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Sample records for cell whole-genome expression

  1. Whole genome expression profile in neuroblastoma cells exposed to 1-methyl-4-phenylpyridine

    OpenAIRE

    Mazzio, E; Soliman, KFA

    2012-01-01

    Mitochondrial dysfunction and subsequent energy failure is a contributing factor to degeneration of the substantia nigra pars compacta associated with Parkinson’s disease (PD). In this study, we investigate molecular events trigger by 1-methyl-4-phenylpyridine (MPP+) using whole genome-expression microarray, western blot and HPLC quantification of metabolites. The data show that MPP+ (500μM) evokes obstruction of mitochondrial respiration/oxidative phosphorylation (OXPHOS) in mouse neuroblast...

  2. A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells

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    Vaudaux Pierre

    2007-06-01

    Full Text Available Abstract Background Staphylococcus aureus, a leading cause of chronic or acute infections, is traditionally considered an extracellular pathogen despite repeated reports of S. aureus internalization by a variety of non-myeloid cells in vitro. This property potentially contributes to bacterial persistence, protection from antibiotics and evasion of immune defenses. Mechanisms contributing to internalization have been partly elucidated, but bacterial processes triggered intracellularly are largely unknown. Results We have developed an in vitro model using human lung epithelial cells that shows intracellular bacterial persistence for up to 2 weeks. Using an original approach we successfully collected and amplified low amounts of bacterial RNA recovered from infected eukaryotic cells. Transcriptomic analysis using an oligoarray covering the whole S. aureus genome was performed at two post-internalization times and compared to gene expression of non-internalized bacteria. No signs of cellular death were observed after prolonged internalization of Staphylococcus aureus 6850 in epithelial cells. Following internalization, extensive alterations of bacterial gene expression were observed. Whereas major metabolic pathways including cell division, nutrient transport and regulatory processes were drastically down-regulated, numerous genes involved in iron scavenging and virulence were up-regulated. This initial adaptation was followed by a transcriptional increase in several metabolic functions. However, expression of several toxin genes known to affect host cell integrity appeared strictly limited. Conclusion These molecular insights correlated with phenotypic observations and demonstrated that S. aureus modulates gene expression at early times post infection to promote survival. Staphylococcus aureus appears adapted to intracellular survival in non-phagocytic cells.

  3. Grouping and Classifying Electrophysiologically-Defined Classes of Neocortical Neurons by Single Cell, Whole-Genome Expression Profiling

    OpenAIRE

    Subkhankulova, Tatiana; Yano, Kojiro; Robinson, Hugh P. C.; Livesey, Frederick J

    2010-01-01

    The diversity of neuronal cell types and how to classify them are perennial questions in neuroscience. The advent of global gene expression analysis raised the possibility that comprehensive transcription profiling will resolve neuronal cell types into groups that reflect some or all aspects of their phenotype. This approach has been successfully used to compare gene expression between groups of neurons defined by a common property. Here we extend this approach to ask whether single neuron ge...

  4. Grouping and classifying electrophysiologically-defined classes of neocortical neurons by single cell, whole-genome expression profiling

    OpenAIRE

    Tatiana Subkhankulova; Kojiro Yano; Hugh Robinson; Livesey, Frederick J

    2010-01-01

    The diversity of neuronal cell types and how to classify them are perennial questions in neuroscience. The advent of global gene expression analysis raised the possibility that comprehensive transcription profiling will resolve neuronal cell types into groups that reflect some or all aspects of their phenotype. This approach has been successfully used to compare gene expression between groups of neurons defined by a common property. Here we extend this approach to ask whether single neuron ge...

  5. Whole-Genome Expression Analysis of Human Mesenchymal Stromal Cells Exposed to Ultrasmooth Tantalum vs. Titanium Oxide Surfaces

    DEFF Research Database (Denmark)

    Stiehler, C.; Bunger, C.; Overall, R. W.;

    2013-01-01

    Durable osseointegration of metallic bone implants requires that progenitor cells attach, proliferate and differentiate on the implant surface. Previously, we demonstrated superior biocompatibility of human mesenchymal stromal cells (MSCs) cultivated on ultrasmooth tantalum (Ta) as compared to...... MSCs cultivated on plain sputter-coated surfaces of Ta or Ti for 1, 2, 4, and 8 days were hybridized to n = 16 U133 Plus 2.0 arrays (Affymetrix(A (R))). Functional annotation, cluster and pathway analyses were performed. The vast majority of genes were differentially regulated after 4 days of...

  6. Rapid Identification of Potential Drugs for Diabetic Nephropathy Using Whole-Genome Expression Profiles of Glomeruli

    OpenAIRE

    Jingsong Shi; Song Jiang; Dandan Qiu; Weibo Le; Xiao Wang; Yinhui Lu; Zhihong Liu

    2016-01-01

    Objective. To investigate potential drugs for diabetic nephropathy (DN) using whole-genome expression profiles and the Connectivity Map (CMAP). Methodology. Eighteen Chinese Han DN patients and six normal controls were included in this study. Whole-genome expression profiles of microdissected glomeruli were measured using the Affymetrix human U133 plus 2.0 chip. Differentially expressed genes (DEGs) between late stage and early stage DN samples and the CMAP database were used to identify pote...

  7. Use of whole genome expression analysis in the toxicity screening of nanoparticles

    International Nuclear Information System (INIS)

    The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays for NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes. - Highlights: • Regulated functions were screened using whole genome expression assays. • Polystyrene particles regulated more genes than short carbon nanotubes. • Protein coating of polystyrene particles did not change regulation pattern. • Functions regulated by microarray were confirmed by cell-based assay

  8. Use of whole genome expression analysis in the toxicity screening of nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Fröhlich, Eleonore, E-mail: eleonore.froehlich@medunigraz.at [Center for Medical Research, Medical University of Graz, Stiftingtalstr. 24, 8010 Graz (Austria); Meindl, Claudia; Wagner, Karin [Center for Medical Research, Medical University of Graz, Stiftingtalstr. 24, 8010 Graz (Austria); Leitinger, Gerd [Center for Medical Research, Medical University of Graz, Stiftingtalstr. 24, 8010 Graz (Austria); Institute for Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, 8010 Graz (Austria); Roblegg, Eva [Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Karl-Franzens-University of Graz, Universitätsplatz 1, 8010 Graz (Austria)

    2014-10-15

    The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays for NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes. - Highlights: • Regulated functions were screened using whole genome expression assays. • Polystyrene particles regulated more genes than short carbon nanotubes. • Protein coating of polystyrene particles did not change regulation pattern. • Functions regulated by microarray were confirmed by cell-based assay.

  9. Current Developments in Prokaryotic Single Cell Whole Genome Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Goudeau, Danielle; Nath, Nandita; Ciobanu, Doina; Cheng, Jan-Fang; Malmstrom, Rex

    2014-03-14

    Our approach to prokaryotic single-cell Whole Genome Amplification at the JGI continues to evolve. To increase both the quality and number of single-cell genomes produced, we explore all aspects of the process from cell sorting to sequencing. For example, we now utilize specialized reagents, acoustic liquid handling, and reduced reaction volumes eliminate non-target DNA contamination in WGA reactions. More specifically, we use a cleaner commercial WGA kit from Qiagen that employs a UV decontamination procedure initially developed at the JGI, and we use the Labcyte Echo for tip-less liquid transfer to set up 2uL reactions. Acoustic liquid handling also dramatically reduces reagent costs. In addition, we are exploring new cell lysis methods including treatment with Proteinase K, lysozyme, and other detergents, in order to complement standard alkaline lysis and allow for more efficient disruption of a wider range of cells. Incomplete lysis represents a major hurdle for WGA on some environmental samples, especially rhizosphere, peatland, and other soils. Finding effective lysis strategies that are also compatible with WGA is challenging, and we are currently assessing the impact of various strategies on genome recovery.

  10. Estrogen receptor-mediated effects of isoflavone supplementation were not observed in whole-genome gene expression profiles of peripheral blood mononuclear cells in postmenopausal, equol-producing women.

    Science.gov (United States)

    van der Velpen, Vera; Geelen, Anouk; Schouten, Evert G; Hollman, Peter C; Afman, Lydia A; van 't Veer, Pieter

    2013-06-01

    Isoflavones (genistein, daidzein, and glycitein) are suggested to have benefits as well as risks for human health. Approximately one-third of the Western population is able to metabolize daidzein into the more potent metabolite equol. Having little endogenous estradiol, equol-producing postmenopausal women who use isoflavone supplements to relieve their menopausal symptoms could potentially be at high risk of adverse effects of isoflavone supplementation. The current trial aimed to study the effects of intake of an isoflavone supplement rich in daidzein compared with placebo on whole-genome gene expression profiles of peripheral blood mononuclear cells (PBMCs) in equol-producing, postmenopausal women. Thirty participants received an isoflavone supplement or a placebo for 8 wk each in a double-blind, randomized cross-over design. The isoflavone supplement was rich in daidzein (60%) and provided 94 mg isoflavones (aglycone equivalents) daily. Gene expression in PBMCs was significantly changed (P isoflavone intervention compared with placebo. Gene set enrichment analysis revealed downregulated clusters of gene sets involved in inflammation, oxidative phosphorylation, and cell cycle. The expression of estrogen receptor (ER) target genes and gene sets related to ER signaling were not significantly altered, which may be explained by the low ERα and ERβ expression in PBMCs. The observed downregulated gene sets point toward potential beneficial effects of isoflavone supplementation with respect to prevention of cancer and cardiovascular disease. However, whether ER-related effects of isoflavones are beneficial or harmful should be studied in tissues that express ERs. PMID:23616509

  11. Whole genome RNA expression profiling for the identification of novel biomarkers in the diagnosis and prognosis of biliary tract cancer

    OpenAIRE

    Chapman, M H

    2011-01-01

    Biliary tract cancer (BTC) is difficult to diagnose, in part related to the lack of reliable tumour markers. The aim of this project was to use whole genome RNA expression profiling in order to identify novel biomarkers for diagnosis and prognosis in biliary tract cancer. Chapter 1 summarises clinical aspects of BTC as well as current diagnostic and prognostic tests. Chapter 2 addresses the identification of circulating tumour cells for the diagnosis of BTC. It includes d...

  12. Pathway Processor: A Tool for Integrating Whole-Genome Expression Results into Metabolic Networks

    OpenAIRE

    Grosu, Paul; Townsend, Jeffrey P.; Hartl, Daniel L.; Cavalieri, Duccio

    2002-01-01

    We have developed a new tool to visualize expression data on metabolic pathways and to evaluate which metabolic pathways are most affected by transcriptional changes in whole-genome expression experiments. Using the Fisher Exact Test, the method scores biochemical pathways according to the probability that as many or more genes in a pathway would be significantly altered in a given experiment by chance alone. This method has been validated on diauxic shift experiments and reproduces well know...

  13. Rapid Identification of Potential Drugs for Diabetic Nephropathy Using Whole-Genome Expression Profiles of Glomeruli

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    Jingsong Shi

    2016-01-01

    Full Text Available Objective. To investigate potential drugs for diabetic nephropathy (DN using whole-genome expression profiles and the Connectivity Map (CMAP. Methodology. Eighteen Chinese Han DN patients and six normal controls were included in this study. Whole-genome expression profiles of microdissected glomeruli were measured using the Affymetrix human U133 plus 2.0 chip. Differentially expressed genes (DEGs between late stage and early stage DN samples and the CMAP database were used to identify potential drugs for DN using bioinformatics methods. Results. (1 A total of 1065 DEGs (FDR 1.5 were found in late stage DN patients compared with early stage DN patients. (2 Piperlongumine, 15d-PGJ2 (15-delta prostaglandin J2, vorinostat, and trichostatin A were predicted to be the most promising potential drugs for DN, acting as NF-κB inhibitors, histone deacetylase inhibitors (HDACIs, PI3K pathway inhibitors, or PPARγ agonists, respectively. Conclusion. Using whole-genome expression profiles and the CMAP database, we rapidly predicted potential DN drugs, and therapeutic potential was confirmed by previously published studies. Animal experiments and clinical trials are needed to confirm both the safety and efficacy of these drugs in the treatment of DN.

  14. Whole-genome expression analysis reveals genes associated with treatment response to escitalopram in major depression.

    Science.gov (United States)

    Pettai, Kristi; Milani, Lili; Tammiste, Anu; Võsa, Urmo; Kolde, Raivo; Eller, Triin; Nutt, David; Metspalu, Andres; Maron, Eduard

    2016-09-01

    The reasons for variability in treatment response in major depressive disorder (MDD) are not fully understood, but there is accumulating evidence suggesting that therapeutic outcomes of antidepressants can be influenced by genetic factors. In the present study we applied the microarray Illumina platform for whole genome expression profiling in depressive patients treated with escitalopram medication in order to identify genes underlying response to antidepressant treatment. The initial study sample consisted of 135 outpatients with major depressive disorder (mean age 31.1±11.6 years, 68% females) treated with escitalopram 10-20mg/day for 12 weeks, from which 87 patients (55 females) were included in gene expression analyzing. The gene expression profiles were measured on peripheral blood cells at baseline, at week 4 and at the end of treatment (week 12) using BeadChips Illumina. The fold change was used to demonstrate rate of changes in average gene expressions between studied groups. Statistical analyses were performed using the false discovery rate (FDR). The most interesting gene, which showed the predictive effect on treatment outcome by delineating low dose responders and treatment-resistant patients at the beginning of medication, was NLGN2, belonging to a family of neuronal cell surface proteins and involving in synapse formation. In addition, the several gene clusters, related to immune response, signal transduction and neurotrophin pathway, have distinguished responders from non-responders at the week 4 of treatment. After 4 weeks of escitalopram treatment (10mg/day), the YWHAZ gene has showed the highest transcriptional change in responders as compared with non-responders. Finally, at the end of the treatment we noticed that at least three genes (NR2C2, ZNF641, FKBP1A) have been strongly associated with resistance to escitalopram. Thus the results of this study support that exploration of peripheral gene expression is a useful tool in the further

  15. Whole-genome fingerprint of the DNA methylome during human B cell differentiation

    OpenAIRE

    Kulis, Marta; Merkel, Angelika; Heath, Simon; Queirós, Ana C; Schuyler, Ronald P.; Castellano, Giancarlo; Beekman, Renée; Raineri, Emanuele; Esteve, Anna; Clot, Guillem; Verdaguer-Dot, Néria; Duran-Ferrer, Martí; Russiñol, Nuria; Vilarrasa-Blasi, Roser; Ecker, Simone

    2015-01-01

    International audience We analyzed the DNA methylome of ten subpopulations spanning the entire B cell differentiation program by whole-genome bisulfite sequencing and high-density microarrays. We observed that non-CpG methylation disappeared upon B cell commitment, whereas CpG methylation changed extensively during B cell maturation, showing an accumulative pattern and affecting around 30% of all measured CpG sites. Early differentiation stages mainly displayed enhancer demethylation, whic...

  16. Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

    DEFF Research Database (Denmark)

    Hou, Yong; Wu, Kui; Shi, Xulian;

    2015-01-01

    -oligonucleotide-primed PCR (DOP-PCR) and multiple annealing and looping-based amplification cycles (MALBAC). However, a comprehensive comparison of variations detection performance between these WGA methods has not yet been performed. RESULTS: We systematically compared the advantages and disadvantages of different WGA...... methods, focusing particularly on variations detection. Low-coverage whole-genome sequencing revealed that DOP-PCR had the highest duplication ratio, but an even read distribution and the best reproducibility and accuracy for detection of copy-number variations (CNVs). However, MDA had significantly......BACKGROUND: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate...

  17. Whole genome amplification from a single cell: implications for genetic analysis.

    OpenAIRE

    Zhang, L; Cui, X.; Schmitt, K.; R.; Hubert; Navidi, W.; Arnheim, N

    1992-01-01

    We have developed an in vitro method for amplifying a large fraction of the DNA sequences present in a single haploid cell by repeated primer extensions using a mixture of 15-base random oligonucleotides. We studied 12 genetic loci and estimate that the probability of amplifying any sequence in the genome to a minimum of 30 copies is not less than 0.78 (95% confidence). Whole genome amplification beginning with a single cell, or other samples with very small amounts of DNA, has significant im...

  18. Whole-genome gene expression profiling of formalin-fixed, paraffin-embedded tissue samples.

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    Craig April

    Full Text Available BACKGROUND: We have developed a gene expression assay (Whole-Genome DASL, capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE specimens. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2 approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2 approximately 0.86 and R(2 approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2>0.98 with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2 approximately 0.92, with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2 approximately 0.80, while still maintaining a correlation of R(2 approximately 0.75 with standard FFPE inputs (200 ng. CONCLUSIONS/SIGNIFICANCE: Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material or when minimally invasive procedures are performed (e

  19. Whole-Genome Sequencing Reveals Diverse Models of Structural Variations in Esophageal Squamous Cell Carcinoma.

    Science.gov (United States)

    Cheng, Caixia; Zhou, Yong; Li, Hongyi; Xiong, Teng; Li, Shuaicheng; Bi, Yanghui; Kong, Pengzhou; Wang, Fang; Cui, Heyang; Li, Yaoping; Fang, Xiaodong; Yan, Ting; Li, Yike; Wang, Juan; Yang, Bin; Zhang, Ling; Jia, Zhiwu; Song, Bin; Hu, Xiaoling; Yang, Jie; Qiu, Haile; Zhang, Gehong; Liu, Jing; Xu, Enwei; Shi, Ruyi; Zhang, Yanyan; Liu, Haiyan; He, Chanting; Zhao, Zhenxiang; Qian, Yu; Rong, Ruizhou; Han, Zhiwei; Zhang, Yanlin; Luo, Wen; Wang, Jiaqian; Peng, Shaoliang; Yang, Xukui; Li, Xiangchun; Li, Lin; Fang, Hu; Liu, Xingmin; Ma, Li; Chen, Yunqing; Guo, Shiping; Chen, Xing; Xi, Yanfeng; Li, Guodong; Liang, Jianfang; Yang, Xiaofeng; Guo, Jiansheng; Jia, JunMei; Li, Qingshan; Cheng, Xiaolong; Zhan, Qimin; Cui, Yongping

    2016-02-01

    Comprehensive identification of somatic structural variations (SVs) and understanding their mutational mechanisms in cancer might contribute to understanding biological differences and help to identify new therapeutic targets. Unfortunately, characterization of complex SVs across the whole genome and the mutational mechanisms underlying esophageal squamous cell carcinoma (ESCC) is largely unclear. To define a comprehensive catalog of somatic SVs, affected target genes, and their underlying mechanisms in ESCC, we re-analyzed whole-genome sequencing (WGS) data from 31 ESCCs using Meerkat algorithm to predict somatic SVs and Patchwork to determine copy-number changes. We found deletions and translocations with NHEJ and alt-EJ signature as the dominant SV types, and 16% of deletions were complex deletions. SVs frequently led to disruption of cancer-associated genes (e.g., CDKN2A and NOTCH1) with different mutational mechanisms. Moreover, chromothripsis, kataegis, and breakage-fusion-bridge (BFB) were identified as contributing to locally mis-arranged chromosomes that occurred in 55% of ESCCs. These genomic catastrophes led to amplification of oncogene through chromothripsis-derived double-minute chromosome formation (e.g., FGFR1 and LETM2) or BFB-affected chromosomes (e.g., CCND1, EGFR, ERBB2, MMPs, and MYC), with approximately 30% of ESCCs harboring BFB-derived CCND1 amplification. Furthermore, analyses of copy-number alterations reveal high frequency of whole-genome duplication (WGD) and recurrent focal amplification of CDCA7 that might act as a potential oncogene in ESCC. Our findings reveal molecular defects such as chromothripsis and BFB in malignant transformation of ESCCs and demonstrate diverse models of SVs-derived target genes in ESCCs. These genome-wide SV profiles and their underlying mechanisms provide preventive, diagnostic, and therapeutic implications for ESCCs. PMID:26833333

  20. Whole-genome sequencing of a malignant granular cell tumor with metabolic response to pazopanib

    Science.gov (United States)

    Wei, Lei; Liu, Song; Conroy, Jeffrey; Wang, Jianmin; Papanicolau-Sengos, Antonios; Glenn, Sean T.; Murakami, Mitsuko; Liu, Lu; Hu, Qiang; Conroy, Jacob; Miles, Kiersten Marie; Nowak, David E.; Liu, Biao; Qin, Maochun; Bshara, Wiam; Omilian, Angela R.; Head, Karen; Bianchi, Michael; Burgher, Blake; Darlak, Christopher; Kane, John; Merzianu, Mihai; Cheney, Richard; Fabiano, Andrew; Salerno, Kilian; Talati, Chetasi; Khushalani, Nikhil I.; Trump, Donald L.; Johnson, Candace S.; Morrison, Carl D.

