Controlling for contamination in re-sequencing studies with a reproducible web-based phylogenetic approach

Polymorphism discovery is a routine application of next-generation sequencing technology where multiple samples are sent to a service provider for library preparation, subsequent sequencing, and bioinformatic analyses. The decreasing cost and advances in multiplexing approaches have made it possible to analyze hundreds of samples at a reasonable cost. However, because of the manual steps involved in the initial processing of samples and handling of sequencing equipment, cross-contamination remains a significant challenge. It is especially problematic in cases where polymorphism frequencies do not adhere to diploid expectation, for example, heterogeneous tumor samples, organellar genomes, as well as during bacterial and viral sequencing. In these instances, low levels of contamination may be readily mistaken for polymorphisms, leading to false results. Here we describe practical steps designed to reliably detect contamination and uncover its origin, and also provide new, Galaxy-based, readily accessible computational tools and workflows for quality control. All results described in this report can be reproduced interactively on the web as described at http://usegalaxy.org/contamination

Immune correction of the dental status of patients with type ii diabetes mellitus using removable orthopedic prostheses

The aim of the study was to improve the effectiveness of therapeutic and preventive manipulations aimed at correcting the dental status of patients with type 2 diabetes mellitus during dental prosthetics with removable types of structures, using immunomodulatory therapy and phytotherap

Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA

The manifestation of mitochondrial DNA (mtDNA) diseases depends on the frequency of heteroplasmy (the presence of several alleles in an individual), yet its transmission across generations cannot be readily predicted owing to a lack of data on the size of the mtDNA bottleneck during oogenesis. For deleterious heteroplasmies, a severe bottleneck may abruptly transform a benign (low) frequency in a mother into a disease-causing (high) frequency in her child. Here we present a high-resolution study of heteroplasmy transmission conducted on blood and buccal mtDNA of 39 healthy mother–child pairs of European ancestry (a total of 156 samples, each sequenced at ∼20,000× per site). On average, each individual carried one heteroplasmy, and one in eight individuals carried a disease-associated heteroplasmy, with minor allele frequency ≥1%. We observed frequent drastic heteroplasmy frequency shifts between generations and estimated the effective size of the germ-line mtDNA bottleneck at only ∼30–35 (interquartile range from 9 to 141). Accounting for heteroplasmies, we estimated the mtDNA germ-line mutation rate at 1.3 × 10−8 (interquartile range from 4.2 × 10−9 to 4.1 × 10−8) mutations per site per year, an order of magnitude higher than for nuclear DNA. Notably, we found a positive association between the number of heteroplasmies in a child and maternal age at fertilization, likely attributable to oocyte aging. This study also took advantage of droplet digital PCR (ddPCR) to validate heteroplasmies and confirm a de novo mutation. Our results can be used to predict the transmission of disease-causing mtDNA variants and illuminate evolutionary dynamics of the mitochondrial genome

Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop

Rationale: In the endothelium, insulin stimulates endothelial NO synthase (eNOS) to generate the antiatherosclerotic signaling radical NO. Insulin-resistant type 2 diabetes mellitus is associated with reduced NO availability and accelerated atherosclerosis. The effect of enhancing endothelial insulin sensitivity on NO availability is unclear. Objective: To answer this question, we generated a mouse with endothelial cell (EC)–specific overexpression of the human insulin receptor (hIRECO) using the Tie2 promoter–enhancer. Methods and Results: hIRECO demonstrated significant endothelial dysfunction measured by blunted endothelium-dependent vasorelaxation to acetylcholine, which was normalized by a specific Nox2 NADPH oxidase inhibitor. Insulin-stimulated phosphorylation of protein kinase B was increased in hIRECO EC as was Nox2 NADPH oxidase–dependent generation of superoxide, whereas insulin-stimulated and shear stress–stimulated eNOS activations were blunted. Phosphorylation at the inhibitory residue Y657 of eNOS and expression of proline-rich tyrosine kinase 2 that phosphorylates this residue were significantly higher in hIRECO EC. Inhibition of proline-rich tyrosine kinase 2 improved insulin-induced and shear stress–induced eNOS activation in hIRECO EC. Conclusions: Enhancing insulin sensitivity specifically in EC leads to a paradoxical decline in endothelial function, mediated by increased tyrosine phosphorylation of eNOS and excess Nox2-derived superoxide. Increased EC insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide. Inhibition of proline-rich tyrosine kinase 2 restores insulin-induced and shear stress–induced NO production. This study demonstrates for the first time that increased endothelial insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide

Evolution and Survival on Eutherian Sex Chromosomes

Since the two eutherian sex chromosomes diverged from an ancestral autosomal pair, the X has remained relatively gene-rich, while the Y has lost most of its genes through the accumulation of deleterious mutations in nonrecombining regions. Presently, it is unclear what is distinctive about genes that remain on the Y chromosome, when the sex chromosomes acquired their unique evolutionary rates, and whether X-Y gene divergence paralleled that of paralogs located on autosomes. To tackle these questions, here we juxtaposed the evolution of X and Y homologous genes (gametologs) in eutherian mammals with their autosomal orthologs in marsupial and monotreme mammals. We discovered that genes on the X and Y acquired distinct evolutionary rates immediately following the suppression of recombination between the two sex chromosomes. The Y-linked genes evolved at higher rates, while the X-linked genes maintained the lower evolutionary rates of the ancestral autosomal genes. These distinct rates have been maintained throughout the evolution of X and Y. Specifically, in humans, most X gametologs and, curiously, also most Y gametologs evolved under stronger purifying selection than similarly aged autosomal paralogs. Finally, after evaluating the current experimental data from the literature, we concluded that unique mRNA/protein expression patterns and functions acquired by Y (versus X) gametologs likely contributed to their retention. Our results also suggest that either the boundary between sex chromosome strata 3 and 4 should be shifted or that stratum 3 should be divided into two strata

