Domenico Simone

HmtDB, a genomic resource for mitochondrion-based human variability studies

HmtDB (http://www.hmtdb.uniba.it:8080/hmdb) is a open resource created to support population genetics and mitochondrial disease studies. The database hosts human mitochondrial genome sequences annotated with population and variability data, the latter being estimated through the application of the SiteVar software based on site-specific nucleotide and amino acid variability calculations. The annotations are manually curated thus adding value to the quality of the information provided to the end-user. Classifier tools implemented in HmtDB allow the prediction of the haplogroup for any human mitochondrial genome currently stored in HmtDB or externally submitted de novo by an end-user. Haplogroup definition is based on the Phylotree system. End-users accessing HmtDB are hence allowed to (i) browse the database through the use of a multi-criterion ‘query’ system; (ii) analyze their own human mitochondrial sequences via the ‘classify’ tool (for complete genomes) or by downloading the ‘fragment-classifier’ tool (for partial sequences); (iii) download multi-alignments with reference genomes as well as variability data.

MToolBox: a highly automated pipeline for heteroplasmy annotation and prioritization analysis of human mitochondrial variants in high-throughput sequencing

Motivation: The increasing availability of mitochondria-targeted and off-target sequencing data in whole-exome and whole-genome sequencing studies (WXS and WGS) has risen the demand of effective pipelines to accurately measure heteroplasmy and to easily recognize the most functionally important mitochondrial variants among a huge number of candidates. To this purpose, we developed MToolBox, a highly automated pipeline to reconstruct and analyze human mitochondrial DNA from high-throughput sequencing data. Results: MToolBox implements an effective computational strategy for mitochondrial genomes assembling and haplogroup assignment also including a prioritization analysis of detected variants. MToolBox provides a Variant Call Format file featuring, for the first time, allele-specific heteroplasmy and annotation files with prioritized variants. MToolBox was tested on simulated samples and applied on 1000 Genomes WXS datasets. Availability and implementation: MToolBox package is available at https://sourceforge.net/projects/mtoolbox/. Contact: marcella.attimonelli@uniba.it Supplementary information: Supplementary data are available at Bioinformatics online.

Extraction and annotation of human mitochondrial genomes from 1000 Genomes Whole Exome Sequencing data

BackgroundWhole Exome Sequencing (WES) is one of the most used and cost-effective next generation technologies that allows sequencing of all nuclear exons. Off-target regions may be captured if they present high sequence similarity with baits. Bioinformatics tools have been optimized to retrieve a large amount of WES off-target mitochondrial DNA (mtDNA), by exploiting the aspecificity of probes, partially overlapping to Nuclear mitochondrial Sequences (NumtS). The 1000 Genomes project represents one of the widest resources to extract mtDNA sequences from WES data, considering the large effort the scientific community is undertaking to reconstruct human population history using mtDNA as marker, and the involvement of mtDNA in pathology.ResultsA previously published pipeline aimed at assembling mitochondrial genomes from off-target WES reads and further improved to detect insertions and deletions (indels) and heteroplasmy in a dataset of 1242 samples from the 1000 Genomes project, enabled to obtain a nearly complete mitochondrial genome from 943 samples (76% analyzed exomes). The robustness of our computational strategy was highlighted by the reduction of reads amount recognized as mitochondrial in the original annotation produced by the Consortium, due to NumtS filtering.An accurate survey was carried out on 1242 individuals. 215 indels, mostly heteroplasmic, and 3407 single base variants were mapped. A homogeneous mismatches distribution was observed along the whole mitochondrial genome, while a lower frequency of indels was found within protein-coding regions, where frameshift mutations may be deleterious. The majority of indels and mismatches found were not previously annotated in mitochondrial databases since conventional sequencing methods were limited to homoplasmy or quasi-homoplasmy detection. Intriguingly, upon filtering out non haplogroup-defining variants, we detected a widespread population occurrence of rare events predicted to be damaging. Eventually, samples were stratified into blood- and lymphoblastoid-derived to detect possibly different trends of mutability in the two datasets, an analysis which did not yield significant discordances.ConclusionsTo the best of our knowledge, this is likely the most extended population-scale mitochondrial genotyping in humans enriched with the estimation of heteroplasmies.

The reference human nuclear mitochondrial sequences compilation validated and implemented on the UCSC genome browser

BackgroundEukaryotic nuclear genomes contain fragments of mitochondrial DNA called NumtS (Nuclear mitochondrial Sequences), whose mode and time of insertion, as well as their functional/structural role within the genome are debated issues. Insertion sites match with chromosomal breaks, revealing that micro-deletions usually occurring at non-homologous end joining loci become reduced in presence of NumtS. Some NumtS are involved in recombination events leading to fragment duplication. Moreover, NumtS are polymorphic, a feature that renders them candidates as population markers. Finally, they are a cause of contamination during human mtDNA sequencing, leading to the generation of false heteroplasmies.ResultsHere we present RHNumtS.2, the most exhaustive human NumtSome catalogue annotating 585 NumtS, 97% of which were here validated in a European individual and in HapMap samples. The NumtS complete dataset and related features have been made available at the UCSC Genome Browser. The produced sequences have been submitted to INSDC databases. The implementation of the RHNumtS.2 tracks within the UCSC Genome Browser has been carried out with the aim to facilitate browsing of the NumtS tracks to be exploited in a wide range of research applications.ConclusionsWe aimed at providing the scientific community with the most exhaustive overview on the human NumtSome, a resource whose aim is to support several research applications, such as studies concerning human structural variation, diversity, and disease, as well as the detection of false heteroplasmic mtDNA variants. Upon implementation of the NumtS tracks, the application of the BLAT program on the UCSC Genome Browser has now become an additional tool to check for heteroplasmic artefacts, supported by data available through the NumtS tracks.