    2015-01-01

    Granular cell tumors are an uncommon soft tissue neoplasm. Malignant granular cell tumors comprise T transitions, particularly when immediately preceded by a 5′ G. A loss-of-function mutation was detected in a newly recognized tumor suppressor candidate, BRD7. No mutations were found in known targets of pazopanib. However, we identified a receptor tyrosine kinase pathway mutation in GFRA2 that warrants further evaluation. To the best of our knowledge, this is only the second reported case of a malignant granular cell tumor exhibiting a response to pazopanib, and the first whole-genome sequencing of this uncommon tumor type. The findings provide insight into the genetic basis of malignant granular cell tumors and identify potential targets for further investigation. PMID:27148567

  1. Gene evolution and gene expression after whole genome duplication in fish: the PhyloFish database.

    Science.gov (United States)

    Pasquier, Jeremy; Cabau, Cédric; Nguyen, Thaovi; Jouanno, Elodie; Severac, Dany; Braasch, Ingo; Journot, Laurent; Pontarotti, Pierre; Klopp, Christophe; Postlethwait, John H; Guiguen, Yann; Bobe, Julien

    2016-01-01

    With more than 30,000 species, ray-finned fish represent approximately half of vertebrates. The evolution of ray-finned fish was impacted by several whole genome duplication (WGD) events including a teleost-specific WGD event (TGD) that occurred at the root of the teleost lineage about 350 million years ago (Mya) and more recent WGD events in salmonids, carps, suckers and others. In plants and animals, WGD events are associated with adaptive radiations and evolutionary innovations. WGD-spurred innovation may be especially relevant in the case of teleost fish, which colonized a wide diversity of habitats on earth, including many extreme environments. Fish biodiversity, the use of fish models for human medicine and ecological studies, and the importance of fish in human nutrition, fuel an important need for the characterization of gene expression repertoires and corresponding evolutionary histories of ray-finned fish genes. To this aim, we performed transcriptome analyses and developed the PhyloFish database to provide (i) de novo assembled gene repertoires in 23 different ray-finned fish species including two holosteans (i.e. a group that diverged from teleosts before TGD) and 21 teleosts (including six salmonids), and (ii) gene expression levels in ten different tissues and organs (and embryos for many) in the same species. This resource was generated using a common deep RNA sequencing protocol to obtain the most exhaustive gene repertoire possible in each species that allows between-species comparisons to study the evolution of gene expression in different lineages. The PhyloFish database described here can be accessed and searched using RNAbrowse, a simple and efficient solution to give access to RNA-seq de novo assembled transcripts. PMID:27189481

  2. Expression profiling of five different xenobiotics using a Caenorhabditis elegans whole genome microarray.

    Science.gov (United States)

    Reichert, Kerstin; Menzel, Ralph

    2005-10-01

    The soil nematode Caenorhabditis elegans is frequently used in ecotoxicological studies due to its wide distribution in terrestrial habitats, its easy handling in the laboratory, and its sensitivity against different kinds of stress. Since its genome has been completely sequenced, more and more studies are investigating the functional relation of gene expression and phenotypic response. For these reasons C. elegans seems to be an attractive animal for the development of a new, genome based, ecotoxicological test system. In recent years, the DNA array technique has been established as a powerful tool to obtain distinct gene expression patterns in response to different experimental conditions. Using a C. elegans whole genome DNA microarray in this study, the effects of five different xenobiotics on the gene expression of the nematode were investigated. The exposure time for the following five applied compounds beta-NF (5 mg/l), Fla (0.5 mg/l), atrazine (25 mg/l), clofibrate (10 mg/l) and DES (0.5 mg/l) was 48+/-5 h. The analysis of the data showed a clear induction of 203 genes belonging to different families like the cytochromes P450, UDP-glucoronosyltransferases (UDPGT), glutathione S-transferases (GST), carboxylesterases, collagenes, C-type lectins and others. Under the applied conditions, fluoranthene was able to induce most of the induceable genes, followed by clofibrate, atrazine, beta-naphthoflavone and diethylstilbestrol. A decreased expression could be shown for 153 genes with atrazine having the strongest effect followed by fluoranthene, diethylstilbestrol, beta-naphthoflavone and clofibrate. For upregulated genes a change ranging from approximately 2.1- till 42.3-fold and for downregulated genes from approximately 2.1 till 6.6-fold of gene expression could be affected through the applied xenobiotics. The results confirm the applicability of the gene expression for the development of an ecotoxicological test system. Compared to classical tests the main

  3. Whole Genome Sequencing

    Science.gov (United States)

    ... you want to learn. Search form Search Whole Genome Sequencing You are here Home Testing & Services Testing ... the full story, click here . What is whole genome sequencing? Whole genome sequencing is the mapping out ...

  4. Implementation of exon arrays: alternative splicing during T-cell proliferation as determined by whole genome analysis

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    Whistler Toni

    2010-09-01

    Full Text Available Abstract Background The contribution of alternative splicing and isoform expression to cellular response is emerging as an area of considerable interest, and the newly developed exon arrays allow for systematic study of these processes. We use this pilot study to report on the feasibility of exon array implementation looking to replace the 3' in vitro transcription expression arrays in our laboratory. One of the most widely studied models of cellular response is T-cell activation from exogenous stimulation. Microarray studies have contributed to our understanding of key pathways activated during T-cell stimulation. We use this system to examine whole genome transcription and alternate exon usage events that are regulated during lymphocyte proliferation in an attempt to evaluate the exon arrays. Results Peripheral blood mononuclear cells form healthy donors were activated using phytohemagglutinin, IL2 and ionomycin and harvested at 5 points over a 7 day period. Flow cytometry measured cell cycle events and the Affymetrix exon array platform was used to identify the gene expression and alternate exon usage changes. Gene expression changes were noted in a total of 2105 transcripts, and alternate exon usage identified in 472 transcript clusters. There was an overlap of 263 transcripts which showed both differential expression and alternate exon usage over time. Gene ontology enrichment analysis showed a broader range of biological changes in biological processes for the differentially expressed genes, which include cell cycle, cell division, cell proliferation, chromosome segregation, cell death, component organization and biogenesis and metabolic process ontologies. The alternate exon usage ontological enrichments are in metabolism and component organization and biogenesis. We focus on alternate exon usage changes in the transcripts of the spliceosome complex. The real-time PCR validation rates were 86% for transcript expression and 71% for

  5. PKS and NRPS gene clusters from microbial symbiont cells of marine sponges by whole genome amplification.

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    Siegl, Alexander; Hentschel, Ute

    2010-08-01

    Whole genome amplification (WGA) approaches provide genomic information on single microbial cells and hold great promise for the field of environmental microbiology. Here, the microbial consortia of the marine sponge Aplysina aerophoba were sorted by fluorescence-activated cell sorting (FACS) and then subjected to WGA. A cosmid library was constructed from the WGA product of a sample containing two bacterial cells, one a member of the candidate phylum Poribacteria and one of a sponge-specific clade of Chloroflexi. Library screening led to the genomic characterization of three cosmid clones, encoding a polyketide synthase (PKS), a non-ribosomal peptide synthetase (NRPS) and the Chloroflexi 16S rRNA gene. PCR screening of WGA products from additional, FACS-sorted single bacterial symbiont cells supports the assignment of the Sup-PKS gene to the Poribacteria and the novel NRPS gene to the Chloroflexi. This promising single-cell genomics approach has permitted cloning of entire gene clusters from single microbial cells of known phylogenetic origin and thus provides a sought-after link between phylogeny and function. PMID:23766222

  6. Whole genome amplification and de novo assembly of single bacterial cells.

    Directory of Open Access Journals (Sweden)

    Sébastien Rodrigue

    Full Text Available BACKGROUND: Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA and complete genome sequencing of individual cells. METHODOLOGY/PRINCIPAL FINDINGS: We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA, and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs. CONCLUSIONS/SIGNIFICANCE: The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.

  7. Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

    Institute of Scientific and Technical Information of China (English)

    徐晨明; 金帆; 黄荷凤; 陶冶; 叶英辉

    2001-01-01

    Objective To develop a reliable and sensitive method for detection of sex and multiloci of Duchenne muscular dystrophy (DMD) gene in single cell Materials & methods Whole genome of single cell were amplified by using 15-base random primers (primer extension preamplification, PEP), then a small aliquot of PEP product were analyzed by using locus-specific nest PCR amplification. The procedure was evaluated by detection dystrophin exons 8, 17, 19, 44, 45, 48 and human testis-determining gene (SRY)in single lymphocytes from known sources and single blastomeres from the couples with no family history of DMD.Results The amplification efficiency rate of six dystrophin exons from single lymphocytes and single blastomeres were 97. 2% (175/180) and 100% (60/60) respectively.Results of SRY showed that 100% (15/15) amplification in single male-derived lymphocytes and 0% (0/15) amplification in single female-derived lymphocytes. Conclusion The technique of single cell PEP-nest PCR for dystrophin exons 8, 17,19, 44, 45, 48 and SRY is highly specifc. PEP-nest PCR is suitable for Preimplantation genetic diagnosis (PGD) of DMD at single cell level.

  8. Whole genome expression profiling using DNA microarray for determining biocompatibility of polymeric surfaces

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Wang, Zhenyu; Kutter, Jörg Peter; Dufva, Hans Martin; Wolff, Anders

    2006-01-01

    There is an ever increasing need to find surfaces that are biocompatible for applications like medical implants and microfluidics-based cell culture systems. The biocompatibility of five different surfaces with different hydrophobicity was determined using gene expression profiling as well as more...... conventional methods to determine biocompatibility such as cellular growth rate, morphology and the hydrophobicity of the surfaces. HeLa cells grown on polymethylmethacrylate (PMMA) or a SU-8 surface treated with HNO3-ceric ammonium nitrate (HNO3-CAN) and ethanolamine showed no differences in growth rate......, morphology or gene expression profiles as compared to HeLa cells grown in cell culture flasks. Cells grown on SU-8 treated with only HNO3-CAN showed almost the same growth rate (36 ¡ 1 h) and similar morphology as cells grown in cell culture flasks (32 ¡ 1 h), indicating good biocompatibility. However, more...

  9. Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains

    OpenAIRE

    Jensen, Taylor J.; Kim, Sung K; Zhu, Zhanyang; Chin, Christine; Gebhard, Claudia; Lu, Tim; Deciu, Cosmin; Van den Boom, Dirk; Ehrich, Mathias

    2015-01-01

    Background Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors. Results We perform whole genome bisulfite sequencing on a set of unmatched sampl...

  10. Whole genome expression profiling using DNA microarray for determining biocompatibility of polymeric surfaces

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Wang, Zhenyu; Kutter, Jörg Peter;

    2006-01-01

    There is an ever increasing need to find surfaces that are biocompatible for applications like medical implants and microfluidics-based cell culture systems. The biocompatibility of five different surfaces with different hydrophobicity was determined using gene expression profiling as well as more...

  11. Microarray analysis of serum mRNA in patients with head and neck squamous cell carcinoma at whole-genome scale

    Czech Academy of Sciences Publication Activity Database

    Čapková, M.; Šáchová, Jana; Strnad, Hynek; Kolář, Michal; Hroudová, Miluše; Chovanec, M.; Čada, Z.; Štefl, M.; Valach, J.; Kastner, J.; Smetana, K. Jr.; Plzák, J.

    -, April 23 (2014). ISSN 2314-6141 R&D Projects: GA MZd(CZ) NT13488 Institutional support: RVO:68378050 Keywords : Microarray Analysis * Head and Neck Squamous Cell Carcinoma * whole-genome scale Subject RIV: EB - Genetics ; Molecular Biology

  12. Classification and toxicity mechanisms of novel flame retardants (NFRs) based on whole genome expression profiling.

    Science.gov (United States)

    Guan, Miao; Su, Guanyong; Giesy, John P; Zhang, Xiaowei

    2016-02-01

    Recently some novel alternative flame retardants (NFRs), which have been widely applied to meet demands for mandated flame retardation of products, have been detected in various matrices of the environment. However, knowledge on toxic effects and associated molecular mechanisms of these chemicals was limited. Here, toxic mechanisms of action of six NFRs, bis (2-ethylhexyl) phosphate (BEHP), chlorendic acid (Het acid), 2,2-bis (bromomethyl)-1,3-propanediol (BMP), tris (2-butoxyethyl) phosphate (TBEP), triethyl phosphate (TEP), tributyl phosphate (TBP) were investigated by use of a library containing ∼1820 modified green fluorescent protein (GFP) expressing promoter reporter vectors constructed from Escherichia coli K12(E.coli). BEHP, Het acid, BMP, TBEP, TEP, TBP inhibited growth of E. coli with 4 h 10%-inhibition concentrations of 53.0-3102.3 μM. A total of 119, 44, 26, 131, 62, 103 genes out of 336 genes selected during preliminary screening were significantly altered with fold-changes greater than 1.5 by BEHP, Het acid, BMP, TBEP, TEP and TBP, respectively. GO analyses of responsive genes suggested that RNA and primary metabolism process were involved in molecular mechanisms of toxicity. Chemical clustering based on expression of 62 multi-responsive genes showed that BEHP, TBP and TBEP were grouped together, which is consistent with similarity of their chemical structures, especially for BEHP and TBP. Clustering by molecular descriptors and molecular activity by use of the multivariate classification system ToxCast was consistent with that by profiles of multi-responsive genes. The results of this study demonstrated the utility of the E. coli, whole-cell assay for determining mechanisms of toxic action of chemicals. PMID:26588597

  13. Analyses and interpretation of whole-genome gene expression from formalin-fixed paraffin-embedded tissue: an illustration with breast cancer tissues

    Directory of Open Access Journals (Sweden)

    Argos Maria

    2010-11-01

    Full Text Available Abstract Background We evaluated (a the feasibility of whole genome cDNA-mediated Annealing, Selection, extension and Ligation (DASL assay on formalin-fixed paraffin-embedded (FFPE tissue and (b whether similar conclusions can be drawn by examining FFPE samples as proxies for fresh frozen (FF tissues. We used a whole genome DASL assay (addressing 18,391 genes on a total of 72 samples from paired breast tumor and surrounding healthy tissues from both FF and FFPE samples. Results Gene detection was very good with comparable success between the FFPE and FF samples. Reproducibility was also high (r2 = 0.98; however, concordance between the two types of samples was low. Only one-third of the differentially expressed genes in tumor tissues (compared to corresponding normal from FF samples could be detected in FFPE samples and conversely only one-fourth of the differentially expressed genes from FFPE samples could be detected in FF samples. GO-enrichment analysis, gene set enrichment analysis (GSEA and GO-ANOVA analyses also suggested small overlap between the lead functional groups that were differentially expressed in tumor detectable by examining FFPE and FF samples. In other words, FFPE samples may not be ideal for picking individual target gene(s, but may be used to identify some of the lead functional group(s of genes that are differentially expressed in tumor. The differentially expressed genes in breast cancer found in our study were biologically meaningful. The "cell cycle" & "cell division" related genes were up-regulated and genes related to "regulation of epithelial cell proliferation" were down-regulated. Conclusions Gene expression experiments using the DASL assay can efficiently handle fragmentation issues in the FFPE tissues. However, formalin fixation seems to change RNA and consequently significantly alters gene expression in a number of genes which may not be uniform between tumor and normal tissues. Therefore, considerable caution

  14. Effect of long real space flight on the whole genome mRNA expression properties in medaka Oryzias latipes

    Science.gov (United States)

    Kozlova, Olga; Gusev, Oleg; Levinskikh, Margarita; Sychev, Vladimir; Poddubko, Svetlana

    The current study is addressed to the complex analysis of whole genome mRNA expression profile and properties of splicing variants formation in different organs of medaka fish exposed to prolonged space flight in the frame of joint Russia-Japan research program “Aquarium-AQH”. The fish were kept in the AQH joint-aquariums system in October-December 2013, followed by fixation in RNA-preserving buffers and freezing during the space flight. The samples we returned to the Earth frozen in March 2013 and mRNAs from four fish were sequenced in organ-specific manner using HiSeq Illumina sequencing platform. The ground group fish treated in the same way was used as a control. The comparison between the groups revealed space group-specific specific mRNA expression pattern. More than 50 genes (including several types of myosins) were down-regulated in the space group. Moreover, we found an evidence for formation of space group-specific splicing variants of mRNA. Taking together, the data suggest that in spite of aquatic environment, space flight-associated factors have a strong effect on the activity of fish genome. This work was supported in part by subsidy of the Russian Government to support the Program of competitive growth of Kazan Federal University among world class academic centres and universities.

  15. Hyperlipidemia-associated gene variations and expression patterns revealed by whole-genome and transcriptome sequencing of rabbit models.

