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Find video protocols related to scientific articles indexed in Pubmed.
The genome of a Mongolian individual reveals the genetic imprints of Mongolians on modern human populations.
Genome Biol Evol
PUBLISHED: 11-08-2014
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Mongolians have played a significant role in modern human evolution, especially after the rise of Genghis Khan (1162?-1227). Although the social cultural impacts of Genghis Khan and the Mongolian population have been well documented, explorations of their genome structure and genetic imprints on other human populations have been lacking. We here present the genome of a Mongolian male individual. The genome was de novo assembled using a total of 130.8-fold genomic data produced from massively parallel whole genome sequencing. We identified high-confidence variation sets, including 3.7 million single nucleotide polymorphisms (SNPs) and 756,234 short insertions and deletions (Indels). Functional SNP analysis predicted the individual has a pathogenic risk for carnitine deficiency. We located the patrilineal inheritance of the Mongolian genome to the lineage D3a through Y haplogroup analysis and inferred that the individual has a common patrilineal ancestor with Tibeto-Burman populations and is likely to be the progeny of the earliest settlers in East Asia. We finally investigated the genetic imprints of Mongolians on other human populations using different approaches. We found varying degrees of gene flows between Mongolians and populations living in Europe, South/Central Asia and the Indian subcontinent. The analyses demonstrate that the genetic impacts of Mongolians likely resulted from the expansion of the Mongolian Empire in the 13(th) century. The genome will be of great help in further explorations of modern human evolution and genetic causes of diseases/traits specific to Mongolians.
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Phylogenomics resolves the timing and pattern of insect evolution.
Bernhard Misof, Shanlin Liu, Karen Meusemann, Ralph S Peters, Alexander Donath, Christoph Mayer, Paul B Frandsen, Jessica Ware, Tomáš Flouri, Rolf G Beutel, Oliver Niehuis, Malte Petersen, Fernando Izquierdo-Carrasco, Torsten Wappler, Jes Rust, Andre J Aberer, Ulrike Aspöck, Horst Aspöck, Daniela Bartel, Alexander Blanke, Simon Berger, Alexander Böhm, Thomas R Buckley, Brett Calcott, Junqing Chen, Frank Friedrich, Makiko Fukui, Mari Fujita, Carola Greve, Peter Grobe, Shengchang Gu, Ying Huang, Lars S Jermiin, Akito Y Kawahara, Lars Krogmann, Martin Kubiak, Robert Lanfear, Harald Letsch, Yiyuan Li, Zhenyu Li, Jiguang Li, Haorong Lu, Ryuichiro Machida, Yuta Mashimo, Pashalia Kapli, Duane D McKenna, Guanliang Meng, Yasutaka Nakagaki, José Luis Navarrete-Heredia, Michael Ott, Yanxiang Ou, Günther Pass, Lars Podsiadlowski, Hans Pohl, Björn M von Reumont, Kai Schütte, Kaoru Sekiya, Shota Shimizu, Adam Slipinski, Alexandros Stamatakis, Wenhui Song, Xu Su, Nikolaus U Szucsich, Meihua Tan, Xuemei Tan, Min Tang, Jingbo Tang, Gerald Timelthaler, Shigekazu Tomizuka, Michelle Trautwein, Xiaoli Tong, Toshiki Uchifune, Manfred G Walzl, Brian M Wiegmann, Jeanne Wilbrandt, Benjamin Wipfler, Thomas K F Wong, Qiong Wu, Gengxiong Wu, Yinlong Xie, Shenzhou Yang, Qing Yang, David K Yeates, Kazunori Yoshizawa, Qing Zhang, Rui Zhang, Wenwei Zhang, Yunhui Zhang, Jing Zhao, Chengran Zhou, Lili Zhou, Tanja Ziesmann, Shijie Zou, Yingrui Li, Xun Xu, Yong Zhang, Huanming Yang, Jian Wang, Jun Wang, Karl M Kjer, Xin Zhou.
Science
PUBLISHED: 11-06-2014
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Insects are the most speciose group of animals, but the phylogenetic relationships of many major lineages remain unresolved. We inferred the phylogeny of insects from 1478 protein-coding genes. Phylogenomic analyses of nucleotide and amino acid sequences, with site-specific nucleotide or domain-specific amino acid substitution models, produced statistically robust and congruent results resolving previously controversial phylogenetic relations hips. We dated the origin of insects to the Early Ordovician [~479 million years ago (Ma)], of insect flight to the Early Devonian (~406 Ma), of major extant lineages to the Mississippian (~345 Ma), and the major diversification of holometabolous insects to the Early Cretaceous. Our phylogenomic study provides a comprehensive reliable scaffold for future comparative analyses of evolutionary innovations among insects.
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Comparative analysis of evolutionarily conserved motifs of epidermal growth factor receptor 2 (HER2) predicts novel potential therapeutic epitopes.
PLoS ONE
PUBLISHED: 09-05-2014
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Overexpression of human epidermal growth factor receptor 2 (HER2) is associated with tumor aggressiveness and poor prognosis in breast cancer. With the availability of therapeutic antibodies against HER2, great strides have been made in the clinical management of HER2 overexpressing breast cancer. However, de novo and acquired resistance to these antibodies presents a serious limitation to successful HER2 targeting treatment. The identification of novel epitopes of HER2 that can be used for functional/region-specific blockade could represent a central step in the development of new clinically relevant anti-HER2 antibodies. In the present study, we present a novel computational approach as an auxiliary tool for identification of novel HER2 epitopes. We hypothesized that the structurally and linearly evolutionarily conserved motifs of the extracellular domain of HER2 (ECD HER2) contain potential druggable epitopes/targets. We employed the PROSITE Scan to detect structurally conserved motifs and PRINTS to search for linearly conserved motifs of ECD HER2. We found that the epitopes recognized by trastuzumab and pertuzumab are located in the predicted conserved motifs of ECD HER2, supporting our initial hypothesis. Considering that structurally and linearly conserved motifs can provide functional specific configurations, we propose that by comparing the two types of conserved motifs, additional druggable epitopes/targets in the ECD HER2 protein can be identified, which can be further modified for potential therapeutic application. Thus, this novel computational process for predicting or searching for potential epitopes or key target sites may contribute to epitope-based vaccine and function-selected drug design, especially when x-ray crystal structure protein data is not available.
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A survey of overlooked viral infections in biological experiment systems.
PLoS ONE
PUBLISHED: 08-21-2014
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It is commonly accepted that there are many unknown viruses on the planet. For the known viruses, do we know their prevalence, even in our experimental systems? Here we report a virus survey using recently published small (s)RNA sequencing datasets. The sRNA reads were assembled and contigs were screened for virus homologues against the NCBI nucleotide (nt) database using the BLASTn program. To our surprise, approximately 30% (28 out of 94) of publications had highly scored viral sequences in their datasets. Among them, only two publications reported virus infections. Though viral vectors were used in some of the publications, virus sequences without any identifiable source appeared in more than 20 publications. By determining the distributions of viral reads and the antiviral RNA interference (RNAi) pathways using the sRNA profiles, we showed evidence that many of the viruses identified were indeed infecting and generated host RNAi responses. As virus infections affect many aspects of host molecular biology and metabolism, the presence and impact of viruses needs to be actively investigated in experimental systems.
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Novel recurrently mutated genes and a prognostic mutation signature in colorectal cancer.
Gut
PUBLISHED: 06-22-2014
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Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication.
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GxE Interactions between FOXO Genotypes and Tea Drinking Are Significantly Associated with Cognitive Disability at Advanced Ages in China.
J. Gerontol. A Biol. Sci. Med. Sci.
PUBLISHED: 06-03-2014
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Logistic regression analysis based on data from 822 Han Chinese oldest old aged 92+ demonstrated that interactions between carrying FOXO1A-266 or FOXO3-310 or FOXO3-292 and tea drinking at around age 60 or at present time were significantly associated with lower risk of cognitive disability at advanced ages. Associations between tea drinking and reduced cognitive disability were much stronger among carriers of the genotypes of FOXO1A-266 or FOXO3-310 or FOXO3-292 compared with noncarriers, and it was reconfirmed by analysis of three-way interactions across FOXO genotypes, tea drinking at around age 60, and at present time. Based on prior findings from animal and human cell models, we postulate that intake of tea compounds may activate FOXO gene expression, which in turn may positively affect cognitive function in the oldest old population. Our empirical findings imply that the health benefits of particular nutritional interventions, including tea drinking, may, in part, depend upon individual genetic profiles.
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Altitude adaptation in Tibetans caused by introgression of Denisovan-like DNA.