Searching for a needle in the haystack: Comparing six methods to evaluate heteroplasmy in difficult sequence context

Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC).

Primates and mouse NumtS in the UCSC Genome Browser

BackgroundNumtS (N uclear M iT ochondrial S equences) are mitochondrial DNA sequences that, after stress events involving the mitochondrion, colonized the nuclear genome. Accurate mapping of NumtS avoids contamination during mtDNA PCR amplification, thus supplying reliable bases for detecting false heteroplasmies. In addition, since they commonly populate mammalian genomes (especially primates) and are polymorphic, in terms of presence/absence and content of SNPs, they may be used as evolutionary markers in intra- and inter-species population analyses.ResultsThe need for an exhaustive NumtS annotation led us to produce the Reference Human NumtS compilation, followed, as reported in this paper, by those for chimpanzee, rhesus macaque and mouse ones. Identification of NumtS inside the UCSC Genome Browser and their inter-species comparison required the design and the implementation of NumtS tracks, starting from the compilation data. NumtS retrieval through the UCSC Genome Browser, in the species examined, is now feasible at a glance.ConclusionsAnalyses involving NumtS tracks, together with other genome element tracks publicly available at the UCSC Genome Browser, can provide deep insight into genome evolution and comparative genomics, thus improving studies dealing with the mechanisms that drove the generation of NumtS. In addition, the NumtS tracks constitute a useful tool in the design of mitochondrial DNA primers.

Polymorphic NumtS trace human population relationships

The human genome is constantly subjected to evolutionary forces which shape its architecture. Insertions of mitochondrial DNA sequences into nuclear genome (NumtS) have been described in several eukaryotic species, including Homo sapiens and other primates. The ongoing process of the generation of NumtS has made them valuable markers in primate phylogenetic studies, as well as potentially informative loci for reconstructing the genetic history of modern humans. Here, we report the identification of 53 human-specific NumtS by inspection of the UCSC genome browser, showing that they may be direct insertions of mitochondrial DNA into the human nuclear DNA after the human-chimpanzee split. In silico analyses allowed us to identify 14 NumtS which are polymorphic in terms of their presence/absence within the human genome in individuals of different ancestry. The allele frequencies of these polymorphic NumtS were calculated for 1000 Genomes Project sequence data from 13 populations worldwide, and principal components analysis and hierarchical clustering methods allowed the detection of strong signals of geographical structure related to the genetic diversity of these loci. All identified polymorphic human-specific NumtS together with a tandemly duplicated NumtS have also been validated by PCR amplification on a panel of 60 samples belonging to five native populations worldwide, confirming the expected NumtS variability. On the basis of these findings, we have succeeded in depicting the landscape of variation of a series of NumtS in several ethnic groups, making an advance in their identification as useful markers in the study on human population genetics.

Genome Sequence of the Polychlorinated-Biphenyl Degrader Pseudomonas pseudoalcaligenes KF707

Pseudomonas pseudoalcaligenes KF707 is a soil polychlorinated biphenyl (PCB) degrader, able to grow both planktonically and as a biofilm in the presence of various toxic metals and metalloids. Here we report the genome sequence (5,957,359 bp) of P. pseudoalcaligenes KF707, which provides insights into metabolic degradation pathways, flagellar motility, and chemotaxis.

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial classes and its specialization may be driven by the substrates it transports and the environment of its host.

Intra-individual purifying selection on mitochondrial DNA variants during human oogenesis

STUDY QUESTION Does selection for mtDNA mutations occur in human oocytes? SUMMARY ANSWER We provide statistical evidence in favor of the existence of purifying selection for mtDNA mutations in human oocytes acting between the expulsion of the first and second polar bodies (PBs). WHAT IS KNOWN ALREADY Several lines of evidence in Metazoa, including humans, indicate that variation within the germline of mitochondrial genomes is under purifying selection. The presence of this internal selection filter in the germline has important consequences for the evolutionary trajectory of mtDNA. However, the nature and localization of this internal filter are still unclear while several hypotheses are proposed in the literature. STUDY DESIGN, SIZE, DURATION In this study, 60 mitochondrial genomes were sequenced from 17 sets of oocytes, first and second PBs, and peripheral blood taken from nine women between 38 and 43 years of age. PARTICIPANTS/MATERIALS, SETTING, METHODS Whole genome amplification was performed only on the single cell samples and Sanger sequencing was performed on amplicons. The comparison of variant profiles between first and second PB sequences showed no difference in substitution rates but displayed instead a sharp difference in pathogenicity scores of protein-coding sequences using three different metrics (MutPred, Polyphen and SNPs&GO). MAIN RESULTS AND THE ROLE OF CHANCE Unlike the first, second PBs showed no significant differences in pathogenic scores with blood and oocyte sequences. This suggests that a filtering mechanism for disadvantageous variants operates during oocyte development between the expulsion of the first and second PB. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION The sample size is small and further studies are needed before this approach can be used in clinical practice. Studies on a model organism would allow the sample size to be increased. WIDER IMPLICATIONS OF THE FINDINGS This work opens the way to the study of the correlation between mtDNA mutations, mitochondrial capacity and viability of oocytes. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by a SISMER grant. Laboratory facilities and skills were freely provided by SISMER, and by the Alma Mater Studiorum, University of Bologna. The authors have no conflict of interest to disclose.