    Science.gov (United States)

    Wang, Zhen; Zhang, Jifeng; Li, Hong; Li, Junyi; Niimi, Manabu; Ding, Guohui; Chen, Haifeng; Xu, Jie; Zhang, Hongjiu; Xu, Ze; Dai, Yulin; Gui, Tuantuan; Li, Shengdi; Liu, Zhi; Wu, Sujuan; Cao, Mushui; Zhou, Lu; Lu, Xingyu; Wang, Junxia; Yang, Jing; Fu, Yunhe; Yang, Dongshan; Song, Jun; Zhu, Tianqing; Li, Shen; Ning, Bo; Wang, Ziyun; Koike, Tomonari; Shiomi, Masashi; Liu, Enqi; Chen, Luonan; Fan, Jianglin; Chen, Y Eugene; Li, Yixue

    2016-01-01

    The rabbit (Oryctolagus cuniculus) is an important experimental animal for studying human diseases, such as hypercholesterolemia and atherosclerosis. Despite this, genetic information and RNA expression profiling of laboratory rabbits are lacking. Here, we characterized the whole-genome variants of three breeds of the most popular experimental rabbits, New Zealand White (NZW), Japanese White (JW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Although the genetic diversity of WHHL rabbits was relatively low, they accumulated a large proportion of high-frequency deleterious mutations due to the small population size. Some of the deleterious mutations were associated with the pathophysiology of WHHL rabbits in addition to the LDLR deficiency. Furthermore, we conducted transcriptome sequencing of different organs of both WHHL and cholesterol-rich diet (Chol)-fed NZW rabbits. We found that gene expression profiles of the two rabbit models were essentially similar in the aorta, even though they exhibited different types of hypercholesterolemia. In contrast, Chol-fed rabbits, but not WHHL rabbits, exhibited pronounced inflammatory responses and abnormal lipid metabolism in the liver. These results provide valuable insights into identifying therapeutic targets of hypercholesterolemia and atherosclerosis with rabbit models. PMID:27245873

  16. Molecular Classifiers for Acute Kidney Transplant Rejection in Peripheral Blood by Whole Genome Gene Expression Profiling

    OpenAIRE

    Kurian, S M; Williams, A N; Gelbart, T.; Campbell, D.; Mondala, T. S.; Head, S. R.; Horvath, S; Gaber, L; R. Thompson; Whisenant, T; Lin, W; Langfelder, P; Robison, E. H.; Schaffer, R. L.; Fisher, J S

    2014-01-01

    There are no minimally invasive diagnostic metrics for acute kidney transplant rejection (AR), especially in the setting of the common confounding diagnosis, acute dysfunction with no rejection (ADNR). Thus, though kidney transplant biopsies remain the gold standard, they are invasive, have substantial risks, sampling error issues and significant costs and are not suitable for serial monitoring. Global gene expression profiles of 148 peripheral blood samples from transplant patients with exce...

  17. Quality assessment metrics for whole genome gene expression profiling of paraffin embedded samples

    OpenAIRE

    Mahoney, Douglas W.; Terry M. Therneau; Anderson, S. Keith; Jen, Jin; Kocher, Jean-Pierre A.; Reinholz, Monica M; Perez, Edith A.; Eckel-Passow, Jeanette E

    2013-01-01

    Background Formalin fixed, paraffin embedded tissues are most commonly used for routine pathology analysis and for long term tissue preservation in the clinical setting. Many institutions have large archives of Formalin fixed, paraffin embedded tissues that provide a unique opportunity for understanding genomic signatures of disease. However, genome-wide expression profiling of Formalin fixed, paraffin embedded samples have been challenging due to RNA degradation. Because of the significant h...

  18. Molecular Classifiers for Acute Kidney Transplant Rejection in Peripheral Blood by Whole Genome Gene Expression Profiling

    Science.gov (United States)

    Kurian, S. M.; Williams, A. N.; Gelbart, T.; Campbell, D.; Mondala, T. S.; Head, S. R.; Horvath, S.; Gaber, L.; Thompson, R.; Whisenant, T.; Lin, W.; Langfelder, P.; Robison, E. H.; Schaffer, R. L.; Fisher, J. S.; Friedewald, J.; Flechner, S. M.; Chan, L. K.; Wiseman, A. C.; Shidban, H.; Mendez, R.; Heilman, R.; Abecassis, M. M.; Marsh, C. L.; Salomon, D. R.

    2015-01-01

    There are no minimally invasive diagnostic metrics for acute kidney transplant rejection (AR), especially in the setting of the common confounding diagnosis, acute dysfunction with no rejection (ADNR). Thus, though kidney transplant biopsies remain the gold standard, they are invasive, have substantial risks, sampling error issues and significant costs and are not suitable for serial monitoring. Global gene expression profiles of 148 peripheral blood samples from transplant patients with excellent function and normal histology (TX; n = 46), AR (n = 63) and ADNR (n = 39), from two independent cohorts were analyzed with DNA microarrays. We applied a new normalization tool, frozen robust multi-array analysis, particularly suitable for clinical diagnostics, multiple prediction tools to discover, refine and validate robust molecular classifiers and we tested a novel one-by-one analysis strategy to model the real clinical application of this test. Multiple three-way classifier tools identified 200 highest value probesets with sensitivity, specificity, positive predictive value, negative predictive value and area under the curve for the validation cohort ranging from 82% to 100%, 76% to 95%, 76% to 95%, 79% to 100%, 84% to 100% and 0.817 to 0.968, respectively. We conclude that peripheral blood gene expression profiling can be used as a minimally invasive tool to accurately reveal TX, AR and ADNR in the setting of acute kidney transplant dysfunction. PMID:24725967

  19. 全基因组表达谱芯片筛选非小细胞肺癌常规分割和大分割放疗差异基因的初步研究*%Identifying the genetic pattern of conventional fractionated and hypofractionated radiotherapy using whole genome expression microarray in a non-small-cell lung cancer cell line

    Institute of Scientific and Technical Information of China (English)

    孙健; 刘宁波; 曲晨慧; 王宝虎; 郭华; 王平

    2013-01-01

    目的:获得稳定的非小细胞肺癌(NSCLC)放射抗拒细胞系,明确常规分割和大分割放疗后肿瘤基因表达改变。方法:采用A549细胞系,6MV X线常规照射(2 Gy×17 f)和大分割照射(4 Gy×7 f),克隆形成实验和γ-H2AX免疫荧光染色结合共聚焦显微镜验证细胞的放射抗拒特性。提取mRNA,全基因组表达谱芯片检测差异基因表达,分析2倍以上改变的基因(P<0.05),同时对芯片结果行Pathway分析(Q<0.05)。结果:获得了2株放疗抗拒细胞系A549R2Gy-R和A549R4Gy-R。表达谱芯片显示,A549与A549R2Gy-R相比,差异表达基因为1701个(357个上调,1344个下调);A549与A549R4Gy-R相比,944个基因上调,2602个基因下调。A549R2Gy-R与A549R4Gy-R相比,318个基因上调,699个基因下调。常规分割照射与大分割照射的pathway显著性富集分析显示,PI3K和Erb B通路等多条信号通路激酶出现显著性差异。结论:多种基因和信号通路参与了NSCLC常规分割和大分割放疗抗拒过程,进一步研究能明确NSCLC放射抗拒机制和为放疗增敏药物开发提供新靶点。%Objective:To obtain stable radioresistant non-small-cell lung cancer (NSCLC) cell lines and identify the genetic pattern of conventional fractioned and hypofractionated radiotherapy. Methods:A549 NSCLC cells were treated with 6 MV of x-rays through conventional fractionated (2 Gy, 17 f) and hypofractionated irradiation (4 Gy, 7 f) to establish a radiation resistance cell model. Tumor cell radioresistance was determined using a clonogenic assay andγ-H2AX immunofluorescence staining combined with confocal microscopy. After extracting total mRNA from the cells, a whole genome expression microarray was applied to detect differential gene expression. The genes with at least a twofold increase in expression (P<0.05) were analyzed, and the pathway (Q<0.05) methods were used to further analyze the chip results

  20. Multi-platform whole-genome microarray analyses refine the epigenetic signature of breast cancer metastasis with gene expression and copy number.

    Directory of Open Access Journals (Sweden)

    Joseph Andrews

    Full Text Available BACKGROUND: We have previously identified genome-wide DNA methylation changes in a cell line model of breast cancer metastasis. These complex epigenetic changes that we observed, along with concurrent karyotype analyses, have led us to hypothesize that complex genomic alterations in cancer cells (deletions, translocations and ploidy are superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes observed in breast cancer metastasis. METHODOLOGY/PRINCIPAL FINDINGS: We undertook simultaneous high-resolution, whole-genome analyses of MDA-MB-468GFP and MDA-MB-468GFP-LN human breast cancer cell lines (an isogenic, paired lymphatic metastasis cell line model using Affymetrix gene expression (U133, promoter (1.0R, and SNP/CNV (SNP 6.0 microarray platforms to correlate data from gene expression, epigenetic (DNA methylation, and combination copy number variant/single nucleotide polymorphism microarrays. Using Partek Software and Ingenuity Pathway Analysis we integrated datasets from these three platforms and detected multiple hypomethylation and hypermethylation events. Many of these epigenetic alterations correlated with gene expression changes. In addition, gene dosage events correlated with the karyotypic differences observed between the cell lines and were reflected in specific promoter methylation patterns. Gene subsets were identified that correlated hyper (and hypo methylation with the loss (or gain of gene expression and in parallel, with gene dosage losses and gains, respectively. Individual gene targets from these subsets were also validated for their methylation, expression and copy number status, and susceptible gene pathways were identified that may indicate how selective advantage drives the processes of tumourigenesis and metastasis. CONCLUSIONS/SIGNIFICANCE: Our approach allows more precisely profiling of functionally relevant epigenetic signatures that are associated with cancer

  1. 哮喘患儿外周血单个核细胞全基因组表达谱的差异研究%Profiling of differential expression in asthma and healthy children' s peripheral blood mononuclear cells by whole-genome microarray

    Institute of Scientific and Technical Information of China (English)

    孔倩; 黄花荣; 吴葆菁; 李雯静; 陈纯

    2013-01-01

    目的:筛选哮喘患儿与对照组儿童外周血的基因表达谱差异,寻找与哮喘防治相关的靶基因.方法:从5名哮喘患儿和5名对照组儿童外周血单个核细胞中提取总RNA,利用全基因组表达谱芯片进行检测,选取经非配对t检验计算所得P≤0.05、同时基因表达变化≥2倍的差异表达基因,以荧光定量PCR(qRT-PCR)验证芯片结果.通过生物信息学软件对初步筛选的差异基因进行层次聚类分析和Gene Ontology (GO)功能分类分析.结果:从45 033条表达基因谱中筛选出哮喘患儿与对照组儿童差异表达2倍以上且P≤0.05的已命名基因758个(含上调基因345个,下调基因413个),其GO生物学过程功能分类主要涉及免疫反应、对外部刺激的反应、信号转导及分子功能的负性调节、细胞死亡、凋亡及其调节等.其中有29个基因与哮喘、气道炎症或气道重构有关,且变化趋势与文献报道一致(含上调基因14个,下调基因15个),并可被层次聚类分析划分为9类.qRT-PCR验证结果与芯片结果一致.结论:用全基因组表达谱芯片可以筛选出哮喘息儿与对照组儿童外周血单个核细胞中的差异表达基因,进一步的数据挖掘很可能寻找到与哮喘防治相关的靶基因或靶通路.%AIM:To compare the differences of whole-genome expression in peripheral blood mononuclear cells (PBMC) between asthma children and healthy controls.METHODS:The subjects were 5 cases of asthma children and 5 cases of healthy controls.Total RNA was extracted from PBMC and subjected to microarray analysis with NimbleG,en human gene expression array.Unpaired t-test algorithm was used to screen the differentially expressed genes when P≤0.05and fold change ≥ 2.Real-time quantitative PCR (qRT-PCR) was performed to verify the microarray results.Classification and function of the differential genes were illustrated by bioinformatic processing including hierarchical clustering and Gene

  2. Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification.

    Directory of Open Access Journals (Sweden)

    Yohei Nishikawa

    Full Text Available Whole genome amplification (WGA is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA, using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.

  3. Whole Genome Selection

    Science.gov (United States)

    Whole genome selection (WGS) is an approach to using DNA markers that are distributed throughout the entire genome. Genes affecting most economically-important traits are distributed throughout the genome and there are relatively few that have large effects with many more genes with progressively sm...

  4. Effect of Wortmannin on the repair profiles of DNA double-strand breaks in the whole genome and in interstitial telomeric sequences of Chinese hamster cells

    International Nuclear Information System (INIS)

    The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was applied to analyze the effect of Wortmannin (WM) in the rejoining kinetics of ionizing radiation-induced DNA double-strand breaks (DSBs) in the whole genome and in the long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cell lines. The results indicate that the ITRS blocks from wild-type Chinese hamster cell lines, CHO9 and V79B, exhibit a slower initial rejoining rate of ionizing radiation-induced DSBs than the genome overall. Neither Rad51C nor the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) activities, involved in homologous recombination (HR) and in non-homologous end-joining (NHEJ) pathways of DSB repair respectively, influenced the rejoining kinetics within ITRS in contrast to DNA sequences in the whole genome. Nevertheless, DSB removal rate within ITRS was decreased in the absence of Ku86 activity, though at a lower affectation level than in the whole genome, thus homogenizing both rejoining kinetics rates. WM treatment slowed down the DSB rejoining kinetics rate in ITRS, this effect being more pronounced in the whole genome, resulting in a similar pattern to that of the Ku86 deficient cells. In fact, no WM effect was detected in the Ku86 deficient Chinese hamster cells, so probably WM does not add further impairment in DSB rejoining than that resulted as a consequence of absence of Ku activity. The same slowing effect was also observed after treatment of Rad51C and DNA-PKcs defective hamster cells by WM, suggesting that: (1) there is no potentiation of the HR when the NHEJ is impaired by WM, either in the whole genome or in the ITRS, and (2) that this impairment may probably involve more targets than DNA-PKcs. These results suggest that there is an intragenomic heterogeneity in DSB repair, as well as in the effect of WM on this process

  5. Whole genome amplification induced bias in the detection of KRAS-mutated cell populations during colorectal carcinoma tissue testing.

    Science.gov (United States)

    Stranska, Jana; Jancik, Sylwia; Slavkovsky, Rastislav; Holinkova, Veronika; Rabcanova, Miroslava; Vojta, Petr; Hajduch, Marian; Drabek, Jiri

    2015-03-01

    Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA. We analyzed the origins of discrepancies in KRAS (Kirsten rat sarcoma viral oncogene homolog gene) mutation detection in six of ten samples amplified using the GenomePlex® Tissue Whole Genome Amplification kit 5 (WGA5; Sigma-Aldrich, St. Louis, MO, USA) and KRAS StripAssay® (KRAS SA; ViennaLab Diagnostics, Vienna, Austria). We undertook reextraction, reamplification, retyping, authentication, reanalysis, and reinterpretation to determine whether the discrepancies originated during the preanalytical, analytical, and/or interpretative phase of genotyping. We conclude that a combination of glass slide/sample heterogeneity and biased amplification due to stochastic effects in the early phases of whole genome amplification (WGA) may have adversely affected the results obtained. Our findings are relevant for both forensic genetics testing and massively parallel sequencing using preamplification. PMID:25655305

  6. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Directory of Open Access Journals (Sweden)

    Pandey Sona

    2010-11-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases (P450s catalyze oxidation of various substrates using oxygen and NAD(PH. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an

  7. Whole-genome expression analysis of Rice black-streaked dwarf virus in different plant hosts and small brown planthopper.

    Science.gov (United States)

    Xu, Qiufang; Ni, Haiping; Zhang, Jinfeng; Lan, Ying; Ren, Chunmei; Zhou, Yijun

    2015-11-10

    Rice black-streaked dwarf virus (RBSDV) can infect a number of gramineous plants and cause severe crop yield losses in southeast Asian countries. The virus is transmitted by small brown planthopper (SBPH) in a persistent circulative manner. The interactions between RBSDV and its different hosts remain unknown. Besides, how the virus adjusts itself to infect different hosts is unclear. In the present study, the relative RNA levels of the thirteen RBSDV genes in rice, maize, wheat, and SBPH were measured by real-time quantitative PCR. P7-1 and P10 genes were predominantly expressed whereas P8 and P7-2 genes were expressed at low levels in plant hosts. Similar to the expression in rice, P7-1 was the most abundantly expressed gene and P8 was expressed at the lowest level in SBPH, indicating that RBSDV adopts the same strategy to infect distinct hosts. The high expression levels of the P7-1 gene in both plants and insect suggest that it can be used as the target gene for disease diagnostics. However, the expression levels of some genes varied from host to host. P5-1, P6 and P9-1, the components of the RBSDV viroplasm, are differentially expressed in different hosts. Moreover, western blot analysis showed that the quantity of the P9-1 protein was more abundant in SBPH than in plant hosts. These data indicate that the virus may adjust its own gene expression to replicate in different hosts. Analysis of time course of gene expression revealed that P7-1 stands out as the only gene highly expressed at the earliest time point and its expression precedes all others throughout infection from 8 to 24days post-inoculation. The high expression levels of the P7-1 gene suggest that it plays a significant role in RBSDV-host interactions. PMID:26149652

  8. Quality control parameters on a large dataset of regionally dissected human control brains for whole genome expression studies

    OpenAIRE

    Trabzuni, Daniah; Ryten, Mina; Walker, Robert; Smith, Colin; Imran, Sabaena; Ramasamy, Adaikalavan; Weale, Michael E; Hardy, John

    2011-01-01

    We are building an open-access database of regional human brain expression designed to allow the genome-wide assessment of genetic variability on expression. Array and RNA sequencing technologies make assessment of genome-wide expression possible. Human brain tissue is a challenging source for this work because it can only be obtained several and variable hours post-mortem and after varying agonal states. These variables alter RNA integrity in a complex manner. In this report, we assess the e...

  9. EFFECTS OF TEMPERATURE ON GENE EXPRESSION PATTERNS IN LEPTOSPIRA INTERROGANS SEROVAR LAI AS ASSESSED BY WHOLE-GENOME MICROARRAYS

    Science.gov (United States)

    The availability of genome sequences for two serovars of Leptospira interrogans, Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor, which is known to affect leptospiral protein expression....

  10. Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing | Office of Cancer Genomics

    Science.gov (United States)

    Abstract: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer comprising at least two molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease.