Nature
PUBLISHED: 04-28-2014
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As modern humans migrated out of Africa, they encountered many new environmental conditions, including greater temperature extremes, different pathogens and higher altitudes. These diverse environments are likely to have acted as agents of natural selection and to have led to local adaptations. One of the most celebrated examples in humans is the adaptation of Tibetans to the hypoxic environment of the high-altitude Tibetan plateau. A hypoxia pathway gene, EPAS1, was previously identified as having the most extreme signature of positive selection in Tibetans, and was shown to be associated with differences in haemoglobin concentration at high altitude. Re-sequencing the region around EPAS1 in 40 Tibetan and 40 Han individuals, we find that this gene has a highly unusual haplotype structure that can only be convincingly explained by introgression of DNA from Denisovan or Denisovan-related individuals into humans. Scanning a larger set of worldwide populations, we find that the selected haplotype is only found in Denisovans and in Tibetans, and at very low frequency among Han Chinese. Furthermore, the length of the haplotype, and the fact that it is not found in any other populations, makes it unlikely that the haplotype sharing between Tibetans and Denisovans was caused by incomplete ancestral lineage sorting rather than introgression. Our findings illustrate that admixture with other hominin species has provided genetic variation that helped humans to adapt to new environments.
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An integrated catalog of reference genes in the human gut microbiome.
Nat. Biotechnol.
PUBLISHED: 04-01-2014
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Many analyses of the human gut microbiome depend on a catalog of reference genes. Existing catalogs for the human gut microbiome are based on samples from single cohorts or on reference genomes or protein sequences, which limits coverage of global microbiome diversity. Here we combined 249 newly sequenced samples of the Metagenomics of the Human Intestinal Tract (MetaHit) project with 1,018 previously sequenced samples to create a cohort from three continents that is at least threefold larger than cohorts used for previous gene catalogs. From this we established the integrated gene catalog (IGC) comprising 9,879,896 genes. The catalog includes close-to-complete sets of genes for most gut microbes, which are also of considerably higher quality than in previous catalogs. Analyses of a group of samples from Chinese and Danish individuals using the catalog revealed country-specific gut microbial signatures. This expanded catalog should facilitate quantitative characterization of metagenomic, metatranscriptomic and metaproteomic data from the gut microbiome to understand its variation across populations in human health and disease.
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Identification of genomic alterations in oesophageal squamous cell cancer.
Nature
PUBLISHED: 02-25-2014
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Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide. Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.
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Camelid genomes reveal evolution and adaptation to desert environments.
Nat Commun
PUBLISHED: 02-06-2014
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Bactrian camel (Camelus bactrianus), dromedary (Camelus dromedarius) and alpaca (Vicugna pacos) are economically important livestock. Although the Bactrian camel and dromedary are large, typically arid-desert-adapted mammals, alpacas are adapted to plateaus. Here we present high-quality genome sequences of these three species. Our analysis reveals the demographic history of these species since the Tortonian Stage of the Miocene and uncovers a striking correlation between large fluctuations in population size and geological time boundaries. Comparative genomic analysis reveals complex features related to desert adaptations, including fat and water metabolism, stress responses to heat, aridity, intense ultraviolet radiation and choking dust. Transcriptomic analysis of Bactrian camels further reveals unique osmoregulation, osmoprotection and compensatory mechanisms for water reservation underpinned by high blood glucose levels. We hypothesize that these physiological mechanisms represent kidney evolutionary adaptations to the desert environment. This study advances our understanding of camelid evolution and the adaptation of camels to arid-desert environments.
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Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing.
Cell Res.
PUBLISHED: 01-22-2014
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Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.
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Direct ChIP-bisulfite sequencing reveals a role of H3K27me3 mediating aberrant hypermethylation of promoter CpG islands in cancer cells.
Genomics
PUBLISHED: 01-07-2014
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The model describing that aberrant CpG island (CGI) methylation leads to repression of tumour suppressor genes in cancers has been influential, but it remains unclear how such aberrancy is induced. Recent studies provided clues indicating that promoter hypermethylation in cancers might be associated with PRC target genes. Here, we used ChIP-BS-seq to examine methylation of the DNA fragments precipitated by the antibodies to both H3K27me3 and H3K4me3 histone modifications. We showed that, for a set of genes highly enriched with H3K27me3 both in cancer and normal cells, CGI promoters were aberrantly hypermethylated only in cancer cells in comparison with normal cells. In contrast, such aberrant CGI hypermethylation in cancer promoters that were deficient of H3K27me3 was not notable. Furthermore, we confirmed that these genes were consistently hypermethylated in TCGA primary cancer cells. These works support the association between H3K27me3 and DNA methylation marks for specific cancer genes and will spur future work on combined histone and DNA methylation that could define cancer's epigenetic abnormalities.
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The co-occurrence of mtDNA mutations on different oxidative phosphorylation subunits, not detected by haplogroup analysis, affects human longevity and is population specific.
Aging Cell
PUBLISHED: 11-14-2013
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To re-examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high-resolution classification, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se have a low, or very low, impact. In particular, the analysis of the mutations occurring in different OXPHOS complex showed a complex scenario with a different mutation burden in 90+ subjects with respect to controls. These findings suggested that mutations in subunits of the OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III (which also occurs in J subhaplogroups involved in LHON) and in complex I and V seemed to be detrimental, likely explaining previous contradictory results. On the whole, our study, which goes beyond haplogroup analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes.
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Genomic insights into salt adaptation in a desert poplar.
Nat Commun
PUBLISHED: 09-01-2013
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Despite the high economic and ecological importance of forests, our knowledge of the genomic evolution of trees under salt stress remains very limited. Here we report the genome sequence of the desert poplar, Populus euphratica, which exhibits high tolerance to salt stress. Its genome is very similar and collinear to that of the closely related mesophytic congener, P. trichocarpa. However, we find that several gene families likely to be involved in tolerance to salt stress contain significantly more gene copies within the P. euphratica lineage. Furthermore, genes showing evidence of positive selection are significantly enriched in functional categories related to salt stress. Some of these genes, and others within the same categories, are significantly upregulated under salt stress relative to their expression in another salt-sensitive poplar. Our results provide an important background for understanding tree adaptation to salt stress and facilitating the genetic improvement of cultivated poplars for saline soils.
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Multilayered molecular profiling supported the monoclonal origin of metastatic renal cell carcinoma.
Int. J. Cancer
PUBLISHED: 07-10-2013
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Primary renal cell carcinomas (pRCCs) have a high degree of intratumoral heterogeneity and are composed of multiple distinct subclones. However, it remains largely unknown that whether metastatic renal cell carcinomas (mRCCs) also have startling intratumoral heterogeneity or whether development of mRCCs is due to early dissemination or late diagnosis. To decipher the evolution of mRCC, we analyzed the multilayered molecular profiles of pRCC, local invasion of the vena cava (IVC), and distant metastasis to the brain (MB) from the same patient using whole-genome sequencing, whole-exome sequencing, DNA methylome profiling, and transcriptome sequencing. We found that mRCC had a lower degree of heterogeneity than pRCC and was likely to result from recent clonal expansion of a rare, advantageous subclone. Consequently, some key pathways that are targeted by clinically available drugs showed distinct expression patterns between pRCC and mRCC. From the genetic distances between different tumor subclones, we estimated that the progeny subclone giving rise to distant metastasis took over half a decade to acquire the full potential of metastasis since the birth of the subclone that evolved into IVC. Our evidence supported that mRCC was monoclonal and distant metastasis occurred late during renal cancer progression. Thus, there was a broad window for early detection of circulating tumor cells and future targeted treatments for patients with mRCCs should rely on the molecular profiles of metastases.
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Integrative analyses of gene expression and DNA methylation profiles in breast cancer cell line models of tamoxifen-resistance indicate a potential role of cells with stem-like properties.
Breast Cancer Res.
PUBLISHED: 05-13-2013
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Development of resistance to tamoxifen is an important clinical issue in the treatment of breast cancer. Tamoxifen resistance may be the result of acquisition of epigenetic regulation within breast cancer cells, such as DNA methylation, resulting in changed mRNA expression of genes pivotal for estrogen-dependent growth. Alternatively, tamoxifen resistance may be due to selection of pre-existing resistant cells, or a combination of the two mechanisms.
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Detection of clinically relevant genetic variants in autism spectrum disorder by whole-genome sequencing.
Am. J. Hum. Genet.