Role of Duplicate Genes in Robustness against Deleterious Human Mutations

It is now widely recognized that robustness is an inherent property of biological systems [1],[2],[3]. The contribution of close sequence homologs to genetic robustness against null mutations has been previously demonstrated in simple organisms [4],[5]. In this paper we investigate in detail the contribution of gene duplicates to back-up against deleterious human mutations. Our analysis demonstrates that the functional compensation by close homologs may play an important role in human genetic disease. Genes with a 90% sequence identity homolog are about 3 times less likely to harbor known disease mutations compared to genes with remote homologs. Moreover, close duplicates affect the phenotypic consequences of deleterious mutations by making a decrease in life expectancy significantly less likely. We also demonstrate that similarity of expression profiles across tissues significantly increases the likelihood of functional compensation by homologs

Identification and Characterization of Lineage-Specific Highly Conserved Noncoding Sequences in Mammalian Genomes

Vertebrate genome comparisons revealed that there are highly conserved noncoding sequences (HCNSs) among a wide range of species and many of which contain regulatory elements. However, recently emerged sequences conserved in specific lineages have not been well studied. Toward this end, we identified 8,198 primate and 21,128 specific HCNSs as representative ones among mammals from human–marmoset and mouse–rat comparisons, respectively. Derived allele frequency analysis of primate-specific HCNSs showed that these HCNSs were under purifying selection, indicating that they may harbor important functions. We selected the top 1,000 largest HCNSs and compared the lineage-specific HCNS-flanking genes (LHF genes) with ultraconserved element (UCE)-flanking genes. Interestingly, the majority of LHF genes were different from UCE-flanking genes. This lineage-specific set of LHF genes was more enriched in protein-binding function. Conversely, the number of LHF genes that were also shared by UCEs was small but significantly larger than random expectation, and many of these genes were involved in anatomical development as transcriptional regulators, suggesting that certain groups of genes preferentially recruit new HCNSs in addition to old HCNSs that are conserved among vertebrates. This group of LHF genes might be involved in the various levels of lineage-specific evolution among vertebrates, mammals, primates, and rodents. If so, the emergence of HCNSs in and around these two groups of LHF genes developed lineage-specific characteristics. Our results provide new insight into lineage-specific evolution through interactions between HCNSs and their LHF genes

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

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

Evolutionary and biomedical insights from the rhesus macaque genome

The rhesus macaque (Macaca mulatta) is an abundant primate species that diverged from the ancestors of Homo sapiens about 25 million years ago. Because they are genetically and physiologically similar to humans, rhesus monkeys are the most widely used nonhuman primate in basic and applied biomedical research. We determined the genome sequence of an Indian-origin Macaca mulatta female and compared the data with chimpanzees and humans to reveal the structure of ancestral primate genomes and to identify evidence for positive selection and lineage-specific expansions and contractions of gene families. A comparison of sequences from individual animals was used to investigate their underlying genetic diversity. The complete description of the macaque genome blueprint enhances the utility of this animal model for biomedical research and improves our understanding of the basic biology of the species

Functional Diversification of Paralogous Transcription Factors via Divergence in DNA Binding Site Motif and in Expression

BACKGROUND: Gene duplication is a major driver of evolutionary innovation as it allows for an organism to elaborate its existing biological functions via specialization or diversification of initially redundant gene paralogs. Gene function can diversify in several ways. Transcription factor gene paralogs in particular, can diversify either by changes in their tissue-specific expression pattern or by changes in the DNA binding site motif recognized by their protein product, which in turn alters their gene targets. The relationship between these two modes of functional diversification of transcription factor paralogs has not been previously investigated, and is essential for understanding adaptive evolution of transcription factor gene families. FINDINGS: Based on a large set of human paralogous transcription factor pairs, we show that when the DNA binding site motifs of transcription factor paralogs are similar, the expressions of the genes that encode the paralogs have diverged, so in general, at most one of the paralogs is highly expressed in a tissue. Moreover, paralogs with diverged DNA binding site motifs tend to be diverged in their function. Conversely, two paralogs that are highly expressed in a tissue tend to have dissimilar DNA binding site motifs. We have also found that in general, within a paralogous family, tissue-specific decrease in gene expression is more frequent than what is expected by chance. CONCLUSIONS: While previous investigations of paralogous gene diversification have only considered coding sequence divergence, by explicitly quantifying divergence in DNA binding site motif, our work presents a new paradigm for investigating functional diversification. Consistent with evolutionary expectation, our quantitative analysis suggests that paralogous transcription factors have survived extinction in part, either through diversification of their DNA binding site motifs or through alterations in their tissue-specific expression levels