  11. Whole genome wide expression profiles of Vitis amurensis grape responding to downy mildew by using Solexa sequencing technology

    Directory of Open Access Journals (Sweden)

    Huang Hong

    2010-10-01

    Full Text Available Abstract Background Downy mildew (DM, caused by pathogen Plasmopara viticola (PV is the single most damaging disease of grapes (Vitis L. worldwide. However, the mechanisms of the disease development in grapes are poorly understood. A method for estimating gene expression levels using Solexa sequencing of Type I restriction-endonuclease-generated cDNA fragments was used for deep sequencing the transcriptomes resulting from PV infected leaves of Vitis amurensis Rupr. cv. Zuoshan-1. Our goal is to identify genes that are involved in resistance to grape DM disease. Results Approximately 8.5 million (M 21-nt cDNA tags were sequenced in the cDNA library derived from PV pathogen-infected leaves, and about 7.5 M were sequenced from the cDNA library constructed from the control leaves. When annotated, a total of 15,249 putative genes were identified from the Solexa sequencing tags for the infection (INF library and 14,549 for the control (CON library. Comparative analysis between these two cDNA libraries showed about 0.9% of the unique tags increased by at least five-fold, and about 0.6% of the unique tags decreased more than five-fold in infected leaves, while 98.5% of the unique tags showed less than five-fold difference between the two samples. The expression levels of 12 differentially expressed genes were confirmed by Real-time RT-PCR and the trends observed agreed well with the Solexa expression profiles, although the degree of change was lower in amplitude. After pathway enrichment analysis, a set of significantly enriched pathways were identified for the differentially expressed genes (DEGs, which associated with ribosome structure, photosynthesis, amino acid and sugar metabolism. Conclusions This study presented a series of candidate genes and pathways that may contribute to DM resistance in grapes, and illustrated that the Solexa-based tag-sequencing approach was a powerful tool for gene expression comparison between control and treated samples.

  12. Effects of Temperature on Gene Expression Patterns in Leptospira interrogans Serovar Lai as Assessed by Whole-Genome Microarrays†

    OpenAIRE

    Lo, Miranda; Bulach, Dieter M.; Powell, David R.; David A Haake; Matsunaga, James; Paustian, Michael L.; Zuerner, Richard L.; Adler, Ben

    2006-01-01

    Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic Leptospira spp. from infected animals or contaminated water or soil. The availability of genome sequences for Leptospira interrogans, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression. Leptospira spp. can grow in a...

  13. Investigation of archived formalin-fixed paraffin-embedded pancreatic tissue with whole-genome gene expression microarray

    DEFF Research Database (Denmark)

    Michelsen, Nete Vinstrup; Brusgaard, Klaus; Tan, Qihua;

    2011-01-01

    The use of formalin-fixed, paraffin-embedded (FFPE) tissue overcomes the most prominent issues related to research on relatively rare diseases: limited sample size, availability of control tissue, and time frame. The use of FFPE pancreatic tissue in GEM may be especially challenging due to its very...... high amounts of ribonucleases compared to other tissues/organs. In choosing pancreatic tissue, we therefore indirectly address the applicability of other FFPE tissues to gene expression microarray (GEM). GEM was performed on archived, routinely fixed, FFPE pancreatic tissue from patients with......, (b) amino acid metabolism, and (c) calcium ion homeostasis. These results should encourage future research and GEM studies on FFPE tissue from the invaluable biobanks available at the departments of pathology worldwide....

  14. Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.

    Directory of Open Access Journals (Sweden)

    Young-Jin Park

    Full Text Available Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes and carbohydrate degradation (392 CAZymes, along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi- cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-cellulose biomass related industries further increase the significance of F. velutipes as a new model.

  15. Analyses and interpretation of whole-genome gene expression from formalin-fixed paraffin-embedded tissue: an illustration with breast cancer tissues

    OpenAIRE

    Argos Maria; Paul-Brutus Rachelle M; Roy Shantanu; Jasmine Farzana; Kibriya Muhammad G; Ahsan Habibul

    2010-01-01

    Abstract Background We evaluated (a) the feasibility of whole genome cDNA-mediated Annealing, Selection, extension and Ligation (DASL) assay on formalin-fixed paraffin-embedded (FFPE) tissue and (b) whether similar conclusions can be drawn by examining FFPE samples as proxies for fresh frozen (FF) tissues. We used a whole genome DASL assay (addressing 18,391 genes) on a total of 72 samples from paired breast tumor and surrounding healthy tissues from both FF and FFPE samples. Results Gene det...

  16. Shared clonal cytogenetic abnormalities in aberrant mast cells and leukemic myeloid blasts detected by single nucleotide polymorphism microarray-based whole-genome scanning.

    Science.gov (United States)

    Frederiksen, John K; Shao, Lina; Bixby, Dale L; Ross, Charles W

    2016-04-01

    Systemic mastocytosis (SM) is characterized by a clonal proliferation of aberrant mast cells within extracutaneous sites. In a subset of SM cases, a second associated hematologic non-mast cell disease (AHNMD) is also present, usually of myeloid origin. Polymerase chain reaction and targeted fluorescence in situ hybridization studies have provided evidence that, in at least some cases, the aberrant mast cells are related clonally to the neoplastic cells of the AHNMD. In this work, a single nucleotide polymorphism microarray (SNP-A) was used to characterize the cytogenetics of the aberrant mast cells from a patient with acute myeloid leukemia and concomitant mast cell leukemia associated with a KIT D816A mutation. The results demonstrate the presence of shared cytogenetic abnormalities between the mast cells and myeloid blasts, as well as additional abnormalities within mast cells (copy-neutral loss of heterozygosity) not detectable by routine karyotypic analysis. To our knowledge, this work represents the first application of SNP-A whole-genome scanning to the detection of shared cytogenetic abnormalities between the two components of a case of SM-AHNMD. The findings provide additional evidence of a frequent clonal link between aberrant mast cells and cells of myeloid AHNMDs, and also highlight the importance of direct sequencing for identifying uncommon activating KIT mutations. PMID:26865278

  17. Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

    Energy Technology Data Exchange (ETDEWEB)

    Catfish Genome Consortium; Wang, Shaolin; Peatman, Eric; Abernathy, Jason; Waldbieser, Geoff; Lindquist, Erika; Richardson, Paul; Lucas, Susan; Wang, Mei; Li, Ping; Thimmapuram, Jyothi; Liu, Lei; Vullaganti, Deepika; Kucuktas, Huseyin; Murdock, Christopher; Small, Brian C; Wilson, Melanie; Liu, Hong; Jiang, Yanliang; Lee, Yoona; Chen, Fei; Lu, Jianguo; Wang, Wenqi; Xu, Peng; Somridhivej, Benjaporn; Baoprasertkul, Puttharat; Quilang, Jonas; Sha, Zhenxia; Bao, Baolong; Wang, Yaping; Wang, Qun; Takano, Tomokazu; Nandi, Samiran; Liu, Shikai; Wong, Lilian; Kaltenboeck, Ludmilla; Quiniou, Sylvie; Bengten, Eva; Miller, Norman; Trant, John; Rokhsar, Daniel; Liu, Zhanjiang

    2010-03-23

    Background-Through the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energy's Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification. Results-A total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35percent of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis. Conclusions-This project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

  18. Whole Genome Sequence of Multiple Myeloma-Prone C57BL/KaLwRij Mouse Strain Suggests the Origin of Disease Involves Multiple Cell Types.

    Directory of Open Access Journals (Sweden)

    Sarah R Amend

    Full Text Available Monoclonal gammopathy of undetermined significance (MGUS is the requisite precursor to multiple myeloma (MM, a malignancy of antibody-producing plasma B-cells. The genetic basis of MGUS and its progression to MM remains poorly understood. C57BL/KaLwRij (KaLwRij is a spontaneously-derived inbred mouse strain with a high frequency of benign idiopathic paraproteinemia (BIP, a phenotype with similarities to MGUS including progression to MM. Using mouse haplotype analysis, human MM SNP array data, and whole exome and whole genome sequencing of KaLwRij mice, we identified novel KaLwRij gene variants, including deletion of Samsn1 and deleterious point mutations in Tnfrsf22 and Tnfrsf23. These variants significantly affected multiple cell types implicated in MM pathogenesis including B-cells, macrophages, and bone marrow stromal cells. These data demonstrate that multiple cell types contribute to MM development prior to the acquisition of somatic driver mutations in KaLwRij mice, and suggest that MM may an inherently non-cell autonomous malignancy.

  19. Whole-genome sequencing of multiple myeloma from diagnosis to plasma cell leukemia reveals genomic initiating events, evolution, and clonal tides.

    Science.gov (United States)

    Egan, Jan B; Shi, Chang-Xin; Tembe, Waibhav; Christoforides, Alexis; Kurdoglu, Ahmet; Sinari, Shripad; Middha, Sumit; Asmann, Yan; Schmidt, Jessica; Braggio, Esteban; Keats, Jonathan J; Fonseca, Rafael; Bergsagel, P Leif; Craig, David W; Carpten, John D; Stewart, A Keith

    2012-08-01

    The longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL. PMID:22529291

  20. Whole Genome and Transcriptome Sequencing of a B3 Thymoma

    OpenAIRE

    Iacopo Petrini; Arun Rajan; Trung Pham; Donna Voeller; Sean Davis; James Gao; Yisong Wang; Giuseppe Giaccone

    2013-01-01

    Molecular pathology of thymomas is poorly understood. Genomic aberrations are frequently identified in tumors but no extensive sequencing has been reported in thymomas. Here we present the first comprehensive view of a B3 thymoma at whole genome and transcriptome levels. A 55-year-old Caucasian female underwent complete resection of a stage IVA B3 thymoma. RNA and DNA were extracted from a snap frozen tumor sample with a fraction of cancer cells over 80%. We performed array comparative genomi...

  1. Interpreting Whole-Genome Marker Data

    OpenAIRE

    Weir, Bruce S.

    2013-01-01

    The challenges of whole-genome data, when genotypes are available from hundreds of thousands of genetic markers, are explored for four topics in statistical genetics: Hardy-Weinberg testing, estimating linkage disequilibrium from unphased genotypic data, association mapping and characterizing population structure.

  2. Whole genome linkage disequilibrium maps in cattle

    Science.gov (United States)

    Bovine whole genome linkage disequilibrium maps were constructed for eight breeds of cattle. These data provide fundamental information concerning bovine genome organization which will allow the design of studies to associate genetic variation with economically important traits and also provides bac...

  3. Whole-genome cartography of estrogen receptor alpha binding sites.

    Directory of Open Access Journals (Sweden)

    Chin-Yo Lin

    2007-06-01

    Full Text Available Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript, suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs, 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha

  4. Whole genome and transcriptome sequencing of a B3 thymoma.

    Directory of Open Access Journals (Sweden)

    Iacopo Petrini

    Full Text Available Molecular pathology of thymomas is poorly understood. Genomic aberrations are frequently identified in tumors but no extensive sequencing has been reported in thymomas. Here we present the first comprehensive view of a B3 thymoma at whole genome and transcriptome levels. A 55-year-old Caucasian female underwent complete resection of a stage IVA B3 thymoma. RNA and DNA were extracted from a snap frozen tumor sample with a fraction of cancer cells over 80%. We performed array comparative genomic hybridization using Agilent platform, transcriptome sequencing using HiSeq 2000 (Illumina and whole genome sequencing using Complete Genomics Inc platform. Whole genome sequencing determined, in tumor and normal, the sequence of both alleles in more than 95% of the reference genome (NCBI Build 37. Copy number (CN aberrations were comparable with those previously described for B3 thymomas, with CN gain of chromosome 1q, 5, 7 and X and CN loss of 3p, 6, 11q42.2-qter and q13. One translocation t(11;X was identified by whole genome sequencing and confirmed by PCR and Sanger sequencing. Ten single nucleotide variations (SNVs and 2 insertion/deletions (INDELs were identified; these mutations resulted in non-synonymous amino acid changes or affected splicing sites. The lack of common cancer-associated mutations in this patient suggests that thymomas may evolve through mechanisms distinctive from other tumor types, and supports the rationale for additional high-throughput sequencing screens to better understand the somatic genetic architecture of thymoma.

  5. Whole-genome shotgun optical mapping of Rhodospirillum rubrum

    Energy Technology Data Exchange (ETDEWEB)

    Reslewic, S. [Univ. Wisc.-Madison; Zhou, S. [Univ. Wisc.-Madison; Place, M. [Univ. Wisc.-Madison; Zhang, Y. [Univ. Wisc.-Madison; Briska, A. [Univ. Wisc.-Madison; Goldstein, S. [Univ. Wisc.-Madison; Churas, C. [Univ. Wisc.-Madison; Runnheim, R. [Univ. Wisc.-Madison; Forrest, D. [Univ. Wisc.-Madison; Lim, A. [Univ. Wisc.-Madison; Lapidus, A. [Univ. Wisc.-Madison; Han, C. S. [Univ. Wisc.-Madison; Roberts, G. P. [Univ. Wisc.-Madison; Schwartz, D. C. [Univ. Wisc.-Madison

    2005-09-01

    Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.

  6. Whole-genome shotgun optical mapping of rhodospirillumrubrum

    Energy Technology Data Exchange (ETDEWEB)

    Reslewic, Susan; Zhou, Shiguo; Place, Mike; Zhang, Yaoping; Briska, Adam; Goldstein, Steve; Churas, Chris; Runnheim, Rod; Forrest,Dan; Lim, Alex; Lapidus, Alla; Han, Cliff S.; Roberts, Gary P.; Schwartz,David C.

    2004-07-01

    Rhodospirillum rubrum is a phototrophic purple non-sulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems, and as a source of hydrogen and biodegradable plastics production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction maps (Xba I, Nhe I, and Hind III) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction maps from randomly sheared genomic DNA molecules extracted directly from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the Hind III map acted as a scaffold for high resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and validation of genome sequence, our work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a ''molecular cytogenetics'' approach to solving problems in genomic analysis.

  7. Whole genome phylogenies for multiple Drosophila species

    Directory of Open Access Journals (Sweden)

    Seetharam Arun

    2012-12-01

    Full Text Available Abstract Background Reconstructing the evolutionary history of organisms using traditional phylogenetic methods may suffer from inaccurate sequence alignment. An alternative approach, particularly effective when whole genome sequences are available, is to employ methods that don’t use explicit sequence alignments. We extend a novel phylogenetic method based on Singular Value Decomposition (SVD to reconstruct the phylogeny of 12 sequenced Drosophila species. SVD analysis provides accurate comparisons for a high fraction of sequences within whole genomes without the prior identification of orthologs or homologous sites. With this method all protein sequences are converted to peptide frequency vectors within a matrix that is decomposed to provide simplified vector representations for each protein of the genome in a reduced dimensional space. These vectors are summed together to provide a vector representation for each species, and the angle between these vectors provides distance measures that are used to construct species trees. Results An unfiltered whole genome analysis (193,622 predicted proteins strongly supports the currently accepted phylogeny for 12 Drosophila species at higher dimensions except for the generally accepted but difficult to discern sister relationship between D. erecta and D. yakuba. Also, in accordance with previous studies, many sequences appear to support alternative phylogenies. In this case, we observed grouping of D. erecta with D. sechellia when approximately 55% to 95% of the proteins were removed using a filter based on projection values or by reducing resolution by using fewer dimensions. Similar results were obtained when just the melanogaster subgroup was analyzed. Conclusions These results indicate that using our novel phylogenetic method, it is possible to consult and interpret all predicted protein sequences within multiple whole genomes to produce accurate phylogenetic estimations of relatedness between

  8. 基因芯片技术研究虚寒证大鼠肝全基因表达谱%Study of Gene Chip Technology on Whole Genome Expression of Liver of Rats with Asthenia Cold Syndrome

    Institute of Scientific and Technical Information of China (English)

    韩冰冰; 王世军; 于华芸; 赵海军; 王媛

    2011-01-01

    Objective;To study the whole genome expression of liver on asthenia cold syndrome rats by gene chip technology. Methods:The Asthenia cold syndrome rats models were induced by compound preparation of traditional Chinese medicine of Raw Gypsum,Radix gentianae、Cortex Phellodendri 、Anemarrhenae Rhizoma.The liver gene expression in each group was detected by gene chip. We selected the differential expression genes and conducted the significant analysis on the genetic function of differential genes. A part of genes were selected to test the accuracy of results by RT - PCR. Results: As compared to the control group,in asthenia cold model group there were 99 strips of differential expression gene, mainly about function of response to stimulus. Conclusion ; Many strips of gene about response to stimulus were down - regulated in asthenia cold syndrome rats, which induced down regulation of function about immune response,defense response,response to other organism. The substance foundation of asthenia cold syndrome was possibly related to these genes.%目的:采用基因芯片技术研究虚寒证大鼠肝全基因表达谱的改变.方法:使用中药复方生石膏、龙胆草、黄柏和知母建立虚寒证大鼠模型,应用基因芯片检测各组大鼠肝脏基因表达,筛选差异表达基因,进行基因功能分类注释.荧光定量PCR验证芯片结果.结果:虚寒模型组与空白对照组比较有99条基因差异表达,主要涉及刺激应答功能.结论:虚寒证可能通过多种刺激应答相关基因的下调,导致免疫应答功能、防御应答功能及对其他生物体刺激应答功能的降低.虚寒证的物质基础可能与此类基因的异常表达相关.

  9. Whole-genome gene expression modifications associated with nitrosamine exposure and micronucleus frequency in human blood cells

    DEFF Research Database (Denmark)

    Hebels, Dennie G A J; Jennen, Danyel G J; van Herwijnen, Marcel H M;

    2011-01-01

    concurrently investigating associated transcriptomic responses may provide crucial information on underlying molecular mechanisms that predispose to carcinogenicity. We used lymphocytes, from adult females participating in the pan-European biomarker research project NewGeneris, as a surrogate tissue for...

  10. Microbial species delineation using whole genome sequences

    Energy Technology Data Exchange (ETDEWEB)

    Kyrpides, Nikos; Mukherjee, Supratim; Ivanova, Natalia; Mavrommatics, Kostas; Pati, Amrita; Konstantinidis, Konstantinos

    2014-10-20

    Species assignments in prokaryotes use a manual, poly-phasic approach utilizing both phenotypic traits and sequence information of phylogenetic marker genes. With thousands of genomes being sequenced every year, an automated, uniform and scalable approach exploiting the rich genomic information in whole genome sequences is desired, at least for the initial assignment of species to an organism. We have evaluated pairwise genome-wide Average Nucleotide Identity (gANI) values and alignment fractions (AFs) for nearly 13,000 genomes using our fast implementation of the computation, identifying robust and widely applicable hard cut-offs for species assignments based on AF and gANI. Using these cutoffs, we generated stable species-level clusters of organisms, which enabled the identification of several species mis-assignments and facilitated the assignment of species for organisms without species definitions.

  11. Strategies and tools for whole genome alignments

    Energy Technology Data Exchange (ETDEWEB)

    Couronne, Olivier; Poliakov, Alexander; Bray, Nicolas; Ishkhanov,Tigran; Ryaboy, Dmitriy; Rubin, Edward; Pachter, Lior; Dubchak, Inna

    2002-11-25

    The availability of the assembled mouse genome makespossible, for the first time, an alignment and comparison of two largevertebrate genomes. We have investigated different strategies ofalignment for the subsequent analysis of conservation of genomes that areeffective for different quality assemblies. These strategies were appliedto the comparison of the working draft of the human genome with the MouseGenome Sequencing Consortium assembly, as well as other intermediatemouse assemblies. Our methods are fast and the resulting alignmentsexhibit a high degree of sensitivity, covering more than 90 percent ofknown coding exons in the human genome. We have obtained such coveragewhile preserving specificity. With a view towards the end user, we havedeveloped a suite of tools and websites for automatically aligning, andsubsequently browsing and working with whole genome comparisons. Wedescribe the use of these tools to identify conserved non-coding regionsbetween the human and mouse genomes, some of which have not beenidentified by other methods.