PUBLISHED: 04-07-2013
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Autism Spectrum Disorder (ASD) demonstrates high heritability and familial clustering, yet the genetic causes remain only partially understood as a result of extensive clinical and genomic heterogeneity. Whole-genome sequencing (WGS) shows promise as a tool for identifying ASD risk genes as well as unreported mutations in known loci, but an assessment of its full utility in an ASD group has not been performed. We used WGS to examine 32 families with ASD to detect de novo or rare inherited genetic variants predicted to be deleterious (loss-of-function and damaging missense mutations). Among ASD probands, we identified deleterious de novo mutations in six of 32 (19%) families and X-linked or autosomal inherited alterations in ten of 32 (31%) families (some had combinations of mutations). The proportion of families identified with such putative mutations was larger than has been previously reported; this yield was in part due to the comprehensive and uniform coverage afforded by WGS. Deleterious variants were found in four unrecognized, nine known, and eight candidate ASD risk genes. Examples include CAPRIN1 and AFF2 (both linked to FMR1, which is involved in fragile X syndrome), VIP (involved in social-cognitive deficits), and other genes such as SCN2A and KCNQ2 (linked to epilepsy), NRXN1, and CHD7, which causes ASD-associated CHARGE syndrome. Taken together, these results suggest that WGS and thorough bioinformatic analyses for de novo and rare inherited mutations will improve the detection of genetic variants likely to be associated with ASD or its accompanying clinical symptoms.
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High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus.
Nat Commun
PUBLISHED: 03-27-2013
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Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat. Here we report a 110-Mb draft sequence of Pst isolate CY32, obtained using a fosmid-to-fosmid strategy, to better understand its race evolution and pathogenesis. The Pst genome is highly heterozygous and contains 25,288 protein-coding genes. Compared with non-obligate fungal pathogens, Pst has a more diverse gene composition and more genes encoding secreted proteins. Re-sequencing analysis indicates significant genetic variation among six isolates collected from different continents. Approximately 35% of SNPs are in the coding sequence regions, and half of them are non-synonymous. High genetic diversity in Pst suggests that sexual reproduction has an important role in the origin of different regional races. Our results show the effectiveness of the fosmid-to-fosmid strategy for sequencing dikaryotic genomes and the feasibility of genome analysis to understand race evolution in Pst and other obligate pathogens.
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Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation.
Epigenetics
PUBLISHED: 03-15-2013
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5-methylcytosine (5-mC) can be oxidized to 5-hydroxymethylcytosine (5-hmC). Genome-wide profiling of 5-hmC thus far indicates 5-hmC may not only be an intermediate form of DNA demethylation but could also constitute an epigenetic mark per se. Here we describe a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in a subset of cytosines in the human genome. This method involves the selective glucosylation of 5-hmC residues, short-sequence tag generation and high-throughput sequencing. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells, and found that differential hydroxymethylation preferentially occurs in bivalent genes during cellular differentiation. Especially, our results support hydroxymethylation can regulate key transcription regulators with bivalent marks through demethylation and affect cellular decision on choosing active or inactive state of these genes upon cellular differentiation. Future application of this technology would enable us to uncover the status of methylation and hydroxymethylation in dynamic biological processes and disease development in multiple biological samples.
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Draft genome sequence of the mulberry tree Morus notabilis.
Nat Commun
PUBLISHED: 03-05-2013
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Human utilization of the mulberry-silkworm interaction started at least 5,000 years ago and greatly influenced world history through the Silk Road. Complementing the silkworm genome sequence, here we describe the genome of a mulberry species Morus notabilis. In the 330-Mb genome assembly, we identify 128 Mb of repetitive sequences and 29,338 genes, 60.8% of which are supported by transcriptome sequencing. Mulberry gene sequences appear to evolve ~3 times faster than other Rosales, perhaps facilitating the species spread worldwide. The mulberry tree is among a few eudicots but several Rosales that have not preserved genome duplications in more than 100 million years; however, a neopolyploid series found in the mulberry tree and several others suggest that new duplications may confer benefits. Five predicted mulberry miRNAs are found in the haemolymph and silk glands of the silkworm, suggesting interactions at molecular levels in the plant-herbivore relationship. The identification and analyses of mulberry genes involved in diversifying selection, resistance and protease inhibitor expressed in the laticifers will accelerate the improvement of mulberry plants.
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Metagenome-wide analysis of antibiotic resistance genes in a large cohort of human gut microbiota.
Nat Commun
PUBLISHED: 02-21-2013
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The human gut microbiota is a reservoir of antibiotic resistance genes, but little is known about their diversity and richness within the gut. Here we analyse the antibiotic resistance genes of gut microbiota from 162 individuals. We identify a total of 1,093 antibiotic resistance genes and find that Chinese individuals harbour the highest number and abundance of antibiotic resistance genes, followed by Danish and Spanish individuals. Single-nucleotide polymorphism-based analysis indicates that antibiotic resistance genes from the two European populations are more closely related while the Chinese ones are clustered separately. We also confirm high abundance of tetracycline resistance genes with this large cohort study. Our study provides a broad view of antibiotic resistance genes in the human gut microbiota.
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Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation.
Nature
PUBLISHED: 02-20-2013
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About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD). Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of bakers flour. Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae.?tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.
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Draft genome of the wheat A-genome progenitor Triticum urartu.
Nature
PUBLISHED: 02-08-2013
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Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A?genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m)A(m)), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A?genome is the diploid wild einkorn wheat T.?urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T.?urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T.?urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.
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Mutations in ABCB6 cause dyschromatosis universalis hereditaria.
J. Invest. Dermatol.
PUBLISHED: 01-24-2013
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Dyschromatosis universalis hereditaria (DUH) is a pigmentary genodermatosis characterized by a mixture of hyperpigmented and hypopigmented macules distributed randomly over the body. No causative genes have been reported to date. In this study, we investigated a large five-generation Chinese family with DUH. After excluding the two known DUH loci, we performed genome-wide linkage analysis and identified a DUH locus on chromosome 2q33.3-q36.1 with a maximum LOD score of 3.49 with marker D2S2382. Exome sequencing identified a c.1067T>C (p.Leu356Pro) mutation in exon 3 of ABCB6 (ATP-binding cassette subfamily B, member 6) in the DUH family. Two additional missense mutations, c.508A>G (p.Ser170Gly) in exon 1 and c.1736G>A (p.Gly579Glu) in exon 12 of ABCB6, were found in two out of six patients by mutational screening using sporadic DUH patients. Immunohistologic examination in biopsy specimens showed that ABCB6 is expressed in the epidermis and had a diffuse cytoplasmic distribution. Examination of subcellular localization of wild-type ABCB6 in a B16 mouse melanoma cell line revealed that it is localized to the endosome-like compartment and dendrite tips, whereas disease-causing mutations of ABCB6 resulted in its retention in the Golgi apparatus. Our studies identified ABCB6 as the first pathogenic gene associated with DUH. These findings suggest that ABCB6 may be a physiological factor for skin pigmentation.
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Genetic basis of Y-linked hearing impairment.
Am. J. Hum. Genet.
PUBLISHED: 01-24-2013
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A single Mendelian trait has been mapped to the human Y chromosome: Y-linked hearing impairment. The molecular basis of this disorder is unknown. Here, we report the detailed characterization of the DFNY1 Y chromosome and its comparison with a closely related Y chromosome from an unaffected branch of the family. The DFNY1 chromosome carries a complex rearrangement, including duplication of several noncontiguous segments of the Y chromosome and insertion of ?160 kb of DNA from chromosome 1, in the pericentric region of Yp. This segment of chromosome 1 is derived entirely from within a known hearing impairment locus, DFNA49. We suggest that a third copy of one or more genes from the shared segment of chromosome 1 might be responsible for the hearing-loss phenotype.
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Whole-genome and whole-exome sequencing of bladder cancer identifies frequent alterations in genes involved in sister chromatid cohesion and segregation.
Nat. Genet.
PUBLISHED: 01-15-2013
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Bladder cancer is one of the most common cancers worldwide, with transitional cell carcinoma (TCC) being the predominant form. Here we report a genomic analysis of TCC by both whole-genome and whole-exome sequencing of 99 individuals with TCC. Beyond confirming recurrent mutations in genes previously identified as being mutated in TCC, we identified additional altered genes and pathways that were implicated in TCC. Notably, we discovered frequent alterations in STAG2 and ESPL1, two genes involved in the sister chromatid cohesion and segregation (SCCS) process. Furthermore, we also detected a recurrent fusion involving FGFR3 and TACC3, another component of SCCS, by transcriptome sequencing of 42 DNA-sequenced tumors. Overall, 32 of the 99 tumors (32%) harbored genetic alterations in the SCCS process. Our analysis provides evidence that genetic alterations affecting the SCCS process may be involved in bladder tumorigenesis and identifies a new therapeutic possibility for bladder cancer.
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A heterozygous moth genome provides insights into herbivory and detoxification.
Nat. Genet.
