previously observed distribution pattern and morbidity of Chagas disease in Gran Chaco, Argentina, we studied two populations (Wichí and Criollos) recruited following an innovative bio-cultural model considering their complex cultural interactions. By reconstructing the genetic background and the structure of these two culturally different populations, the pattern of admixture, the correspondence between genealogical and genetic relationships, this integrated perspective had the power to validate data and to link the gap usually relying on a singular discipline. Although Wichí and Criollos share the same area, these sympatric populations are differentiated from the genetic point of view as revealed by Non Recombinant Y Chromosome genotyping resulting in significantly high Fst values and in a lower genetic variability in the Wichí population. Surprisingly, the Amerindian and the European components emerged with comparable amounts (20%) among Criollos and Wichí respectively. The detailed analysis of mitochondrial DNA showed that the two populations have as much as 87% of private haplotypes. Moreover, from the maternal perspective, despite a common Amerindian origin, an Andean and an Amazonian component emerged in Criollos and in Wichí respectively. Our approach allowed us to highlight that quite frequently there is a discrepancy between self-reported and genetic kinship. Indeed, if self-reported identity and kinship are usually utilized in population genetics as a reliable proxy for genetic identity and parental relationship, in our model populations appear to be the result not only and not simply of the genetic background but also of complex cultural determinants. This integrated approach paves the way to a rigorous reconstruction of demographic and cultural history as well as of bioancestry and propensity to diseases of Wichí and Criollos.</p></div