  12. Whole Genome Epidemiological Typing of Escherichia coli

    DEFF Research Database (Denmark)

    Kaas, Rolf Sommer

    is in general expensive and to some extent unreliable. Next generation sequencing has quickly become a tool widely available and has enabled even smaller laboratories to do whole genome sequencing (WGS). Having the entire genome available provides the opportunity to create the ultimate typing method. This Ph......D thesis attempts to take the first steps toward such a method. In Kaas I all publicly available E. coli genomes sequenced (186) are analyzed. 1,702 core genes were found in all genomes. 3,051 genes were found in 95% of the genomes. The pan genome was found to consist of 16,373 genes. The overall phylogeny...... was inferred from the core genome and also set into context of the Escherichia genus. The variance within each gene cluster was calculated in order to compare the variance between genes and possibly identify typing targets for further study. The variance scores calculated was also used to compare the three...

  13. Whole Genome Epidemiological Typing of Salmonella

    DEFF Research Database (Denmark)

    Leekitcharoenphon, Pimlapas

    . Technological advances and effective price in high throughput genome sequencing are making whole genome sequencing (WGS) available as a routine tool for bacterial typing. Typing of Salmonella, especially sub-typing within the same serotype or even the same clone, the genetic variation of the target genes being...... used for typing is crucial for successful discrimination. The core genes or the genes that are conserved in all members of a genus or species are potentially good candidates for investigating genomic variation in phylogeny and epidemiology. A total of 2,882 core genes have been observed among 73...... available Salmonella enterica genomes (accessed in April 2011). A consensus tree based on variation of the core genes gives better resolution than 16S rRNA and MLST that rarely provide separation between closely related strains. The performance of the pan-genome tree which is based on the presence...

  14. Whole-genome sequence-based analysis of thyroid function

    DEFF Research Database (Denmark)

    Taylor, Peter N.; Porcu, Eleonora; Chew, Shelby;

    2015-01-01

    Normal thyroid function is essential for health, but its genetic architecture remains poorly understood. Here, for the heritable thyroid traits thyrotropin (TSH) and free thyroxine (FT4), we analyse whole-genome sequence data from the UK10K project (N = 2,287). Using additional whole-genome...... association with FT4 in NRG1. Our results demonstrate that increased coverage in whole-genome sequence association studies identifies novel variants associated with thyroid function....

  15. Tolerance of Whole-Genome Doubling Propagates Chromosomal Instability and Accelerates Cancer Genome Evolution

    DEFF Research Database (Denmark)

    Dewhurst, Sally M.; McGranahan, Nicholas; Burrell, Rebecca A.; Rowan, Andrew J.; Grönroos, Eva; Endesfelder, David; Joshi, Tejal; Mouradov, Dmitri; Gibbs, Peter; Ward, Robyn L.; Hawkins, Nicholas J.; Szallasi, Zoltan Imre; Sieber, Oliver M.; Swanton, Charles

    2014-01-01

    The contribution of whole-genome doubling to chromosomal instability (CIN) and tumor evolution is unclear. We use long-term culture of isogenic tetraploid cells from a stable diploid colon cancer progenitor to investigate how a genome-doubling event affects genome stability over time. Rare cells ...

  16. Whole genome analysis of a Vietnamese trio

    Indian Academy of Sciences (India)

    Dang Thanh Hai; Nguyen Dai Thanh; Pham Thi Minh Trang; Le Si Quang; Phan Thi Thu Hang; Dang Cao Cuong; Hoang Kim Phuc; Nguyen Huu Duc; Do Duc Dong; Bui Quang Minh; Pham Bao Son; Le Sy Vinh

    2015-03-01

    We here present the first whole genome analysis of an anonymous Kinh Vietnamese (KHV) trio whose genomes were deeply sequenced to 30-fold average coverage. The resulting short reads covered 99.91% of the human reference genome (GRCh37d5). We identified 4,719,412 SNPs and 827,385 short indels that satisfied the Mendelian inheritance law. Among them, 109,914 (2.3%) SNPs and 59,119 (7.1%) short indels were novel. We also detected 30,171 structural variants of which 27,604 (91.5%) were large indels. There were 6,681 large indels in the range 0.1–100 kbp occurring in the child genome that were also confirmed in either the father or mother genome.We compared these large indels against the DGV database and found that 1,499 (22.44%) were KHV specific. De novo assembly of high-quality unmapped reads yielded 789 contigs with the length ≥ 300 bp. There were 235 contigs from the child genome of which 199 (84.7%) were significantly matched with at least one contig from the father or mother genome. Blasting these 199 contigs against other alternative human genomes revealed 4 novel contigs. The novel variants identified from our study demonstrated the necessity of conducting more genome-wide studies not only for Kinh but also for other ethnic groups in Vietnam.

  17. Small Sample Whole-Genome Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Hara, C A; Nguyen, C P; Wheeler, E K; Sorensen, K J; Arroyo, E S; Vrankovich, G P; Christian, A T

    2005-09-20

    Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.

  18. Integrated clinical, whole-genome, and transcriptome analysis of multisampled lethal metastatic prostate cancer.

    Science.gov (United States)

    Bova, G Steven; Kallio, Heini M L; Annala, Matti; Kivinummi, Kati; Högnäs, Gunilla; Häyrynen, Sergei; Rantapero, Tommi; Kivinen, Virpi; Isaacs, William B; Tolonen, Teemu; Nykter, Matti; Visakorpi, Tapio

    2016-05-01

    We report the first combined analysis of whole-genome sequence, detailed clinical history, and transcriptome sequence of multiple prostate cancer metastases in a single patient (A21). Whole-genome and transcriptome sequence was obtained from nine anatomically separate metastases, and targeted DNA sequencing was performed in cancerous and noncancerous foci within the primary tumor specimen removed 5 yr before death. Transcriptome analysis revealed increased expression of androgen receptor (AR)-regulated genes in liver metastases that harbored an AR p.L702H mutation, suggesting a dominant effect by the mutation despite being present in only one of an estimated 16 copies per cell. The metastases harbored several alterations to the PI3K/AKT pathway, including a clonal truncal mutation in PIK3CG and present in all metastatic sites studied. The list of truncal genomic alterations shared by all metastases included homozygous deletion of TP53, hemizygous deletion of RB1 and CHD1, and amplification of FGFR1. If the patient were treated today, given this knowledge, the use of second-generation androgen-directed therapies, cessation of glucocorticoid administration, and therapeutic inhibition of the PI3K/AKT pathway or FGFR1 receptor could provide personalized benefit. Three previously unreported truncal clonal missense mutations (ABCC4 p.R891L, ALDH9A1 p.W89R, and ASNA1 p.P75R) were expressed at the RNA level and assessed as druggable. The truncal status of mutations may be critical for effective actionability and merit further study. Our findings suggest that a large set of deeply analyzed cases could serve as a powerful guide to more effective prostate cancer basic science and personalized cancer medicine clinical trials. PMID:27148588

  19. Integrated clinical, whole-genome, and transcriptome analysis of multisampled lethal metastatic prostate cancer

    Science.gov (United States)

    Bova, G. Steven; Kallio, Heini M.L.; Annala, Matti; Kivinummi, Kati; Högnäs, Gunilla; Häyrynen, Sergei; Rantapero, Tommi; Kivinen, Virpi; Isaacs, William B.; Tolonen, Teemu; Nykter, Matti; Visakorpi, Tapio

    2016-01-01

    We report the first combined analysis of whole-genome sequence, detailed clinical history, and transcriptome sequence of multiple prostate cancer metastases in a single patient (A21). Whole-genome and transcriptome sequence was obtained from nine anatomically separate metastases, and targeted DNA sequencing was performed in cancerous and noncancerous foci within the primary tumor specimen removed 5 yr before death. Transcriptome analysis revealed increased expression of androgen receptor (AR)-regulated genes in liver metastases that harbored an AR p.L702H mutation, suggesting a dominant effect by the mutation despite being present in only one of an estimated 16 copies per cell. The metastases harbored several alterations to the PI3K/AKT pathway, including a clonal truncal mutation in PIK3CG and present in all metastatic sites studied. The list of truncal genomic alterations shared by all metastases included homozygous deletion of TP53, hemizygous deletion of RB1 and CHD1, and amplification of FGFR1. If the patient were treated today, given this knowledge, the use of second-generation androgen-directed therapies, cessation of glucocorticoid administration, and therapeutic inhibition of the PI3K/AKT pathway or FGFR1 receptor could provide personalized benefit. Three previously unreported truncal clonal missense mutations (ABCC4 p.R891L, ALDH9A1 p.W89R, and ASNA1 p.P75R) were expressed at the RNA level and assessed as druggable. The truncal status of mutations may be critical for effective actionability and merit further study. Our findings suggest that a large set of deeply analyzed cases could serve as a powerful guide to more effective prostate cancer basic science and personalized cancer medicine clinical trials.

  20. Allelic imbalance analysis by high-density single-nucleotide polymorphic allele (SNP) array with whole genome amplified DNA

    OpenAIRE

    Wong, Kwong-Kwok; Tsang, Yvonne T.M.; Shen, Jianhe; Cheng, Rita S.; Chang, Yi-Mieng; Man, Tsz-Kwong; Lau, Ching C.

    2004-01-01

    Besides their use in mRNA expression profiling, oligonucleotide microarrays have also been applied to single-nucleotide polymorphism (SNP) and loss of heterozygosity (LOH) or allelic imbalance studies. In this report, we evaluate the reliability of using whole genome amplified DNA for analysis with an oligonucleotide microarray containing 11 560 SNPs to detect allelic imbalance and chromosomal copy number abnormalities. Whole genome SNP analyses were performed with DNA extracted from osteosar...

  1. Whole Genome Sequencing: Cracking the Genetic Code for Foodborne Illness

    Science.gov (United States)

    ... For Consumers Home For Consumers Consumer Updates Whole Genome Sequencing: Cracking the Genetic Code for Foodborne Illness ... Bacteria that cause disease have millions of different genomes, or sequences of genetic code, each as unique ...

  2. Comparative Copy Number Variation From Whole Genome Sequencing

    OpenAIRE

    Janevski, A.; Varadan, V.; Kamalakaran, S.; Banerjee, N.; Dimitrova, D

    2011-01-01

    Whole genome sequencing enables a high resolution view of the humangenome and enables unique insights into copy number variations in anunprecedented scale. Numerous tools and studies have already been introduced that provide confirmatory and new genomic variability datain individuals and across populations. We investigate two such methods, CNV-seq and FREEC and compare their outputs when applied to five whole genome sequences representing four populations. We focus onthe ability of these tool...

  3. Mapping Challenging Mutations by Whole-Genome Sequencing

    OpenAIRE

    Smith, Harold E.; Fabritius, Amy S.; Aimee Jaramillo-Lambert; Andy Golden

    2016-01-01

    Whole-genome sequencing provides a rapid and powerful method for identifying mutations on a global scale, and has spurred a renewed enthusiasm for classical genetic screens in model organisms. The most commonly characterized category of mutation consists of monogenic, recessive traits, due to their genetic tractability. Therefore, most of the mapping methods for mutation identification by whole-genome sequencing are directed toward alleles that fulfill those criteria (i.e., single-gene, homoz...

  4. Bioinformatics for Whole-Genome Shotgun Sequencing of Microbial Communities

    OpenAIRE

    Chen, Kevin; Pachter, Lior

    2005-01-01

    The application of whole-genome shotgun sequencing to microbial communities represents a major development in metagenomics, the study of uncultured microbes via the tools of modern genomic analysis. In the past year, whole-genome shotgun sequencing projects of prokaryotic communities from an acid mine biofilm, the Sargasso Sea, Minnesota farm soil, three deep-sea whale falls, and deep-sea sediments have been reported, adding to previously published work on viral communities from marine and fe...

  5. Comparative analysis of whole genome structure of Streptococcus suis using whole genome PCR scanning

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    An outbreak associated with Streptococcus suis infection in humans emerged in Sichuan province, China in 2005. The outbreak is atypical for the apparent large number of human cases, high fatality rate and geographical spread. To determine whether the bacterium has changed, we compared both human and animal isolates from the Sichuan outbreak with those collected previously within China and in other countries using whole genome PCR scanning (WGPScaning) comparative sequencing of several known virulence factor genes and multilocus sequence typing (MLST) analysis. WGPScanning analysis showed that all primer pairs yielded PCR products of the expected sizes in all four strains tested. The nucleotide sequences of all the detected virulence factor genes are identical in the four strains and MLST results showed that the four isolates studied and reference strain all belonged to the ST1 com-plex. No new genetic changes were found in the genome structure of the isolates from this Sichuan outbreak.

  6. Comparative analysis of whole genome structure of Streptococcus suis using whole genome PCR scanning

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    An outbreak associated with Streptococcus suis infection in humans emerged in Sichuan province, China in 2005. The outbreak is atypical for the apparent large number of human cases, high fatality rate and geographical spread. To determine whether the bacterium has changed, we compared both human and animal isolates from the Sichuan outbreak with those collected previously within China and in other countries using whole genome PCR scanning (WGPScaning) comparative sequencing of several known virulence factor genes and multilocus sequence typing (MLST) analysis. WGPScanning analysis showed that all primer pairs yielded PCR products of the expected sizes in all four strains tested. The nucleotide sequences of all the detected virulence factor genes are identical in the four strains and MLST results showed that the four isolates studied and reference strain all belonged to the ST1 complex. No new genetic changes were found in the genome structure of the isolates from this Sichuan outbreak.

  7. New wheat microRNA using whole-genome sequence.

    Science.gov (United States)

    Kurtoglu, Kuaybe Yucebilgili; Kantar, Melda; Budak, Hikmet

    2014-06-01

    MicroRNAs are post-transcriptional regulators of gene expression, taking roles in a variety of fundamental biological processes. Hence, their identification, annotation and characterization are of great significance, especially in bread wheat, one of the main food sources for humans. The recent availability of 5× coverage Triticum aestivum L. whole-genome sequence provided us with the opportunity to perform a systematic prediction of a complete catalogue of wheat microRNAs. Using an in silico homology-based approach, stem-loop coding regions were derived from two assemblies, constructed from wheat 454 reads. To avoid the presence of pseudo-microRNAs in the final data set, transposable element related stem-loops were eliminated by repeat analysis. Overall, 52 putative wheat microRNAs were predicted, including seven, which have not been previously published. Moreover, with distinct analysis of the two different assemblies, both variety and representation of putative microRNA-coding stem-loops were found to be predominant in the intergenic regions. By searching available expressed sequences and small RNA library databases, expression evidence for 39 (out of 52) putative wheat microRNAs was provided. Expression of three of the predicted microRNAs (miR166, miR396 and miR528) was also comparatively quantified with real-time quantitative reverse transcription PCR. This is the first report on in silico prediction of a whole repertoire of bread wheat microRNAs, supported by the wet-lab validation. PMID:24395439

  8. Information recovery from low coverage whole-genome bisulfite sequencing

    Science.gov (United States)

    Libertini, Emanuele; Heath, Simon C.; Hamoudi, Rifat A.; Gut, Marta; Ziller, Michael J.; Czyz, Agata; Ruotti, Victor; Stunnenberg, Hendrik G.; Frontini, Mattia; Ouwehand, Willem H.; Meissner, Alexander; Gut, Ivo G.; Beck, Stephan

    2016-01-01

    The cost of whole-genome bisulfite sequencing (WGBS) remains a bottleneck for many studies and it is therefore imperative to extract as much information as possible from a given dataset. This is particularly important because even at the recommend 30X coverage for reference methylomes, up to 50% of high-resolution features such as differentially methylated positions (DMPs) cannot be called with current methods as determined by saturation analysis. To address this limitation, we have developed a tool that dynamically segments WGBS methylomes into blocks of comethylation (COMETs) from which lost information can be recovered in the form of differentially methylated COMETs (DMCs). Using this tool, we demonstrate recovery of ∼30% of the lost DMP information content as DMCs even at very low (5X) coverage. This constitutes twice the amount that can be recovered using an existing method based on differentially methylated regions (DMRs). In addition, we explored the relationship between COMETs and haplotypes in lymphoblastoid cell lines of African and European origin. Using best fit analysis, we show COMETs to be correlated in a population-specific manner, suggesting that this type of dynamic segmentation may be useful for integrated (epi)genome-wide association studies in the future. PMID:27346250

  9. Whole genome transcript profiling from fingerstick blood samples: a comparison and feasibility study

    OpenAIRE

    Williams Adam R; Mondala Tony S; Robison Elizabeth H; Head Steven R; Salomon Daniel R; Kurian Sunil M

    2009-01-01

    Abstract Background Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a veno...

  10. Fish-oil supplementation induces antiinflammatory gene expression profiles in human blood mononuclear cells

    OpenAIRE

    Bouwens, M.; Rest, van de, O.; Dellschaft, N.; Grootte Bromhaar, M.M.; Groot, de, W.T.; Geleijnse, J M; Müller, M.R.; Afman, L.A.

    2009-01-01

    Background: Polyunsaturated fatty acids can have beneficial effects on human immune cells, such as peripheral blood mononuclear cells (PBMCs). However, the mechanisms of action of polyunsaturated fatty acids on immune cells are still largely unknown. Objective: The objective was to examine the effects of supplementation with the polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on whole-genome PBMC gene expression profiles, in healthy Dutch elderly subject...

  11. Whole-Genome Sequencing of Two Bartonella bacilliformis Strains

    Science.gov (United States)

    Guillen, Yolanda; Casadellà, Maria; García-de-la-Guarda, Ruth; Espinoza-Culupú, Abraham; Paredes, Roger; Ruiz, Joaquim

    2016-01-01

    Bartonella bacilliformis is the causative agent of Carrion’s disease, a highly endemic human bartonellosis in Peru. We performed a whole-genome assembly of two B. bacilliformis strains isolated from the blood of infected patients in the acute phase of Carrion’s disease from the Cusco and Piura regions in Peru. PMID:27389274

  12. Whole-Genome Sequences of Three Symbiotic Endozoicomonas Bacteria

    KAUST Repository

    Neave, Matthew J.

    2014-08-14

    Members of the genus Endozoicomonas associate with a wide range of marine organisms. Here, we report on the whole-genome sequencing, assembly, and annotation of three Endozoicomonas type strains. These data will assist in exploring interactions between Endozoicomonas organisms and their hosts, and it will aid in the assembly of genomes from uncultivated Endozoicomonas spp.