PUBLISHED: 01-13-2013
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How an insect evolves to become a successful herbivore is of profound biological and practical importance. Herbivores are often adapted to feed on a specific group of evolutionarily and biochemically related host plants, but the genetic and molecular bases for adaptation to plant defense compounds remain poorly understood. We report the first whole-genome sequence of a basal lepidopteran species, Plutella xylostella, which contains 18,071 protein-coding and 1,412 unique genes with an expansion of gene families associated with perception and the detoxification of plant defense compounds. A recent expansion of retrotransposons near detoxification-related genes and a wider system used in the metabolism of plant defense compounds are shown to also be involved in the development of insecticide resistance. This work shows the genetic and molecular bases for the evolutionary success of this worldwide herbivore and offers wider insights into insect adaptation to plant feeding, as well as opening avenues for more sustainable pest management.
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Deep sequencing identifies regulated small RNAs in Dugesia japonica.
Mol. Biol. Rep.
PUBLISHED: 01-12-2013
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MicroRNAs (miRNAs), which are 18 ~ 24 nucleotides length, play important roles in regulating the expression of gene at the post-transcription level. Dugesia japonica is a branch of planarian organism. It is a model organism for studying the role of miRNAs in stem cell function. Next generation sequencing technology was used to identify the miRNAs of D. japonica. Bioinformatic analysis showed that 262 miRNA and miRNA* sequences were discovered, of which 102 miRNAs were the same as Schmidtea mediterranea and 160 miRNAs were related to other animals. There were 21 miRNAs expressed differentially after amputation. Results also revealed that some key miRNAs might play essential roles in the regeneration progress and some miRNAs might take part in the regulation progress of polarity regeneration in D. japonica.
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TIMP-1 overexpression does not affect sensitivity to HER2-targeting drugs in the HER2-gene-amplified SK-BR-3 human breast cancer cell line.
Tumour Biol.
PUBLISHED: 01-09-2013
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Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been suggested as a marker of prognosis and response to treatment in breast cancer. In vitro, TIMP-1 can regulate shedding of the extracellular domain of HER2 and signalling via the Akt pathway, and we hypothesize that TIMP-1 therefore can affect sensitivity to the HER2-targeting drugs trastuzumab and lapatinib. SK-BR-3 human breast cancer cells were stably transfected with TIMP-1, characterized with regard to TIMP-1 protein expression, proliferation, and functionality of the secreted TIMP-1, and the sensitivity to trastuzumab and lapatinib was studied in five selected single-cell subclones expressing TIMP-1 protein at various levels plus the parental SK-BR-3 cell line. Both trastuzumab and lapatinib reduced cell viability, as determined by MTT assay, but the sensitivity to the drugs was not associated with the expression level of TIMP-1 protein. Western blotting showed that the activation of Akt, PTEN, and HER2 as well as ADAM10 was similar in all clones. In conclusion, in this model, TIMP-1 overexpression does not affect HER2 cleavage by ADAM10 or signalling via the Akt pathway, and TIMP-1 does not influence sensitivity to trastuzumab and lapatinib.
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Exome capture sequencing of adenoma reveals genetic alterations in multiple cellular pathways at the early stage of colorectal tumorigenesis.
PLoS ONE
PUBLISHED: 01-02-2013
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Most of colorectal adenocarcinomas are believed to arise from adenomas, which are premalignant lesions. Sequencing the whole exome of the adenoma will help identifying molecular biomarkers that can predict the occurrence of adenocarcinoma more precisely and help understanding the molecular pathways underlying the initial stage of colorectal tumorigenesis. We performed the exome capture sequencing of the normal mucosa, adenoma and adenocarcinoma tissues from the same patient and sequenced the identified mutations in additional 73 adenomas and 288 adenocarcinomas. Somatic single nucleotide variations (SNVs) were identified in both the adenoma and adenocarcinoma by comparing with the normal control from the same patient. We identified 12 nonsynonymous somatic SNVs in the adenoma and 42 nonsynonymous somatic SNVs in the adenocarcinoma. Most of these mutations including OR6X1, SLC15A3, KRTHB4, RBFOX1, LAMA3, CDH20, BIRC6, NMBR, GLCCI1, EFR3A, and FTHL17 were newly reported in colorectal adenomas. Functional annotation of these mutated genes showed that multiple cellular pathways including Wnt, cell adhesion and ubiquitin mediated proteolysis pathways were altered genetically in the adenoma and that the genetic alterations in the same pathways persist in the adenocarcinoma. CDH20 and LAMA3 were mutated in the adenoma while NRXN3 and COL4A6 were mutated in the adenocarcinoma from the same patient, suggesting for the first time that genetic alterations in the cell adhesion pathway occur as early as in the adenoma. Thus, the comparison of genomic mutations between adenoma and adenocarcinoma provides us a new insight into the molecular events governing the early step of colorectal tumorigenesis.
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Development of transgenic minipigs with expression of antimorphic human cryptochrome 1.
PLoS ONE
PUBLISHED: 01-01-2013
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Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3-20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1(AP)). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-?, known to be risk factors in cancer and metabolic disorders.
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Exome sequencing and linkage analysis identified tenascin-C (TNC) as a novel causative gene in nonsyndromic hearing loss.
PLoS ONE
PUBLISHED: 01-01-2013
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In this study, a five-generation Chinese family (family F013) with progressive autosomal dominant hearing loss was mapped to a critical region spanning 28.54 Mb on chromosome 9q31.3-q34.3 by linkage analysis, which was a novel DFNA locus, assigned as DFNA56. In this interval, there were 398 annotated genes. Then, whole exome sequencing was applied in three patients and one normal individual from this family. Six single nucleotide variants and two indels were found co-segregated with the phenotypes. Then using mass spectrum (Sequenom, Inc.) to rank the eight sites, we found only the TNC gene be co-segregated with hearing loss in 53 subjects of F013. And this missense mutation (c.5317G>A, p.V1773M ) of TNC located exactly in the critical linked interval. Further screening to the coding region of this gene in 587 subjects with nonsyndromic hearing loss (NSHL) found a second missense mutation, c.5368A>T (p. T1796S), co-segregating with phenotype in the other family. These two mutations located in the conserved region of TNC and were absent in the 387 normal hearing individuals of matched geographical ancestry. Functional effects of the two mutations were predicted using SIFT and both mutations were deleterious. All these results supported that TNC may be the causal gene for the hearing loss inherited in these families. TNC encodes tenascin-C, a member of the extracellular matrix (ECM), is present in the basilar membrane (BM), and the osseous spiral lamina of the cochlea. It plays an important role in cochlear development. The up-regulated expression of TNC gene in tissue repair and neural regeneration was seen in human and zebrafish, and in sensory receptor recovery in the vestibular organ after ototoxic injury in birds. Then the absence of normal tenascin-C was supposed to cause irreversible injuries in cochlea and caused hearing loss.
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Haplotype-assisted accurate non-invasive fetal whole genome recovery through maternal plasma sequencing.
Genome Med
PUBLISHED: 01-01-2013
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BACKGROUND: The applications of massively parallel sequencing technology to fetal cell-free DNA (cff-DNA) have brought new insight to non-invasive prenatal diagnosis. However, most previous research based on maternal plasma sequencing has been restricted to fetal aneuploidies. To detect specific parentally inherited mutations, invasive approaches to obtain fetal DNA are the current standard in the clinic because of the experimental complexity and resource consumption of previously reported non-invasive approaches. METHODS: Here, we present a simple and effective non-invasive method for accurate fetal genome recovery-assisted with parental haplotypes. The parental haplotype were firstly inferred using a combination strategy of trio and unrelated individuals. Assisted with the parental haplotype, we then employed a hidden Markov model to non-invasively recover the fetal genome through maternal plasma sequencing. RESULTS: Using a sequence depth of approximately 44X against a an approximate 5.69% cff-DNA concentration, we non-invasively inferred fetal genotype and haplotype under different situations of parental heterozygosity. Our data show that 98.57%, 95.37%, and 98.45% of paternal autosome alleles, maternal autosome alleles, and maternal chromosome X in the fetal haplotypes, respectively, were recovered accurately. Additionally, we obtained efficient coverage or strong linkage of 96.65% of reported Mendelian-disorder genes and 98.90% of complex disease-associated markers. CONCLUSIONS: Our method provides a useful strategy for non-invasive whole fetal genome recovery.
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Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4.
N. Engl. J. Med.
PUBLISHED: 07-27-2011
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An outbreak caused by Shiga-toxin–producing Escherichia coli O104:H4 occurred in Germany in May and June of 2011, with more than 3000 persons infected. Here, we report a cluster of cases associated with a single family and describe an open-source genomic analysis of an isolate from one member of the family. This analysis involved the use of rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses. In less than a week, these studies revealed that the outbreak strain belonged to an enteroaggregative E. coli lineage that had acquired genes for Shiga toxin 2 and for antibiotic resistance.