  13. Whole-genome bisulfite DNA sequencing of a DNMT3B mutant patient

    Science.gov (United States)

    Heyn, Holger; Vidal, Enrique; Sayols, Sergi; Sanchez-Mut, Jose V.; Moran, Sebastian; Medina, Ignacio; Sandoval, Juan; Simó-Riudalbas, Laia; Szczesna, Karolina; Huertas, Dori; Gatto, Sole; Matarazzo, Maria R.; Dopazo, Joaquin; Esteller, Manel

    2012-01-01

    The immunodeficiency, centromere instability and facial anomalies (ICF) syndrome is associated to mutations of the DNA methyl-transferase DNMT3B, resulting in a reduction of enzyme activity. Aberrant expression of immune system genes and hypomethylation of pericentromeric regions accompanied by chromosomal instability were determined as alterations driving the disease phenotype. However, so far only technologies capable to analyze single loci were applied to determine epigenetic alterations in ICF patients. In the current study, we performed whole-genome bisulphite sequencing to assess alteration in DNA methylation at base pair resolution. Genome-wide we detected a decrease of methylation level of 42%, with the most profound changes occurring in inactive heterochromatic regions, satellite repeats and transposons. Interestingly, transcriptional active loci and ribosomal RNA repeats escaped global hypomethylation. Despite a genome-wide loss of DNA methylation the epigenetic landscape and crucial regulatory structures were conserved. Remarkably, we revealed a mislocated activity of mutant DNMT3B to H3K4me1 loci resulting in hypermethylation of active promoters. Functionally, we could associate alterations in promoter methylation with the ICF syndrome immunodeficient phenotype by detecting changes in genes related to the B-cell receptor mediated maturation pathway. PMID:22595875

  14. Whole Genome Sequencing Identifies a Novel Factor Required for Secretory Granule Maturation in Tetrahymena thermophila

    Science.gov (United States)

    Kontur, Cassandra; Kumar, Santosh; Lan, Xun; Pritchard, Jonathan K.; Turkewitz, Aaron P.

    2016-01-01

    Unbiased genetic approaches have a unique ability to identify novel genes associated with specific biological pathways. Thanks to next generation sequencing, forward genetic strategies can be expanded to a wider range of model organisms. The formation of secretory granules, called mucocysts, in the ciliate Tetrahymena thermophila relies, in part, on ancestral lysosomal sorting machinery, but is also likely to involve novel factors. In prior work, multiple strains with defects in mucocyst biogenesis were generated by nitrosoguanidine mutagenesis, and characterized using genetic and cell biological approaches, but the genetic lesions themselves were unknown. Here, we show that analyzing one such mutant by whole genome sequencing reveals a novel factor in mucocyst formation. Strain UC620 has both morphological and biochemical defects in mucocyst maturation—a process analogous to dense core granule maturation in animals. Illumina sequencing of a pool of UC620 F2 clones identified a missense mutation in a novel gene called MMA1 (Mucocyst maturation). The defects in UC620 were rescued by expression of a wild-type copy of MMA1, and disrupting MMA1 in an otherwise wild-type strain phenocopies UC620. The product of MMA1, characterized as a CFP-tagged copy, encodes a large soluble cytosolic protein. A small fraction of Mma1p-CFP is pelletable, which may reflect association with endosomes. The gene has no identifiable homologs except in other Tetrahymena species, and therefore represents an evolutionarily recent innovation that is required for granule maturation. PMID:27317773

  15. Post-Fragmentation Whole Genome Amplification-Based Method

    Science.gov (United States)

    Benardini, James; LaDuc, Myron T.; Langmore, John

    2011-01-01

    This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at

  16. Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Z. He; Q. He; L. Wu; M.E. Clark; J.D. Wall; Jizhong Zhou; Matthew W. Fields

    2004-03-17

    ,4-cyclodiphosphate (MECDP) synthase. Spermidines are polyamines that are typically abundant in rapidly dividing cells and are essential growth factors in eukaryotic organisms. Polyamines are thought to stabilize DNA by the association of the amino groups with the phosphate residues of DNA and can also enhance tRNA and ribosome stability. The MECDP synthase enzyme is essential in Escherichia coli and participates in the nonmevalonate pathway of isoprenoid biosynthesis, a critical pathway present in some bacteria and apicomplexans but distinct from that used by mammals. Several of the highly up-regulated ORFs were annotated as conserved hypothetical proteins. Interestingly, an ORF that was predicted to contain a flocculin repeat domain was almost 9-fold up-regulated in stationary phase cells compared to logarithmically growing cells. The flocculin domain is commonly observed in fungi, and is thought to play a role during flocculation (non-sexual aggregation of single-cell microorganisms). These preliminary results have identified possible responses of D. vulgaris cells to stationary phase growth and suggest that polyamine production as well as cell aggregation and/or extracellular polymer production are responses of D. vulgaris during stationary phase. The initial microarray results indicate that the recently produced oligonucleotide microarrays are functional. We are currently optimizing growth conditions in order to culture D. vulgaris cells in the presence of uranium(VI) and to monitor whole-genome expression levels.

  17. When aging meets microgravity: whole genome promoters and enchancers transcription landscape in zebrafish onboard ISS

    Science.gov (United States)

    Arshanovskii, Kirill; Gusev, Oleg; Sychev, Vladimir; Poddubko, Svetlana; Deviatiiarov, Ruslan

    2016-07-01

    In order to gen new insights of gene regulation changes under conditions of real spaceflight, we have conducted whole-genome analysis of dynamic of promotes and enhancers transcriptional changes in zebrafish during prolonged exposure to real spaceflight. In the frame of Russia-Japan joint experiments "Aquatic Habitat"-"Aquarium" we have conducted Cap Analysis of Gene Expression (CAGE) assay of zebrafish in the rage from 7 to 40 days of real spaceflight onboard ISS. The analysis showed that both gene expression patterns and architecture of shapes and types of the promoters are affected by spaceflight environment.

  18. Bioinformatics for whole-genome shotgun sequencing of microbial communities.

    Directory of Open Access Journals (Sweden)

    Kevin Chen

    2005-07-01

    Full Text Available The application of whole-genome shotgun sequencing to microbial communities represents a major development in metagenomics, the study of uncultured microbes via the tools of modern genomic analysis. In the past year, whole-genome shotgun sequencing projects of prokaryotic communities from an acid mine biofilm, the Sargasso Sea, Minnesota farm soil, three deep-sea whale falls, and deep-sea sediments have been reported, adding to previously published work on viral communities from marine and fecal samples. The interpretation of this new kind of data poses a wide variety of exciting and difficult bioinformatics problems. The aim of this review is to introduce the bioinformatics community to this emerging field by surveying existing techniques and promising new approaches for several of the most interesting of these computational problems.

  19. Whole genome amplification - Review of applications and advances

    Energy Technology Data Exchange (ETDEWEB)

    Hawkins, Trevor L.; Detter, J.C.; Richardson, Paul

    2001-11-15

    The concept of Whole Genome Amplification is something that has arisen in the past few years as modifications to the polymerase chain reaction (PCR) have been adapted to replicate regions of genomes which are of biological interest. The applications here are many--forensics, embryonic disease diagnosis, bio terrorism genome detection, ''imoralization'' of clinical samples, microbial diversity, and genotyping. The key question is if DNA can be replicated a genome at a time without bias or non random distribution of the target. Several papers published in the last year and currently in preparation may lead to the conclusion that whole genome amplification may indeed be possible and therefore open up a new avenue to molecular biology.

  20. Whole genome sequencing in clinical and public health microbiology

    OpenAIRE

    Kwong, J. C.; McCallum, N; Sintchenko, V.; Howden, B. P.

    2015-01-01

    SummaryGenomics and whole genome sequencing (WGS) have the capacity to greatly enhance knowledge and understanding of infectious diseases and clinical microbiology. The growth and availability of bench-top WGS analysers has facilitated the feasibility of genomics in clinical and public health microbiology. Given current resource and infrastructure limitations, WGS is most applicable to use in public health laboratories, reference laboratories, and hospital infection control-affiliated laborat...

  1. Whole Genome Sequencing: Innovation Dream or Privacy Nightmare?

    OpenAIRE

    De Cristofaro, Emiliano

    2012-01-01

    Over the past several years, DNA sequencing has emerged as one of the driving forces in life-sciences, paving the way for affordable and accurate whole genome sequencing. As genomes represent the entirety of an organism's hereditary information, the availability of complete human genomes prompts a wide range of revolutionary applications. The hope for improving modern healthcare and better understanding the human genome propels many interesting and challenging research frontiers. Unfortunatel...

  2. Whole genome amplification of DNA for genotyping pharmacogenetics candidate genes.

    Directory of Open Access Journals (Sweden)

    Santosh ePhilips

    2012-03-01

    Full Text Available Whole genome amplification (WGA technologies can be used to amplify genomic DNA when only small amounts of DNA are available. The Multiple Displacement Amplification Phi polymerase based amplification has been shown to accurately amplify DNA for a variety of genotyping assays; however, it has not been tested for genotyping many of the clinically relevant genes important for pharmacogenetic studies, such as the cytochrome P450 genes, that are typically difficult to genotype due to multiple pseudogenes, copy number variations, and high similarity to other related genes. We evaluated whole genome amplified samples for Taqman™ genotyping of SNPs in a variety of pharmacogenetic genes. In 24 DNA samples from the Coriell human diversity panel, the call rates and concordance between amplified (~200-fold amplification and unamplified samples was 100% for two SNPs in CYP2D6 and one in ESR1. In samples from a breast cancer clinical trial (Trial 1, we compared the genotyping results in samples before and after WGA for four SNPs in CYP2D6, one SNP in CYP2C19, one SNP in CYP19A1, two SNPs in ESR1, and two SNPs in ESR2. The concordance rates were all >97%. Finally, we compared the allele frequencies of 143 SNPs determined in Trial 1 (whole genome amplified DNA to the allele frequencies determined in unamplified DNA samples from a separate trial (Trial 2 that enrolled a similar population. The call rates and allele frequencies between the two trials were 98% and 99.7%, respectively. We conclude that the whole genome amplified DNA is suitable for Taqman™ genotyping for a wide variety of pharmacogenetically relevant SNPs.

  3. Whole genome sequencing of clinical isolates of Giardia lamblia

    OpenAIRE

    Hanevik, Kurt; Bakken, R.; Brattbakk, Hans-Richard; Saghaug, Christina Skår; Langeland, Nina

    2015-01-01

    Clinical isolates from protozoan parasites such as Giardia lamblia are at present practically impossible to culture. By using simple cyst purification methods, we show that Giardia whole genome sequencing of clinical stool samples is possible. Immunomagnetic separation after sucrose gradient flotation gave superior results compared to sucrose gradient flotation alone. The method enables detailed analysis of a wide range of genes of interest for genotyping, virulence and drug resistance.

  4. Physical map-assisted whole-genome shotgun sequence assemblies

    OpenAIRE

    Warren, René L.; Varabei, Dmitry; Platt, Darren; Huang, Xiaoqiu; Messina, David; Yang, Shiaw-Pyng; Kronstad, James W.; Krzywinski, Martin; Warren, Wesley C; Wallis, John W.; Hillier, LaDeana W.; Chinwalla, Asif T.; Schein, Jacqueline E.; Siddiqui, Asim S.; Marra, Marco A.

    2006-01-01

    We describe a targeted approach to improve the contiguity of whole-genome shotgun sequence (WGS) assemblies at run-time, using information from Bacterial Artificial Chromosome (BAC)-based physical maps. Clone sizes and overlaps derived from clone fingerprints are used for the calculation of length constraints between any two BAC neighbors sharing 40% of their size. These constraints are used to promote the linkage and guide the arrangement of sequence contigs within a sequence scaffold at the...

  5. Epigenetic regulation of subgenome dominance following whole genome triplication in Brassica rapa.

    Science.gov (United States)

    Cheng, Feng; Sun, Chao; Wu, Jian; Schnable, James; Woodhouse, Margaret R; Liang, Jianli; Cai, Chengcheng; Freeling, Michael; Wang, Xiaowu

    2016-07-01

    Subgenome dominance is an important phenomenon observed in allopolyploids after whole genome duplication, in which one subgenome retains more genes as well as contributes more to the higher expressing gene copy of paralogous genes. To dissect the mechanism of subgenome dominance, we systematically investigated the relationships of gene expression, transposable element (TE) distribution and small RNA targeting, relating to the multicopy paralogous genes generated from whole genome triplication in Brassica rapa. The subgenome dominance was found to be regulated by a relatively stable factor established previously, then inherited by and shared among B. rapa varieties. In addition, we found a biased distribution of TEs between flanking regions of paralogous genes. Furthermore, the 24-nt small RNAs target TEs and are negatively correlated to the dominant expression of individual paralogous gene pairs. The biased distribution of TEs among subgenomes and the targeting of 24-nt small RNAs together produce the dominant expression phenomenon at a subgenome scale. Based on these findings, we propose a bucket hypothesis to illustrate subgenome dominance and hybrid vigor. Our findings and hypothesis are valuable for the evolutionary study of polyploids, and may shed light on studies of hybrid vigor, which is common to most species. PMID:26871271

  6. Whole genome sequence of Pantoea ananatis R100, an antagonistic bacterium isolated from rice seed.

    Science.gov (United States)

    Wu, Liwen; Liu, Ruifang; Niu, Yaofang; Lin, Haiyan; Ye, Weijun; Guo, Longbiao; Hu, Xingming

    2016-05-10

    Pantoea ananatis is a group of bacteria, which was first reported as plant pathogen. Recently, several papers also described its biocontrol ability. In 2003, P. ananatis R100, which showed strong antagonism against several plant pathogens, was isolated from rice seeds. In this study, whole genome sequence of this strain was determined by SMRT Cell technology. The total genome size of R100 is 4,857,861bp with 4659 coding genes (CDS), 82 tRNAs and 22 rRNAs. The genome sequence of R100 may shed a light on the research of antagonism P. ananatis. PMID:26965742

  7. Genomic prediction using QTL derived from whole genome sequence data

    DEFF Research Database (Denmark)

    Brøndum, Rasmus Froberg; Su, Guosheng; Janss, Luc;

    This study investigated the gain in accuracy of genomic prediction when a small number of significant variants from single marker analysis based on whole genome sequence data were added to the regular 54k SNP data. Analyses were performed for Nordic Holstein and Danish Jersey animals, using either...... a genomic BLUP or a Bayesian variable selection model. When using the genomic BLUP model, results showed increases in accuracy of up to two percentage points for production traits in both Holstein and Jersey animals by including the extra variants in the analysis, and an extra 1.5 percentage points...

  8. Whole-Genome Sequences of Thirteen Isolates of Borrelia burgdorferi

    Energy Technology Data Exchange (ETDEWEB)

    Schutzer S. E.; Dunn J.; Fraser-Liggett, C. M.; Casjens, S. R.; Qiu, W.-G.; Mongodin, E. F.; Luft, B. J.

    2011-02-01

    Borrelia burgdorferi is a causative agent of Lyme disease in North America and Eurasia. The first complete genome sequence of B. burgdorferi strain 31, available for more than a decade, has assisted research on the pathogenesis of Lyme disease. Because a single genome sequence is not sufficient to understand the relationship between genotypic and geographic variation and disease phenotype, we determined the whole-genome sequences of 13 additional B. burgdorferi isolates that span the range of natural variation. These sequences should allow improved understanding of pathogenesis and provide a foundation for novel detection, diagnosis, and prevention strategies.

  9. Integration of transcriptome and whole genomic resequencing data to identify key genes affecting swine fat deposition.

    Directory of Open Access Journals (Sweden)

    Kai Xing

    Full Text Available Fat deposition is highly correlated with the growth, meat quality, reproductive performance and immunity of pigs. Fatty acid synthesis takes place mainly in the adipose tissue of pigs; therefore, in this study, a high-throughput massively parallel sequencing approach was used to generate adipose tissue transcriptomes from two groups of Songliao black pigs that had opposite backfat thickness phenotypes. The total number of paired-end reads produced for each sample was in the range of 39.29-49.36 millions. Approximately 188 genes were differentially expressed in adipose tissue and were enriched for metabolic processes, such as fatty acid biosynthesis, lipid synthesis, metabolism of fatty acids, etinol, caffeine and arachidonic acid and immunity. Additionally, many genetic variations were detected between the two groups through pooled whole-genome resequencing. Integration of transcriptome and whole-genome resequencing data revealed important genomic variations among the differentially expressed genes for fat deposition, for example, the lipogenic genes. Further studies are required to investigate the roles of candidate genes in fat deposition to improve pig breeding programs.