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Draft genome sequence of pigeonpea (Cajanus cajan), an orphan legume crop of resource-poor farmers.
Nat. Biotechnol.
PUBLISHED: 07-19-2011
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Pigeonpea is an important legume food crop grown primarily by smallholder farmers in many semi-arid tropical regions of the world. We used the Illumina next-generation sequencing platform to generate 237.2 Gb of sequence, which along with Sanger-based bacterial artificial chromosome end sequences and a genetic map, we assembled into scaffolds representing 72.7% (605.78 Mb) of the 833.07 Mb pigeonpea genome. Genome analysis predicted 48,680 genes for pigeonpea and also showed the potential role that certain gene families, for example, drought tolerance-related genes, have played throughout the domestication of pigeonpea and the evolution of its ancestors. Although we found a few segmental duplication events, we did not observe the recent genome-wide duplication events observed in soybean. This reference genome sequence will facilitate the identification of the genetic basis of agronomically important traits, and accelerate the development of improved pigeonpea varieties that could improve food security in many developing countries.
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A systematic analysis on DNA methylation and the expression of both mRNA and microRNA in bladder cancer.
PLoS ONE
PUBLISHED: 07-05-2011
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DNA methylation aberration and microRNA (miRNA) deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported.
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Association of PDE4B polymorphisms and schizophrenia in Northwestern Han Chinese.
Hum. Genet.
PUBLISHED: 06-21-2011
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The phosphodiesterase 4B (PDE4B) is a candidate susceptibility gene for schizophrenia (SCZ), interacting with DISC1, a known genetic risk factor for SCZ. To examine if variants within PDE4B gene are associated with SCZ in Northwestern Han Chinese, and if these effects vary in gender-specific subgroup, we analyzed 20 SNPs, selected from previous studies and preliminary HapMap data analyses with minor allele frequency (MAF) ? 20%, in a cohort of 428 cases and 572 controls from genetically independent Northwestern Han Chinese. Single SNP association, haplotype association and sex-specific association analysis were performed. We found that rs472952 is significantly associated with SCZ and rs7537440 is associated with SCZ in females. Further analysis indicated that a haplotype block spanning PDE4B2 splice site is highly associated with SCZ and several haplotypes in this block have about twofold to threefold increase in cases. Our results provide further evidence that PDE4B may play important roles in the etiology of SCZ.
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Ascaris suum draft genome.
Nature
PUBLISHED: 06-16-2011
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Parasitic diseases have a devastating, long-term impact on human health, welfare and food production worldwide. More than two billion people are infected with geohelminths, including the roundworms Ascaris (common roundworm), Necator and Ancylostoma (hookworms), and Trichuris (whipworm), mainly in developing or impoverished nations of Asia, Africa and Latin America. In humans, the diseases caused by these parasites result in about 135,000 deaths annually, with a global burden comparable with that of malaria or tuberculosis in disability-adjusted life years. Ascaris alone infects around 1.2 billion people and, in children, causes nutritional deficiency, impaired physical and cognitive development and, in severe cases, death. Ascaris also causes major production losses in pigs owing to reduced growth, failure to thrive and mortality. The Ascaris-swine model makes it possible to study the parasite, its relationship with the host, and ascariasis at the molecular level. To enable such molecular studies, we report the 273 megabase draft genome of Ascaris suum and compare it with other nematode genomes. This genome has low repeat content (4.4%) and encodes about 18,500 protein-coding genes. Notably, the A. suum secretome (about 750 molecules) is rich in peptidases linked to the penetration and degradation of host tissues, and an assemblage of molecules likely to modulate or evade host immune responses. This genome provides a comprehensive resource to the scientific community and underpins the development of new and urgently needed interventions (drugs, vaccines and diagnostic tests) against ascariasis and other nematodiases.
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Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma.
Nat. Genet.
PUBLISHED: 06-03-2011
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We sequenced whole exomes of ten clear cell renal cell carcinomas (ccRCCs) and performed a screen of ?1,100 genes in 88 additional ccRCCs, from which we discovered 12 previously unidentified genes mutated at elevated frequencies in ccRCC. Notably, we detected frequent mutations in the ubiquitin-mediated proteolysis pathway (UMPP), and alterations in the UMPP were significantly associated with overexpression of HIF1? and HIF2? in the tumors (P = 0.01 and 0.04, respectively). Our findings highlight the potential contribution of UMPP to ccRCC tumorigenesis through the activation of the hypoxia regulatory network.
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Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder.
Nat. Genet.
PUBLISHED: 05-13-2011
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Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Here we sequenced the exomes of nine individuals with TCC and screened all the somatically mutated genes in a prevalence set of 88 additional individuals with TCC with different tumor stages and grades. In our study, we discovered a variety of genes previously unknown to be mutated in TCC. Notably, we identified genetic aberrations of the chromatin remodeling genes (UTX, MLL-MLL3, CREBBP-EP300, NCOR1, ARID1A and CHD6) in 59% of our 97 subjects with TCC. Of these genes, we showed UTX to be altered substantially more frequently in tumors of low stages and grades, highlighting its potential role in the classification and diagnosis of bladder cancer. Our results provide an overview of the genetic basis of TCC and suggest that aberration of chromatin regulation might be a hallmark of bladder cancer.
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Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls.
PLoS ONE
PUBLISHED: 04-27-2011
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Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.
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Comparative mRNA and microRNA expression profiling of three genitourinary cancers reveals common hallmarks and cancer-specific molecular events.
PLoS ONE
PUBLISHED: 04-07-2011
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Genome-wide gene expression profile using deep sequencing technologies can drive the discovery of cancer biomarkers and therapeutic targets. Such efforts are often limited to profiling the expression signature of either mRNA or microRNA (miRNA) in a single type of cancer.
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Aseptic minimum volume vitrification technique for porcine parthenogenetically activated blastocyst.
Cryo Letters
PUBLISHED: 04-07-2011
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Minimum volume vitrification may provide extremely high cooling and warming rates if the sample and the surrounding medium contacts directly with the respective liquid nitrogen and warming medium. However, this direct contact may result in microbial contamination. In this work, an earlier aseptic technique was applied for minimum volume vitrification. After equilibration, samples were loaded on a plastic film, immersed rapidly into factory derived, filter-sterilized liquid nitrogen, and sealed into sterile, pre-cooled straws. At warming, the straw was cut, the filmstrip was immersed into a 39 degree C warming medium, and the sample was stepwise rehydrated. Cryosurvival rates of porcine blastocysts produced by parthenogenetical activation did not differ from control, vitrified blastocysts with Cryotop. This approach can be used for minimum volume vitrification methods and may be suitable to overcome the biological dangers and legal restrictions that hamper the application of open vitrification techniques.
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Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques.
Nat. Biotechnol.
PUBLISHED: 03-08-2011
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The nonhuman primates most commonly used in medical research are from the genus Macaca. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.
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Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly.
Nat. Biotechnol.
PUBLISHED: 03-04-2011
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Here we use whole-genome de novo assembly of second-generation sequencing reads to map structural variation (SV) in an Asian genome and an African genome. Our approach identifies small- and intermediate-size homozygous variants (1-50 kb) including insertions, deletions, inversions and their precise breakpoints, and in contrast to other methods, can resolve complex rearrangements. In total, we identified 277,243 SVs ranging in length from 1-23 kb. Validation using computational and experimental methods suggests that we achieve overall <6% false-positive rate and <10% false-negative rate in genomic regions that can be assembled, which outperforms other methods. Analysis of the SVs in the genomes of 106 individuals sequenced as part of the 1000 Genomes Project suggests that SVs account for a greater fraction of the diversity between individuals than do single-nucleotide polymorphisms (SNPs). These findings demonstrate that whole-genome de novo assembly is a feasible approach to deriving more comprehensive maps of genetic variation.
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Multiple gene dysfunctions lead to high cancer-susceptibility: evidences from a whole-exome sequencing study.
Am J Cancer Res
PUBLISHED: 02-22-2011
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A total of $275 million has been launched to The Cancer Genome Atlas Project for genomic mapping of more than 20 types of cancers. The major challenge is to develop high throughput and cost-effective techniques for human genome sequencing. We developed a targeted exome sequencing technology to routinely determine human exome sequence. As a proof-of-concept, we chose a unique patient, who underwent three high mortalities cancers, i.e., breast, gallbladder and lung cancers, to reveal the genetic cause of high-cancer-susceptibility. Total 24,545 SNPs were detected. 10,868 (44.27%) SNPs were within coding regions, and 1,077 (4.38%) located in the UTRs. 3367 genes were hit by 4480 non-sysnonymous mutations in CDS with truncation of 30 proteins; and 10 mutations occurred at the splice sites that would generate different protein isoforms. Substitutions or premature terminations occurred in 132 proteins encoded by cancer-associated genes. CARD8 was completely loss; ANAPC1 was pre-translationally terminated from the transcripts of one allele. On the Ras-MAPK pathway, 18 genes were homozygously mutated. 15 growth factors/cytokines and their receptors, 9 transcription factors, 6 proteins on WNT signaling pathway, and 16 cell surface and extracellular proteins may be dysfunctioned. Exome sequencing made it possible for individualized cancer therapy.