  10. Whole Genome Re-Sequencing of Three Domesticated Chicken Breeds.

    Science.gov (United States)

    Oh, Dongyep; Son, Bongjun; Mun, Seyoung; Oh, Man Hwan; Oh, Sejong; Ha, Jaejung; Yi, Junkoo; Lee, Seunguk; Han, Kyudong

    2016-02-01

    Chicken is one of the most popular domesticated species worldwide, as it can serve an important role in agricultural as well as biomedical research fields. Because it inhabits almost every continent and presents diverse morphology and traits, the need of genetic markers for distinguishing each breed for various purposes has increased. The whole genome sequencing of three different breeds (White Leghorn, Korean domestic, and Araucana) that show similar coloring patterns, with the exception of the White Leghorn breed, have confirmed previously reported genomic alterations and identified many novel variants. Additionally, the Whole Genome Re-Sequencing (WGRS) approach identified an approximately 4 kb insert within SLCO1B3 responsible for blue egg shell color. Targeted investigation of pigment-related genes corroborated previously reported non-synonymous mutations, and provided deeper insight into chicken coloring, where not a single but a combination of non-synonymous mutations in the MC1R gene is likely to be responsible for altered feather coloring. PMID:26853871

  11. Whole-genome validation of high-information-content fingerprinting.

    Science.gov (United States)

    Nelson, William M; Bharti, Arvind K; Butler, Ed; Wei, Fusheng; Fuks, Galina; Kim, Hyeran; Wing, Rod A; Messing, Joachim; Soderlund, Carol

    2005-09-01

    Fluorescent-based high-information-content fingerprinting (HICF) techniques have recently been developed for physical mapping. These techniques make use of automated capillary DNA sequencing instruments to enable both high-resolution and high-throughput fingerprinting. In this article, we report the construction of a whole-genome HICF FPC map for maize (Zea mays subsp. mays cv B73), using a variant of HICF in which a type IIS restriction enzyme is used to generate the fluorescently labeled fragments. The HICF maize map was constructed from the same three maize bacterial artificial chromosome libraries as previously used for the whole-genome agarose FPC map, providing a unique opportunity for direct comparison of the agarose and HICF methods; as a result, it was found that HICF has substantially greater sensitivity in forming contigs. An improved assembly procedure is also described that uses automatic end-merging of contigs to reduce the effects of contamination and repetitive bands. Several new features in FPC v7.2 are presented, including shared-memory multiprocessing, which allows dramatically faster assemblies, and automatic end-merging, which permits more accurate assemblies. It is further shown that sequenced clones may be digested in silico and located accurately on the HICF assembly, despite size deviations that prevent the precise prediction of experimental fingerprints. Finally, repetitive bands are isolated, and their effect on the assembly is studied. PMID:16166258

  12. 基因芯片技术研究附子对虚寒证大鼠肝全基因表达谱的影响%Influenceof Aconiti Lateralis Radix Praeparata on asthenia cold syndrome rats with whole genome gene expression of liver by gene chip technique

    Institute of Scientific and Technical Information of China (English)

    韩冰冰; 王世军; 张发艳; 赵海军; 王成岗

    2012-01-01

    Objective; To study the influence of Aconiti Lateralis Radix Praeparata on asthenia cold syndrome rats with whole genome gene expression of liver by gene chip technique. Method: The asthenia cold syndrome rat models were established by administering traditional Chinese medicine raw Gypsum Fibrosum, Gentianae Radix, Phellodendri Chinensis Cortex and Anemarrhenae Rhizo-ma. After treated with Aconiti Lateralis Radix Praeparata, the rats' liver gene expressions were detected using gene chip. Differential expression genes were screened for gene function annotation, and some genes were selected to check the accuracy of the results by RT-PCR. Result; Compared with the asthenia cold model group, the asthenia cold treatment group showed 212 differential expression genes, mainly involving function of immune response and oxidoreductase activity. Conclusion: Aconiti Lateralis Radix Praeparata is proved to have an effect on up-regulating immune response-related genes and oxidizing oxidoreductase activity-related genes of asthenia cold syndrome rats and may be a molecular mechanism for classical warm-nature medicine Aconiti Lateralis Radix Praeparata in warming meridians and dissipating cold.%目的:采用基因芯片技术研究附子对虚寒证大鼠肝全基因表达谱的影响.方法:使用中药复方生石膏、龙胆草、黄柏、知母建立虚寒证大鼠模型,附子治疗后应用基因芯片检测大鼠肝脏基因表达,筛选差异表达基因,进行基因功能注释,荧光定量PCR验证芯片结果.结果:虚寒治疗组与虚寒模型组比较有212条基因差异表达,主要涉及免疫应答及氧化还原酶活性相关基因.结论:附子能够上调虚寒证大鼠的免疫应答相关基因及氧化还原酶活性相关基因,可能是经典热药附子温阳散寒作用的分子机制.

  13. Lysis of a Single Cyanobacterium for Whole Genome Amplification

    Directory of Open Access Journals (Sweden)

    Richard N. Zare

    2013-08-01

    Full Text Available Bacterial species from natural environments, exhibiting a great degree of genetic diversity that has yet to be characterized, pose a specific challenge to whole genome amplification (WGA from single cells. A major challenge is establishing an effective, compatible, and controlled lysis protocol. We present a novel lysis protocol that can be used to extract genomic information from a single cyanobacterium of Synechocystis sp. PCC 6803 known to have multilayer cell wall structures that resist conventional lysis methods. Simple but effective strategies for releasing genomic DNA from captured cells while retaining cellular identities for single-cell analysis are presented. Successful sequencing of genetic elements from single-cell amplicons prepared by multiple displacement amplification (MDA is demonstrated for selected genes (15 loci nearly equally spaced throughout the main chromosome.

  14. Comparative whole genome sequence analysis of wild-type and cidofovir-resistant monkeypoxvirus

    Directory of Open Access Journals (Sweden)

    Huggins John

    2010-05-01

    Full Text Available Abstract We performed whole genome sequencing of a cidofovir {[(S-1-(3-hydroxy-2-phosphonylmethoxy-propyl cytosine] [HPMPC]}-resistant (CDV-R strain of Monkeypoxvirus (MPV. Whole-genome comparison with the wild-type (WT strain revealed 55 single-nucleotide polymorphisms (SNPs and one tandem-repeat contraction. Over one-third of all identified SNPs were located within genes comprising the poxvirus replication complex, including the DNA polymerase, RNA polymerase, mRNA capping methyltransferase, DNA processivity factor, and poly-A polymerase. Four polymorphic sites were found within the DNA polymerase gene. DNA polymerase mutations observed at positions 314 and 684 in MPV were consistent with CDV-R loci previously identified in Vaccinia virus (VACV. These data suggest the mechanism of CDV resistance may be highly conserved across Orthopoxvirus (OPV species. SNPs were also identified within virulence genes such as the A-type inclusion protein, serine protease inhibitor-like protein SPI-3, Schlafen ATPase and thymidylate kinase, among others. Aberrant chain extension induced by CDV may lead to diverse alterations in gene expression and viral replication that may result in both adaptive and attenuating mutations. Defining the potential contribution of substitutions in the replication complex and RNA processing machinery reported here may yield further insight into CDV resistance and may augment current therapeutic development strategies.

  15. Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing

    DEFF Research Database (Denmark)

    Sydenham, Thomas Vognbjerg; Sóki, József; Hasman, Henrik;

    2015-01-01

    Bacteroides fragilis constitutes the most frequent anaerobic bacterium causing bacteremia in humans. The genetic background for antimicrobial resistance in B. fragilis is diverse with some genes requiring insertion sequence (IS) elements inserted upstream for increased expression. To evaluate whole...... genome shotgun sequencing as a method for predicting antimicrobial resistance properties, one meropenem resistant and five multidrug-resistant blood culture isolates were sequenced and antimicrobial resistance genes and IS elements identified using ResFinder 2.1 (http...

  16. Divergent Whole-Genome Methylation Maps of Human and Chimpanzee Brains Reveal Epigenetic Basis of Human Regulatory Evolution

    OpenAIRE

    Zeng, Jia; Konopka, Genevieve; Hunt, Brendan G.; Preuss, Todd M.; Geschwind, Dan; Yi, Soojin V.

    2012-01-01

    DNA methylation is a pervasive epigenetic DNA modification that strongly affects chromatin regulation and gene expression. To date, it remains largely unknown how patterns of DNA methylation differ between closely related species and whether such differences contribute to species-specific phenotypes. To investigate these questions, we generated nucleotide-resolution whole-genome methylation maps of the prefrontal cortex of multiple humans and chimpanzees. Levels and patterns of DNA methylatio...

  17. Characterization of Genomic Variants Associated with Scout and Recruit Behavioral Castes in Honey Bees Using Whole-Genome Sequencing

    OpenAIRE

    Southey, Bruce R.; Ping Zhu; Carr-Markell, Morgan K.; Liang, Zhengzheng S.; Amro Zayed; Ruiqiang Li; Robinson, Gene E.; Rodriguez-Zas, Sandra L.

    2016-01-01

    Among forager honey bees, scouts seek new resources and return to the colony, enlisting recruits to collect these resources. Differentially expressed genes between these behaviors and genetic variability in scouting phenotypes have been reported. Whole-genome sequencing of 44 Apis mellifera scouts and recruits was undertaken to detect variants and further understand the genetic architecture underlying the behavioral differences between scouts and recruits. The median coverage depth in recruit...

  18. SNP annotation-based whole genomic prediction and selection

    DEFF Research Database (Denmark)

    Do, Duy Ngoc; Janss, Luc; Jensen, Just;

    2015-01-01

    into a training (968 pigs) and a validation dataset (304 pigs) by assigning records as before and after January 1, 2012, respectively. SNP were annotated by 14 different classes using Ensembl variant effect prediction. Predictive accuracy and prediction bias were calculated using Bayesian Power LASSO...... SNP to total genomic variance was similar among annotated classes across different traits. Predictive performance of SNP classes did not significantly differ from randomized SNP groups. Genomic prediction has accuracy comparable to observed phenotype, and use of genomic prediction can be cost...... effective by replacing feed intake measurement. Genomic annotation had less impact on predictive accuracy traits considered here but may be different for other traits. It is the first study to provide useful insights into biological classes of SNP driving the whole genomic prediction for complex traits in...

  19. Whole-genome characterization of chemoresistant ovarian cancer.

    Science.gov (United States)

    Patch, Ann-Marie; Christie, Elizabeth L; Etemadmoghadam, Dariush; Garsed, Dale W; George, Joshy; Fereday, Sian; Nones, Katia; Cowin, Prue; Alsop, Kathryn; Bailey, Peter J; Kassahn, Karin S; Newell, Felicity; Quinn, Michael C J; Kazakoff, Stephen; Quek, Kelly; Wilhelm-Benartzi, Charlotte; Curry, Ed; Leong, Huei San; Hamilton, Anne; Mileshkin, Linda; Au-Yeung, George; Kennedy, Catherine; Hung, Jillian; Chiew, Yoke-Eng; Harnett, Paul; Friedlander, Michael; Quinn, Michael; Pyman, Jan; Cordner, Stephen; O'Brien, Patricia; Leditschke, Jodie; Young, Greg; Strachan, Kate; Waring, Paul; Azar, Walid; Mitchell, Chris; Traficante, Nadia; Hendley, Joy; Thorne, Heather; Shackleton, Mark; Miller, David K; Arnau, Gisela Mir; Tothill, Richard W; Holloway, Timothy P; Semple, Timothy; Harliwong, Ivon; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Idrisoglu, Senel; Bruxner, Timothy J C; Christ, Angelika N; Poudel, Barsha; Holmes, Oliver; Anderson, Matthew; Leonard, Conrad; Lonie, Andrew; Hall, Nathan; Wood, Scott; Taylor, Darrin F; Xu, Qinying; Fink, J Lynn; Waddell, Nick; Drapkin, Ronny; Stronach, Euan; Gabra, Hani; Brown, Robert; Jewell, Andrea; Nagaraj, Shivashankar H; Markham, Emma; Wilson, Peter J; Ellul, Jason; McNally, Orla; Doyle, Maria A; Vedururu, Ravikiran; Stewart, Collin; Lengyel, Ernst; Pearson, John V; Waddell, Nicola; deFazio, Anna; Grimmond, Sean M; Bowtell, David D L

    2015-05-28

    Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1. PMID:26017449

  20. Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing.

    Directory of Open Access Journals (Sweden)

    Adriano C Coelho

    Full Text Available BACKGROUND: Miltefosine (MF is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance. METHODS/PRINCIPAL FINDINGS: Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins-pyridoxal kinase and α-adaptin like protein-were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. CONCLUSIONS/SIGNIFICANCE: Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.

  1. Plantagora: modeling whole genome sequencing and assembly of plant genomes.

    Directory of Open Access Journals (Sweden)

    Roger Barthelson

    Full Text Available BACKGROUND: Genomics studies are being revolutionized by the next generation sequencing technologies, which have made whole genome sequencing much more accessible to the average researcher. Whole genome sequencing with the new technologies is a developing art that, despite the large volumes of data that can be produced, may still fail to provide a clear and thorough map of a genome. The Plantagora project was conceived to address specifically the gap between having the technical tools for genome sequencing and knowing precisely the best way to use them. METHODOLOGY/PRINCIPAL FINDINGS: For Plantagora, a platform was created for generating simulated reads from several different plant genomes of different sizes. The resulting read files mimicked either 454 or Illumina reads, with varying paired end spacing. Thousands of datasets of reads were created, most derived from our primary model genome, rice chromosome one. All reads were assembled with different software assemblers, including Newbler, Abyss, and SOAPdenovo, and the resulting assemblies were evaluated by an extensive battery of metrics chosen for these studies. The metrics included both statistics of the assembly sequences and fidelity-related measures derived by alignment of the assemblies to the original genome source for the reads. The results were presented in a website, which includes a data graphing tool, all created to help the user compare rapidly the feasibility and effectiveness of different sequencing and assembly strategies prior to testing an approach in the lab. Some of our own conclusions regarding the different strategies were also recorded on the website. CONCLUSIONS/SIGNIFICANCE: Plantagora provides a substantial body of information for comparing different approaches to sequencing a plant genome, and some conclusions regarding some of the specific approaches. Plantagora also provides a platform of metrics and tools for studying the process of sequencing and assembly

  2. Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing

    Directory of Open Access Journals (Sweden)

    Plant Ramona N

    2006-08-01

    Full Text Available Abstract Background Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs or large scale (CGH array, FISH methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls. Results All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR. Conclusion Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.

  3. A whole genome screen for HIV restriction factors

    Directory of Open Access Journals (Sweden)

    Liu Li

    2011-11-01

    Full Text Available Abstract Background Upon cellular entry retroviruses must avoid innate restriction factors produced by the host cell. For human immunodeficiency virus (HIV human restriction factors, APOBEC3 (apolipoprotein-B-mRNA-editing-enzyme, p21 and tetherin are well characterised. Results To identify intrinsic resistance factors to HIV-1 replication we screened 19,121 human genes and identified 114 factors with significant inhibition of infection. Those with a known function are involved in a broad spectrum of cellular processes including receptor signalling, vesicle trafficking, transcription, apoptosis, cross-nuclear membrane transport, meiosis, DNA damage repair, ubiquitination and RNA processing. We focused on the PAF1 complex which has been previously implicated in gene transcription, cell cycle control and mRNA surveillance. Knockdown of all members of the PAF1 family of proteins enhanced HIV-1 reverse transcription and integration of provirus. Over-expression of PAF1 in host cells renders them refractory to HIV-1. Simian Immunodeficiency Viruses and HIV-2 are also restricted in PAF1 expressing cells. PAF1 is expressed in primary monocytes, macrophages and T-lymphocytes and we demonstrate strong activity in MonoMac1, a monocyte cell line. Conclusions We propose that the PAF1c establishes an anti-viral state to prevent infection by incoming retroviruses. This previously unrecognised mechanism of restriction could have implications for invasion of cells by any pathogen.

  4. Whole-genome sequence variation, population structure and demographic history of the Dutch population

    NARCIS (Netherlands)

    The Genome of the Netherlands Consortium; Marschall, T.; Schoenhuth, A.

    2014-01-01

    Whole-genome sequencing enables complete characterization of genetic variation, but geographic clustering of rare alleles demands many diverse populations be studied. Here we describe the Genome of the Netherlands (GoNL) Project, in which we sequenced the whole genomes of 250 Dutch parent-offspring

  5. Use of metaphors about exome and whole genome sequencing.

    Science.gov (United States)

    Nelson, Sarah C; Crouch, Julia M; Bamshad, Michael J; Tabor, Holly K; Yu, Joon-Ho

    2016-05-01

    Clinical and research uses of exome and whole genome sequencing (ES/WGS) are growing rapidly. An enhanced understanding of how individuals conceptualize and communicate about sequencing results is needed to ensure effective, mutual exchange of information between care providers and patients and between researchers and participants. Focus groups and interviews participants were recruited to discuss their attitudes and preferences for receiving hypothetical results from ES/WGS. African Americans were intentionally oversampled. We qualitatively analyzed participants' speech to identify unsolicited metaphorical language pertaining to genes and health, and grouped these occurrences into metaphorical concepts. Participants compared genetic information to physical objects including tools, weapons, contents of boxes, and formal documents or reports. These metaphorical concepts centered on several key themes, including locus of control; containment versus release of information; and desirability, usability, interpretability, and ownership of genetic results. Metaphorical language is often used intentionally or unintentionally in discussions about receiving results from ES/WGS in both clinical and research settings. Awareness of the use of metaphorical language and attention to its varied meanings facilitates effective communication about return of ES/WGS results. In turn, both should foster shared and informed decision-making and improve the translation of genetic information by clinicians and researchers. © 2016 Wiley Periodicals, Inc. PMID:26822973

  6. Whole genome shotgun assembly in theory and practice

    Science.gov (United States)

    Chapman, Jarrod Andrew

    The subject of this dissertation is the development of novel analytical and algorithmic approaches to the fragment assembly problem in the context of the Whole Genome Shotgun (WGS) DNA sequencing strategy. A collection of analyses and methods centered on the computational reconstruction of genomic DNA sequence from randomly sampled genome fragments, with particular focus on applications to large, polymorphic, and inhomogeneous datasets are presented. Several novel pre-assembly WGS data analyses are described including assessment of genome size, sequence uniformity, and repetitive element content with particular emphasis on the establishment of standardized quality assurance metrics for large WGS sequencing projects. A theoretical framework for understanding the statistical properties of WGS assemblies in the presence of paired-end sequence data is discussed and the algorithmic sub-problems of quality-based sequence trimming, global pairwise alignment detection, and consensus sequence generation are treated. Finally, as a novel application of these analyses and methods, the results of a collaboration to produce the first WGS sequence reconstruction of a community sample from a natural environment are presented.

  7. Complete Sequence Construction of the Highly Repetitive Ribosomal RNA Gene Repeats in Eukaryotes Using Whole Genome Sequence Data.

    Science.gov (United States)

    Agrawal, Saumya; Ganley, Austen R D

    2016-01-01

    The ribosomal RNA genes (rDNA) encode the major rRNA species of the ribosome, and thus are essential across life. These genes are highly repetitive in most eukaryotes, forming blocks of tandem repeats that form the core of nucleoli. The primary role of the rDNA in encoding rRNA has been long understood, but more recently the rDNA has been implicated in a number of other important biological phenomena, including genome stability, cell cycle, and epigenetic silencing. Noncoding elements, primarily located in the intergenic spacer region, appear to mediate many of these phenomena. Although sequence information is available for the genomes of many organisms, in almost all cases rDNA repeat sequences are lacking, primarily due to problems in assembling these intriguing regions during whole genome assemblies. Here, we present a method to obtain complete rDNA repeat unit sequences from whole genome assemblies. Limitations of next generation sequencing (NGS) data make them unsuitable for assembling complete rDNA unit sequences; therefore, the method we present relies on the use of Sanger whole genome sequence data. Our method makes use of the Arachne assembler, which can assemble highly repetitive regions such as the rDNA in a memory-efficient way. We provide a detailed step-by-step protocol for generating rDNA sequences from whole genome Sanger sequence data using Arachne, for refining complete rDNA unit sequences, and for validating the sequences obtained. In principle, our method will work for any species where the rDNA is organized into tandem repeats. This will help researchers working on species without a complete rDNA sequence, those working on evolutionary aspects of the rDNA, and those interested in conducting phylogenetic footprinting studies with the rDNA. PMID:27576718

  8. Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments

    Energy Technology Data Exchange (ETDEWEB)

    Abulencia, C.B.; Wyborski, D.L.; Garcia, J.; Podar, M.; Chen, W.; Chang, S.H.; Chang, H.W.; Watson, D.; Brodie,E.I.; Hazen, T.C.; Keller, M.

    2005-12-10

    Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using ?29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2 percent genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9 percent of the sequences had significant similarities to known proteins, and ''clusters of orthologous groups'' (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.