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Exome sequencing identifies ZNF644 mutations in high myopia.
PLoS Genet.
PUBLISHED: 01-18-2011
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Myopia is the most common ocular disorder worldwide, and high myopia in particular is one of the leading causes of blindness. Genetic factors play a critical role in the development of myopia, especially high myopia. Recently, the exome sequencing approach has been successfully used for the disease gene identification of Mendelian disorders. Here we show a successful application of exome sequencing to identify a gene for an autosomal dominant disorder, and we have identified a gene potentially responsible for high myopia in a monogenic form. We captured exomes of two affected individuals from a Han Chinese family with high myopia and performed sequencing analysis by a second-generation sequencer with a mean coverage of 30× and sufficient depth to call variants at ?97% of each targeted exome. The shared genetic variants of these two affected individuals in the family being studied were filtered against the 1000 Genomes Project and the dbSNP131 database. A mutation A672G in zinc finger protein 644 isoform 1 (ZNF644) was identified as being related to the phenotype of this family. After we performed sequencing analysis of the exons in the ZNF644 gene in 300 sporadic cases of high myopia, we identified an additional five mutations (I587V, R680G, C699Y, 3UTR+12 C>G, and 3UTR+592 G>A) in 11 different patients. All these mutations were absent in 600 normal controls. The ZNF644 gene was expressed in human retinal and retinal pigment epithelium (RPE). Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, mutation may cause the axial elongation of eyeball found in high myopia patients. Our results suggest that ZNF644 might be a causal gene for high myopia in a monogenic form.
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High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer.
Transgenic Res.
PUBLISHED: 10-07-2010
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Germline inactivating mutations of the breast cancer associated gene 1 (BRCA1) predispose to breast cancer and account for most cases of familiar breast and/or ovarian cancer. The pig is an excellent model for medical research as well as testing of new methods and drugs for disease prevention and treatment. We have generated cloned BRCA1 knockout (KO) Yucatan miniature piglets by targeting exon 11 using recombinant adeno-associated virus (rAAV)-mediated gene targeting and somatic cell nuclear transfer by Handmade Cloning (HMC). We found a very high targeting rate of rAAV-mediated BRCA1 KO. Approximately 35% of the selected cells were BRCA1 targeted. One BRCA1 KO cell clone (5D1), identified by PCR and Southern blot, was used as nuclear donor for HMC. Reconstructed embryos were transferred to three recipient sows which gave birth to 8 piglets in total. Genotyping identified seven piglets as BRCA1 heterozygotes (BRCA1(+/?11)), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1(+/?11) fibroblasts. However, all BRCA1(+/?11) piglets died within 18 days after birth. The causes of perinatal mortality remain unclear. Possible explanations may include a combination of the BRCA1 haploinsufficiency, problems of epigenetic reprogramming, presence of the marker gene, single cell clone effects, and/or the special genetic background of the minipigs.
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The DNA methylome of human peripheral blood mononuclear cells.
PLoS Biol.
PUBLISHED: 09-21-2010
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DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and <0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine methylation is minor in human PBMC. Analysis of the PBMC methylome revealed a rich epigenomic landscape for 20 distinct genomic features, including regulatory, protein-coding, non-coding, RNA-coding, and repeat sequences. Integration of our methylome data with the YH genome sequence enabled a first comprehensive assessment of allele-specific methylation (ASM) between the two haploid methylomes of any individual and allowed the identification of 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2 kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.
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Genome-wide patterns of genetic variation among elite maize inbred lines.
Nat. Genet.
PUBLISHED: 09-13-2010
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We have resequenced a group of six elite maize inbred lines, including the parents of the most productive commercial hybrid in China. This effort uncovered more than 1,000,000 SNPs, 30,000 indel polymorphisms and 101 low-sequence-diversity chromosomal intervals in the maize genome. We also identified several hundred complete genes that show presence/absence variation among these resequenced lines. We discuss the potential roles of complementation of presence/absence variations and other deleterious mutations in contributing to heterosis. High-density SNP and indel polymorphism markers reported here are expected to be a valuable resource for future genetic studies and the molecular breeding of this important crop.
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High hydrostatic pressure treatment of porcine oocytes induces parthenogenetic activation.
Cell Reprogram
PUBLISHED: 08-12-2010
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An innovative technique called high hydrostatic pressure (HHP) treatment has recently been reported to improve the cryosurvival of gametes and embryos in certain mammalian species, including the mouse, pig, and cattle. In the present study the parthenogenetic activation (PA) of pig oocytes caused by HHP treatment was investigated in different holding media with or without Ca(2+). The efficiency of activation was tested at different pressure levels and media including T2 (HEPES-buffered TCM-199 containing 2% cattle serum), and mannitol-PVA fusion medium with (MPVA + Ca(2+)) or without Ca(2+) and Mg(2+)(MPVA). The results showed that HHP cannot induce PA in T2, but only in MPVA + Ca(2+) with low Ca(2+) concentration and MPVA without Ca(2+). The highest activation efficiency was achieved with 10 min HHP treatment using 100 or 200 bars for oocytes in MPVA + Ca(2+) or MPVA, respectively. In the light of these results, the possible source of Ca(2+) during activation was investigated. It was found that even after a total of 30-min wash with TL-HEPES-PVA buffer without Ca(2+) before HHP treatment in MPVA, the oocytes could still be activated, indicating the possibility of an intracellular Ca(2+) source caused cytoplasmic free Ca(2+) elevation. In conclusion, parthenogenetic activation could be induced by HHP in certain holding media with low or zero Ca(2+) content. Further experiments are needed to identify the exact mechanisms of activation.
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Integrated profiling of microRNAs and mRNAs: microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma.
PLoS ONE
PUBLISHED: 08-04-2010
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With the advent of second-generation sequencing, the expression of gene transcripts can be digitally measured with high accuracy. The purpose of this study was to systematically profile the expression of both mRNA and miRNA genes in clear cell renal cell carcinoma (ccRCC) using massively parallel sequencing technology.
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Evolutionary transients in the rice transcriptome.
Genomics Proteomics Bioinformatics
PUBLISHED: 07-21-2010
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In the canonical version of evolution by gene duplication, one copy is kept unaltered while the other is free to evolve. This process of evolutionary experimentation can persist for millions of years. Since it is so short lived in comparison to the lifetime of the core genes that make up the majority of most genomes, a substantial fraction of the genome and the transcriptome may-in principle-be attributable to what we will refer to as "evolutionary transients", referring here to both the process and the genes that have gone or are undergoing this process. Using the rice gene set as a test case, we argue that this phenomenon goes a long way towards explaining why there are so many more rice genes than Arabidopsis genes, and why most excess rice genes show low similarity to eudicots.
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Sequencing of 50 human exomes reveals adaptation to high altitude.
Science
PUBLISHED: 07-03-2010
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Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency difference between Tibetan and Han samples, representing the fastest allele frequency change observed at any human gene to date. This SNPs association with erythrocyte abundance supports the role of EPAS1 in adaptation to hypoxia. Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.
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The Trw type IV secretion system of Bartonella mediates host-specific adhesion to erythrocytes.
PLoS Pathog.
PUBLISHED: 05-10-2010
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Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The alpha-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.
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International network of cancer genome projects.
, Thomas J Hudson, Warwick Anderson, Axel Artez, Anna D Barker, Cindy Bell, Rosa R Bernabé, M K Bhan, Fabien Calvo, Iiro Eerola, Daniela S Gerhard, Alan Guttmacher, Mark Guyer, Fiona M Hemsley, Jennifer L Jennings, David Kerr, Peter Klatt, Patrik Kolar, Jun Kusada, David P Lane, Frank Laplace, Lu Youyong, Gerd Nettekoven, Brad Ozenberger, Jane Peterson, T S Rao, Jacques Remacle, Alan J Schafer, Tatsuhiro Shibata, Michael R Stratton, Joseph G Vockley, Koichi Watanabe, Huanming Yang, Matthew M F Yuen, Bartha M Knoppers, Martin Bobrow, Anne Cambon-Thomsen, Lynn G Dressler, Stephanie O M Dyke, Yann Joly, Kazuto Kato, Karen L Kennedy, Pilar Nicolás, Michael J Parker, Emmanuelle Rial-Sebbag, Carlos M Romeo-Casabona, Kenna M Shaw, Susan Wallace, Georgia L Wiesner, Nikolajs Zeps, Peter Lichter, Andrew V Biankin, Christian Chabannon, Lynda Chin, Bruno Clément, Enrique De Alava, Françoise Degos, Martin L Ferguson, Peter Geary, D Neil Hayes, Amber L Johns, Arek Kasprzyk, Hidewaki Nakagawa, Robert Penny, Miguel A Piris, Rajiv Sarin, Aldo Scarpa, Marc van de Vijver, P Andrew Futreal, Hiroyuki Aburatani, Mònica Bayés, David D L Botwell, Peter J Campbell, Xavier Estivill, Sean M Grimmond, Ivo Gut, Martin Hirst, Carlos Lopez-Otin, Partha Majumder, Marco Marra, John D McPherson, Zemin Ning, Xose S Puente, Yijun Ruan, Hendrik G Stunnenberg, Harold Swerdlow, Victor E Velculescu, Richard K Wilson, Hong H Xue, Liu Yang, Paul T Spellman, Gary D Bader, Paul C Boutros, Paul Flicek, Gad Getz, Roderic Guigo, Guangwu Guo, David Haussler, Simon Heath, Tim J Hubbard, Tao Jiang, Steven M Jones, Qibin Li, Nuria López-Bigas, Ruibang Luo, Lakshmi Muthuswamy, B F Francis Ouellette, John V Pearson, Víctor Quesada, Benjamin J Raphael, Chris Sander, Terence P Speed, Lincoln D Stein, Joshua M Stuart, Jon W Teague, Yasushi Totoki, Tatsuhiko Tsunoda, Alfonso Valencia, David A Wheeler, Honglong Wu, Shancen Zhao, Guangyu Zhou, Mark Lathrop, Gilles Thomas, Teruhiko Yoshida, Myles Axton, Chris Gunter, Linda J Miller, Junjun Zhang, Syed A Haider, Jianxin Wang, Christina K Yung, Anthony Cros, Anthony Cross, Yong Liang, Saravanamuttu Gnaneshan, Jonathan Guberman, Jack Hsu, Don R C Chalmers, Karl W Hasel, Terry S H Kaan, William W Lowrance, Tohru Masui, Laura Lyman Rodriguez, Catherine Vergely, David D L Bowtell, Nicole Cloonan, Anna deFazio, James R Eshleman, Dariush Etemadmoghadam, Brooke B Gardiner, Brooke A Gardiner, James G Kench, Robert L Sutherland, Margaret A Tempero, Nicola J Waddell, Peter J Wilson, Steve Gallinger, Ming-Sound Tsao, Patricia A Shaw, Gloria M Petersen, Debabrata Mukhopadhyay, Ronald A DePinho, Sarah Thayer, Kamran Shazand, Timothy Beck, Michelle Sam, Lee Timms, Vanessa Ballin, Youyong Lu, Jiafu Ji, Xiuqing Zhang, Feng Chen, Xueda Hu, Qi Yang, Geng Tian, Lianhai Zhang, Xiaofang Xing, Xianghong Li, Zhenggang Zhu, Yingyan Yu, Jun Yu, Jörg Tost, Paul Brennan, Ivana Holcatova, David Zaridze, Alvis Brazma, Lars Egevard, Egor Prokhortchouk, Rosamonde Elizabeth Banks, Mathias Uhlén, Juris Viksna, Fredrik Ponten, Konstantin Skryabin, Ewan Birney, Ake Borg, Anne-Lise Børresen-Dale, Carlos Caldas, John A Foekens, Sancha Martin, Jorge S Reis-Filho, Andrea L Richardson, Christos Sotiriou, Giles Thoms, Laura van't Veer, Daniel Birnbaum, Hélène Blanché, Pascal Boucher, Sandrine Boyault, Jocelyne D Masson-Jacquemier, Iris Pauporté, Xavier Pivot, Anne Vincent-Salomon, Eric Tabone, Charles Theillet, Isabelle Treilleux, Paulette Bioulac-Sage, Thomas Decaens, Dominique Franco, Marta Gut, Didier Samuel, Jessica Zucman-Rossi, Roland Eils, Benedikt Brors, Jan O Korbel, Andrey Korshunov, Pablo Landgraf, Hans Lehrach, Stefan Pfister, Bernhard Radlwimmer, Guido Reifenberger, Michael D Taylor, Christof von Kalle, Partha P Majumder, Paolo Pederzoli, Rita A Lawlor, Massimo Delledonne, Alberto Bardelli, Thomas Gress, David Klimstra, Giuseppe Zamboni, Yusuke Nakamura, Satoru Miyano, Akihiro Fujimoto, Elias Campo, Silvia de Sanjosé, Emili Montserrat, Marcos Gonzalez-Díaz, Pedro Jares, Heinz Himmelbauer, Heinz Himmelbaue, Sílvia Beà, Samuel Aparicio, Douglas F Easton, Francis S Collins, Carolyn C Compton, Eric S Lander, Wylie Burke, Anthony R Green, Stanley R Hamilton, Olli P Kallioniemi, Timothy J Ley, Edison T Liu, Brandon J Wainwright.
Nature
PUBLISHED: 04-16-2010
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The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.
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Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome.
Genome Res.
PUBLISHED: 03-19-2010
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Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative splicing in the rice transcriptome revealed that alternative cis-splicing occurred in approximately 33% of all rice genes. This is far more than previously reported. In addition, we also identified 234 putative chimeric transcripts that seem to be produced by trans-splicing, indicating that transcript fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell. Taken together, our data provide extensive evidence that transcriptional regulation in rice is vastly more complex than previously believed.
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A human gut microbial gene catalogue established by metagenomic sequencing.
Nature
PUBLISHED: 03-06-2010
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To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.
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Resequencing of 200 human exomes identifies an excess of low-frequency non-synonymous coding variants.
Nat. Genet.
PUBLISHED: 02-08-2010
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Targeted capture combined with massively parallel exome sequencing is a promising approach to identify genetic variants implicated in human traits. We report exome sequencing of 200 individuals from Denmark with targeted capture of 18,654 coding genes and sequence coverage of each individual exome at an average depth of 12-fold. On average, about 95% of the target regions were covered by at least one read. We identified 121,870 SNPs in the sample population, including 53,081 coding SNPs (cSNPs). Using a statistical method for SNP calling and an estimation of allelic frequencies based on our population data, we derived the allele frequency spectrum of cSNPs with a minor allele frequency greater than 0.02. We identified a 1.8-fold excess of deleterious, non-syonomyous cSNPs over synonymous cSNPs in the low-frequency range (minor allele frequencies between 2% and 5%). This excess was more pronounced for X-linked SNPs, suggesting that deleterious substitutions are primarily recessive.
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Codon Usage Patterns in Corynebacterium glutamicum: Mutational Bias, Natural Selection and Amino Acid Conservation.
Comp. Funct. Genomics
PUBLISHED: 01-29-2010
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The alternative synonymous codons in Corynebacterium glutamicum, a well-known bacterium used in industry for the production of amino acid, have been investigated by multivariate analysis. As C. glutamicum is a GC-rich organism, G and C are expected to predominate at the third position of codons. Indeed, overall codon usage analyses have indicated that C and/or G ending codons are predominant in this organism. Through multivariate statistical analysis, apart from mutational selection, we identified three other trends of codon usage variation among the genes. Firstly, the majority of highly expressed genes are scattered towards the positive end of the first axis, whereas the majority of lowly expressed genes are clustered towards the other end of the first axis. Furthermore, the distinct difference in the two sets of genes was that the C ending codons are predominate in putatively highly expressed genes, suggesting that the C ending codons are translationally optimal in this organism. Secondly, the majority of the putatively highly expressed genes have a tendency to locate on the leading strand, which indicates that replicational and transciptional selection might be invoked. Thirdly, highly expressed genes are more conserved than lowly expressed genes by synonymous and nonsynonymous substitutions among orthologous genes fromthe genomes of C. glutamicum and C. diphtheriae. We also analyzed other factors such as the length of genes and hydrophobicity that might influence codon usage and found their contributions to be weak.
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De novo assembly of human genomes with massively parallel short read sequencing.
Genome Res.
PUBLISHED: 12-17-2009
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Next-generation massively parallel DNA sequencing technologies provide ultrahigh throughput at a substantially lower unit data cost; however, the data are very short read length sequences, making de novo assembly extremely challenging. Here, we describe a novel method for de novo assembly of large genomes from short read sequences. We successfully assembled both the Asian and African human genome sequences, achieving an N50 contig size of 7.4 and 5.9 kilobases (kb) and scaffold of 446.3 and 61.9 kb, respectively. The development of this de novo short read assembly method creates new opportunities for building reference sequences and carrying out accurate analyses of unexplored genomes in a cost-effective way.
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Building the sequence map of the human pan-genome.
Nat. Biotechnol.
PUBLISHED: 10-21-2009
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Here we integrate the de novo assembly of an Asian and an African genome with the NCBI reference human genome, as a step toward constructing the human pan-genome. We identified approximately 5 Mb of novel sequences not present in the reference genome in each of these assemblies. Most novel sequences are individual or population specific, as revealed by their comparison to all available human DNA sequence and by PCR validation using the human genome diversity cell line panel. We found novel sequences present in patterns consistent with known human migration paths. Cross-species conservation analysis of predicted genes indicated that the novel sequences contain potentially functional coding regions. We estimate that a complete human pan-genome would contain approximately 19-40 Mb of novel sequence not present in the extant reference genome. The extensive amount of novel sequence contributing to the genetic variation of the pan-genome indicates the importance of using complete genome sequencing and de novo assembly.
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Complete resequencing of 40 genomes reveals domestication events and genes in silkworm (Bombyx).
Science
PUBLISHED: 08-27-2009
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A single-base pair resolution silkworm genetic variation map was constructed from 40 domesticated and wild silkworms, each sequenced to approximately threefold coverage, representing 99.88% of the genome. We identified ~16 million single-nucleotide polymorphisms, many indels, and structural variations. We find that the domesticated silkworms are clearly genetically differentiated from the wild ones, but they have maintained large levels of genetic variability, suggesting a short domestication event involving a large number of individuals. We also identified signals of selection at 354 candidate genes that may have been important during domestication, some of which have enriched expression in the silk gland, midgut, and testis. These data add to our understanding of the domestication processes and may have applications in devising pest control strategies and advancing the use of silkworms as efficient bioreactors.
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The sequence and de novo assembly of the giant panda genome.
Ruiqiang Li, Wei Fan, Geng Tian, Hongmei Zhu, Lin He, Jing Cai, Quanfei Huang, Qingle Cai, Bo Li, Yinqi Bai, Zhihe Zhang, Yaping Zhang, Wen Wang, Jun Li, Fuwen Wei, Heng Li, Min Jian, Jianwen Li, Zhaolei Zhang, Rasmus Nielsen, Dawei Li, Wanjun Gu, Zhentao Yang, Zhaoling Xuan, Oliver A Ryder, Frederick Chi-Ching Leung, Yan Zhou, Jianjun Cao, Xiao Sun, Yonggui Fu, Xiaodong Fang, Xiaosen Guo, Bo Wang, Rong Hou, Fujun Shen, Bo Mu, Peixiang Ni, Runmao Lin, Wubin Qian, Guodong Wang, Chang Yu, Wenhui Nie, Jinhuan Wang, Zhigang Wu, Huiqing Liang, Jiumeng Min, Qi Wu, Shifeng Cheng, Jue Ruan, Mingwei Wang, Zhongbin Shi, Ming Wen, Binghang Liu, Xiaoli Ren, Huisong Zheng, Dong Dong, Kathleen Cook, Gao Shan, Hao Zhang, Carolin Kosiol, Xueying Xie, Zuhong Lu, Hancheng Zheng, Yingrui Li, Cynthia C Steiner, Tommy Tsan-Yuk Lam, Siyuan Lin, Qinghui Zhang, Guoqing Li, Jing Tian, Timing Gong, Hongde Liu, Dejin Zhang, Lin Fang, Chen Ye, Juanbin Zhang, Wenbo Hu, Anlong Xu, Yuanyuan Ren, Guojie Zhang, Michael W Bruford, Qibin Li, Lijia Ma, Yiran Guo, Na An, Yujie Hu, Yang Zheng, Yongyong Shi, Zhiqiang Li, Qing Liu, Yanling Chen, Jing Zhao, Ning Qu, Shancen Zhao, Feng Tian, Xiaoling Wang, Haiyin Wang, Lizhi Xu, Xiao Liu, Tomás Vinar, Yajun Wang, Tak-Wah Lam, Siu-Ming Yiu, Shiping Liu, Hemin Zhang, Desheng Li, Yan Huang, Xia Wang, Guohua Yang, Zhi Jiang, Junyi Wang, Nan Qin, Li Li, Jingxiang Li, Lars Bolund, Karsten Kristiansen, Gane Ka-Shu Wong, Maynard Olson, Xiuqing Zhang, Songgang Li, Huanming Yang, Jian Wang, Jun Wang.
Nature
PUBLISHED: 08-19-2009
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Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes.
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DNA copy number aberrations in breast cancer by array comparative genomic hybridization.
Genomics Proteomics Bioinformatics
PUBLISHED: 07-14-2009
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Array comparative genomic hybridization (CGH) has been popularly used for analyzing DNA copy number variations in diseases like cancer. In this study, we investigated 82 sporadic samples from 49 breast cancer patients using 1-Mb resolution bacterial artificial chromosome CGH arrays. A number of highly frequent genomic aberrations were discovered, which may act as "drivers" of tumor progression. Meanwhile, the genomic profiles of four "normal" breast tissue samples taken at least 2 cm away from the primary tumor sites were also found to have some genomic aberrations that recurred with high frequency in the primary tumors, which may have important implications for clinical therapy. Additionally, we performed class comparison and class prediction for various clinicopathological parameters, and a list of characteristic genomic aberrations associated with different clinicopathological phenotypes was compiled. Our study provides clues for further investigations of the underlying mechanisms of breast carcinogenesis.
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Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions.
Sci. China, C, Life Sci.
PUBLISHED: 05-27-2009
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To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, havent benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation synthesis by sequencing technology Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection effectiveness) of using the LBA protocol in facilitating PCR-based re-sequencing and genetic-variant-detection studies on the next-generation sequencing machine, raising the prospect of various PCR-based genomic/genetic studies using this strategy.
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An improved method for genome wide DNA methylation profiling correlated to transcription and genomic instability in two breast cancer cell lines.
BMC Genomics
PUBLISHED: 05-13-2009
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DNA methylation is a widely studied epigenetic mechanism known to correlate with gene repression and genomic stability. Development of sensitive methods for global detection of DNA methylation events is of particular importance.
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The genome of the cucumber, Cucumis sativus L.
Nat. Genet.
PUBLISHED: 05-06-2009
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Cucumber is an economically important crop as well as a model system for sex determination studies and plant vascular biology. Here we report the draft genome sequence of Cucumis sativus var. sativus L., assembled using a novel combination of traditional Sanger and next-generation Illumina GA sequencing technologies to obtain 72.2-fold genome coverage. The absence of recent whole-genome duplication, along with the presence of few tandem duplications, explains the small number of genes in the cucumber. Our study establishes that five of the cucumbers seven chromosomes arose from fusions of ten ancestral chromosomes after divergence from Cucumis melo. The sequenced cucumber genome affords insight into traits such as its sex expression, disease resistance, biosynthesis of cucurbitacin and fresh green odor. We also identify 686 gene clusters related to phloem function. The cucumber genome provides a valuable resource for developing elite cultivars and for studying the evolution and function of the plant vascular system.
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SNP detection for massively parallel whole-genome resequencing.
Genome Res.
PUBLISHED: 05-06-2009
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Next-generation massively parallel sequencing technologies provide ultrahigh throughput at two orders of magnitude lower unit cost than capillary Sanger sequencing technology. One of the key applications of next-generation sequencing is studying genetic variation between individuals using whole-genome or target region resequencing. Here, we have developed a consensus-calling and SNP-detection method for sequencing-by-synthesis Illumina Genome Analyzer technology. We designed this method by carefully considering the data quality, alignment, and experimental errors common to this technology. All of this information was integrated into a single quality score for each base under Bayesian theory to measure the accuracy of consensus calling. We tested this methodology using a large-scale human resequencing data set of 36x coverage and assembled a high-quality nonrepetitive consensus sequence for 92.25% of the diploid autosomes and 88.07% of the haploid X chromosome. Comparison of the consensus sequence with Illumina human 1M BeadChip genotyped alleles from the same DNA sample showed that 98.6% of the 37,933 genotyped alleles on the X chromosome and 98% of 999,981 genotyped alleles on autosomes were covered at 99.97% and 99.84% consistency, respectively. At a low sequencing depth, we used prior probability of dbSNP alleles and were able to improve coverage of the dbSNP sites significantly as compared to that obtained using a nonimputation model. Our analyses demonstrate that our method has a very low false call rate at any sequencing depth and excellent genome coverage at a high sequencing depth.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.