  9. Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response

    Directory of Open Access Journals (Sweden)

    Brucklacher Robert M

    2008-06-01

    Full Text Available Abstract Background Despite advances in the understanding of diabetic retinopathy, the nature and time course of molecular changes in the retina with diabetes are incompletely described. This study characterized the functional and molecular phenotype of the retina with increasing durations of diabetes. Results Using the streptozotocin-induced rat model of diabetes, levels of retinal permeability, caspase activity, and gene expression were examined after 1 and 3 months of diabetes. Gene expression changes were identified by whole genome microarray and confirmed by qPCR in the same set of animals as used in the microarray analyses and subsequently validated in independent sets of animals. Increased levels of vascular permeability and caspase-3 activity were observed at 3 months of diabetes, but not 1 month. Significantly more and larger magnitude gene expression changes were observed after 3 months than after 1 month of diabetes. Quantitative PCR validation of selected genes related to inflammation, microvasculature and neuronal function confirmed gene expression changes in multiple independent sets of animals. Conclusion These changes in permeability, apoptosis, and gene expression provide further evidence of progressive retinal malfunction with increasing duration of diabetes. The specific gene expression changes confirmed in multiple sets of animals indicate that pro-inflammatory, anti-vascular barrier, and neurodegenerative changes occur in tandem with functional increases in apoptosis and vascular permeability. These responses are shared with the clinically documented inflammatory response in diabetic retinopathy suggesting that this model may be used to test anti-inflammatory therapeutics.

  10. A whole genome association study on meat palatability in hanwoo.

    Science.gov (United States)

    Hyeong, K-E; Lee, Y-M; Kim, Y-S; Nam, K C; Jo, C; Lee, K-H; Lee, J-E; Kim, J-J

    2014-09-01

    A whole genome association (WGA) study was carried out to find quantitative trait loci (QTL) for sensory evaluation traits in Hanwoo. Carcass samples of 250 Hanwoo steers were collected from National Agricultural Cooperative Livestock Research Institute, Ansung, Gyeonggi province, Korea, between 2011 and 2012 and genotyped with the Affymetrix Bovine Axiom Array 640K single nucleotide polymorphism (SNP) chip. Among the SNPs in the chip, a total of 322,160 SNPs were chosen after quality control tests. After adjusting for the effects of age, slaughter-year-season, and polygenic effects using genome relationship matrix, the corrected phenotypes for the sensory evaluation measurements were regressed on each SNP using a simple linear regression additive based model. A total of 1,631 SNPs were detected for color, aroma, tenderness, juiciness and palatability at 0.1% comparison-wise level. Among the significant SNPs, the best set of 52 SNP markers were chosen using a forward regression procedure at 0.05 level, among which the sets of 8, 14, 11, 10, and 9 SNPs were determined for the respectively sensory evaluation traits. The sets of significant SNPs explained 18% to 31% of phenotypic variance. Three SNPs were pleiotropic, i.e. AX-26703353 and AX-26742891 that were located at 101 and 110 Mb of BTA6, respectively, influencing tenderness, juiciness and palatability, while AX-18624743 at 3 Mb of BTA10 affected tenderness and palatability. Our results suggest that some QTL for sensory measures are segregating in a Hanwoo steer population. Additional WGA studies on fatty acid and nutritional components as well as the sensory panels are in process to characterize genetic architecture of meat quality and palatability in Hanwoo. PMID:25178363

  11. Challenges in Whole-Genome Annotation of Pyrosequenced Eukaryotic Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kuo, Alan; Grigoriev, Igor

    2009-04-17

    Pyrosequencing technologies such as 454/Roche and Solexa/Illumina vastly lower the cost of nucleotide sequencing compared to the traditional Sanger method, and thus promise to greatly expand the number of sequenced eukaryotic genomes. However, the new technologies also bring new challenges such as shorter reads and new kinds and higher rates of sequencing errors, which complicate genome assembly and gene prediction. At JGI we are deploying 454 technology for the sequencing and assembly of ever-larger eukaryotic genomes. Here we describe our first whole-genome annotation of a purely 454-sequenced fungal genome that is larger than a yeast (>30 Mbp). The pezizomycotine (filamentous ascomycote) Aspergillus carbonarius belongs to the Aspergillus section Nigri species complex, members of which are significant as platforms for bioenergy and bioindustrial technology, as members of soil microbial communities and players in the global carbon cycle, and as agricultural toxigens. Application of a modified version of the standard JGI Annotation Pipeline has so far predicted ~;;10k genes. ~;;12percent of these preliminary annotations suffer a potential frameshift error, which is somewhat higher than the ~;;9percent rate in the Sanger-sequenced and conventionally assembled and annotated genome of fellow Aspergillus section Nigri member A. niger. Also,>90percent of A. niger genes have potential homologs in the A. carbonarius preliminary annotation. Weconclude, and with further annotation and comparative analysis expect to confirm, that 454 sequencing strategies provide a promising substrate for annotation of modestly sized eukaryotic genomes. We will also present results of annotation of a number of other pyrosequenced fungal genomes of bioenergy interest.

  12. Whole genome sequencing of field isolates provides robust characterization of genetic diversity in Plasmodium vivax.

    Directory of Open Access Journals (Sweden)

    Ernest R Chan

    Full Text Available BACKGROUND: An estimated 2.85 billion people live at risk of Plasmodium vivax transmission. In endemic countries vivax malaria causes significant morbidity and its mortality is becoming more widely appreciated, drug-resistant strains are increasing in prevalence, and an increasing number of reports indicate that P. vivax is capable of breaking through the Duffy-negative barrier long considered to confer resistance to blood stage infection. Absence of robust in vitro propagation limits our understanding of fundamental aspects of the parasite's biology, including the determinants of its dormant hypnozoite phase, its virulence and drug susceptibility, and the molecular mechanisms underlying red blood cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report results from whole genome sequencing of five P. vivax isolates obtained from Malagasy and Cambodian patients, and of the monkey-adapted Belem strain. We obtained an average 70-400 X coverage of each genome, resulting in more than 93% of the Sal I reference sequence covered by 20 reads or more. Our study identifies more than 80,000 SNPs distributed throughout the genome which will allow designing association studies and population surveys. Analysis of the genome-wide genetic diversity in P. vivax also reveals considerable allele sharing among isolates from different continents. This observation could be consistent with a high level of gene flow among parasite strains distributed throughout the world. CONCLUSIONS: Our study shows that it is feasible to perform whole genome sequencing of P. vivax field isolates and rigorously characterize the genetic diversity of this parasite. The catalogue of polymorphisms generated here will enable large-scale genotyping studies and contribute to a better understanding of P. vivax traits such as drug resistance or erythrocyte invasion, partially circumventing the lack of laboratory culture that has hampered vivax research for years.

  13. Gene expression profile of renal cell carcinoma clear cell type

    Directory of Open Access Journals (Sweden)

    Marcos F. Dall’Oglio

    2010-08-01

    Full Text Available PURPOSE: The determination of prognosis in patients with renal cell carcinoma (RCC is based, classically, on stage and histopathological aspects. The metastatic disease develops in one third of patients after surgery, even in localized tumors. There are few options for treating those patients, and even the new target designed drugs have shown low rates of success in controlling disease progression. Few studies used high throughput genomic analysis in renal cell carcinoma for determination of prognosis. This study is focused on the identification of gene expression signatures in tissues of low-risk, high-risk and metastatic RCC clear cell type (RCC-CCT. MATERIALS AND METHODS: We analyzed the expression of approximately 55,000 distinct transcripts using the Whole Genome microarray platform hybridized with RNA extracted from 19 patients submitted to surgery to treat RCC-CCT with different clinical outcomes. They were divided into three groups (1 low risk, characterized by pT1, Fuhrman grade 1 or 2, no microvascular invasion RCC; (2 high risk, pT2-3, Fuhrman grade 3 or 4 with, necrosis and microvascular invasion present and (3 metastatic RCC-CCT. Normal renal tissue was used as control. RESULTS: After comparison of differentially expressed genes among low-risk, high-risk and metastatic groups, we identified a group of common genes characterizing metastatic disease. Among them Interleukin-8 and Heat shock protein 70 were over-expressed in metastasis and validated by real-time polymerase chain reaction. CONCLUSION: These findings can be used as a starting point to generate molecular markers of RCC-CCT as well as a target for the development of innovative therapies.

  14. 三倍化复制对白菜花粉特异表达候选基因的影响%The Influence of Whole-Genome Triplication (WGT) on the Candidate Genes of Pollen Specific Expression in Brassica rapa

    Institute of Scientific and Technical Information of China (English)

    王晓波; 马原; 程锋; 武剑; 梁建丽; 王晓武

    2015-01-01

    [Objective] The objective of this study is to research the evolution of the candidate genes of pollen specific expression in Brassica rapa after whole-genome triplication (WGT). This study will provide a theoretical basis for pollen specific expressed genes in B. rapa in the future research.[Method]The genes of pollen specific expression in B. rapa were acquired by SynOrths referred to Arabidopsis thaliana based on their syntenic relationship. Interpro Scan was used to get their Gene Ontology(GO), which were grouped into 13 categories related to pollen. To compare among three duplicated categories, the ratio of gene number of each GO to total number of genes in these categories was counted. Furthermore, these genes were grouped into three subgenomes based on their explicit dataset of B. rapa genes. Based on the syntenic relationship of tandem genes of pollen specific expression in B. rapa with genes in A. thaliana,whether these genes were generated before WGT or after the event was determined.[Result] Totally, 1962 candidate genes of pollen specific expression in B. rapa were verified via the syntenic relationship with 1651 genes in A. thaliana. There are 182 tandem genes in A. thaliana, while 137 tandem genes in B. rapa. The result showed that the number of pollen specific expressed genes between A. thaliana and B. rapa was almost equivalent, so it could be inferred that most of copy genes of B. rapa were lost after WGT event occurs. A total of 549 genes of pollen specific expression in A. thaliana were not found syntenic counterparts in B. rapa, so these genes might be also lost after WGT. There are 898 genes of pollen specific expression in A. thaliana corresponding to these genes with single-copy, double-copies or triple-copies in B. rapa. A total of 480 genes in A. thaliana with single-copy in B. rapa, accounting for 53.5%, which is the largest proportion. While 322 genes with double-copies account for about 35.8%, and only 96 genes with triple-copies account

  15. Assessment of Whole-Genome Mapping in a Well-Defined Outbreak of Salmonella enterica Serotype Saintpaul

    OpenAIRE

    Fey, P. D.; Iwen, P C; Zentz, E. B.; Briska, A. M.; Henkhaus, J. K.; Bryant, K.A.; Larson, M. A; Noel, R. K.; Hinrichs, S. H.

    2012-01-01

    We investigated the use of whole-genome mapping and pulsed-field gel electrophoresis (PFGE) with isolates from an outbreak of Salmonella enterica serotype Saintpaul. PFGE and whole-genome mapping were concordant with 22 of 23 isolates. Whole-genome mapping is a viable alternative tool for the epidemiological analysis of Salmonella food-borne disease investigations.

  16. SOX4 expression in bladder carcinoma

    DEFF Research Database (Denmark)

    Aaboe, Mads; Birkenkamp-Demtroder, Karin; Wiuf, Carsten;

    2006-01-01

    The human transcription factor SOX4 was 5-fold up-regulated in bladder tumors compared with normal tissue based on whole-genome expression profiling of 166 clinical bladder tumor samples and 27 normal urothelium samples. Using a SOX4-specific antibody, we found that the cancer cells expressed the...

  17. Maps of cis-Regulatory Nodes in Megabase Long Genome Segments are an Inevitable Intermediate Step Toward Whole Genome Functional Mapping.

    Science.gov (United States)

    Nikolaev, Lev G; Akopov, Sergey B; Chernov, Igor P; Sverdlov, Eugene D

    2007-04-01

    The availability of complete human and other metazoan genome sequences has greatly facilitated positioning and analysis of various genomic functional elements, with initial emphasis on coding sequences. However, complete functional maps of sequenced eukaryotic genomes should include also positions of all non-coding regulatory elements. Unfortunately, experimental data on genomic positions of a multitude of regulatory sequences, such as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited, especially at the whole genome level. Since most genomic regulatory elements (e.g. enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements by computational methods is difficult and often ambiguous. Therefore, the development of high-throughput experimental approaches for identifying and mapping genomic functional elements is highly desirable. At the same time, the creation of whole-genome map of hundreds of thousands of regulatory elements in several hundreds of tissue/cell types is presently far beyond our capabilities. A possible alternative for the whole genome approach is to concentrate efforts on individual genomic segments and then to integrate the data obtained into a whole genome functional map. Moreover, the maps of polygenic fragments with functional cis-regulatory elements would provide valuable data on complex regulatory systems, including their variability and evolution. Here, we reviewed experimental approaches to the realization of these ideas, including our own developments of experimental techniques for selection of cis-acting functionally active DNA fragments from large (megabase-sized) segments of mammalian genomes. PMID:18660850

  18. Maps of cis-Regulatory Nodes in Megabase Long Genome Segments are an Inevitable Intermediate Step Toward Whole Genome Functional Mapping

    Science.gov (United States)

    Nikolaev, Lev G; Akopov, Sergey B; Chernov, Igor P; Sverdlov, Eugene D

    2007-01-01

    The availability of complete human and other metazoan genome sequences has greatly facilitated positioning and analysis of various genomic functional elements, with initial emphasis on coding sequences. However, complete functional maps of sequenced eukaryotic genomes should include also positions of all non-coding regulatory elements. Unfortunately, experimental data on genomic positions of a multitude of regulatory sequences, such as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited, especially at the whole genome level. Since most genomic regulatory elements (e.g. enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements by computational methods is difficult and often ambiguous. Therefore, the development of high-throughput experimental approaches for identifying and mapping genomic functional elements is highly desirable. At the same time, the creation of whole-genome map of hundreds of thousands of regulatory elements in several hundreds of tissue/cell types is presently far beyond our capabilities. A possible alternative for the whole genome approach is to concentrate efforts on individual genomic segments and then to integrate the data obtained into a whole genome functional map. Moreover, the maps of polygenic fragments with functional cis-regulatory elements would provide valuable data on complex regulatory systems, including their variability and evolution. Here, we reviewed experimental approaches to the realization of these ideas, including our own developments of experimental techniques for selection of cis-acting functionally active DNA fragments from large (megabase-sized) segments of mammalian genomes. PMID:18660850

  19. Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data

    NARCIS (Netherlands)

    Bryant, J.M.; Schürch, A.C.; Deutekom, van H.; Harris, S.R.; Beer, de J.L.; Jager, de V.C.L.; Kremer, K.; Hijum, van S.A.F.T.; Siezen, R.J.; Borgdorff, M.; Bentley, S.D.; Parkhill, J.; Soolingen, van D.

    2013-01-01

    BACKGROUND: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of th

  20. Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data

    NARCIS (Netherlands)

    J.M. Bryant (Josephine); A. Schürch (Anita); H. van Deutekom (Henk); S.R. Harris (Simon); J.L. de Beer (Jessica); V. de Jager (Victor); K. Kremer (Kristin); S.A.F.T. van Hijum (Sacha); R.J. Siezen (Roland); M.W. Borgdorff (Martien ); S.D. Bentley (Stephen); J. Parkhill (Julian); D. van Soolingen (Dick)

    2013-01-01

    textabstractBackground: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate kno

  1. Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data.

    NARCIS (Netherlands)

    Bryant, J.M.; Schurch, A.C.; Deutekom, H. van; Harris, S.R.; Beer, J.L. de; Jager, V. de; Kremer, K.; Hijum, S.A.F.T. van; Siezen, R.J.; Borgdorff, M.; Bentley, S.D.; Parkhill, J.; Soolingen, D. van

    2013-01-01

    BACKGROUND: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of th

  2. Whole-Genome Shotgun Sequence of Arthrospira platensis Strain Paraca, a Cultivated and Edible Cyanobacterium

    OpenAIRE

    Lefort, Francois; Calmin, Gautier; Crovadore, Julien; Falquet, Jacques; Hurni, Jean-Pierre; Osteras, Magne; Haldemann, Francois; Farinelli, Laurent

    2014-01-01

    Here we report the whole-genome shotgun sequence of a Peruvian strain of Arthrospira platensis (Paraca), a cultivated and edible haloalkaliphilic cyanobacterium of great scientific, technical, and economic potential.

  3. Whole-Genome Sequencing of Micrococcus luteus Strain Modasa, of Indian Origin

    OpenAIRE

    A. Ghosh; Chaudhary, S. A.; Apurva, S. R.; T. Tiwari; Gupta, S.; A.K. Singh; Katudia, K. H.; Patel, M.P.; Chikara, S. K.

    2013-01-01

    The hydrocarbon-degrading bacterium Micrococcus luteus strain Modasa was isolated from contaminated soil from Modasa, North Gujarat, India. Whole-genome sequencing and analysis provide an insight into the potentially important genes responsible for bioremediation.

  4. New perspectives on microbial community distortion after whole-genome amplification

    Science.gov (United States)

    Whole-genome amplification (WGA) has become an important tool to explore the genomic information of microorganisms in an environmental sample with limited biomass, however potential selective biases during the amplification processes are poorly understood. Here, we describe the e...

  5. Clinical Diagnosis by Whole-Genome Sequencing of a Prenatal Sample

    OpenAIRE

    Talkowski, Michael E.; Ordulu, Zehra; Pillalamarri, Vamsee; Benson, Carol B.; Blumenthal, Ian; Connolly, Susan; Hanscom, Carrie; Hussain, Naveed; Pereira, Shahrin; Picker, Jonathan; Rosenfeld, Jill A.; Shaffer, Lisa G.; Wilkins-Haug, Louise E.; Gusella, James F.; Morton, Cynthia C.

    2012-01-01

    Conventional cytogenetic testing offers low-resolution detection of balanced karyotypic abnormalities but cannot provide the precise, gene-level knowledge required to predict outcomes. The use of high-resolution whole-genome deep sequencing is currently impractical for the purpose of routine clinical care. We show here that whole-genome “jumping libraries” can offer an immediately applicable, nucleotide-level complement to conventional genetic diagnostics within a time frame that allows for c...

  6. Discovery of new Mycoplasma pneumoniae antigens by use of a whole-genome lambda display library.

    Science.gov (United States)

    Beghetto, Elisa; De Paolis, Francesca; Montagnani, Francesca; Cellesi, Carla; Gargano, Nicola

    2009-01-01

    Mycoplasma pneumoniae is the leading cause of atypical pneumonia in children and young adults. Bacterial colonization can occur in both the upper and the lower respiratory tracts and take place both endemically and epidemically worldwide. Characteristically, the infection is chronic in onset and recovery and both humoral and cell-mediated mechanisms are involved in the response to bacterial colonization. To identify bacterial proteins recognized by host antibody responses, a whole-genome M. pneumoniae library was created and displayed on lambda bacteriophage. The challenge of such a library with sera from individuals hospitalized for mycoplasmal pneumonia allowed the identification of a panel of recombinant bacteriophages carrying B-cell epitopes. Among the already known M. pneumoniae B-cell antigens, our results confirmed the immunogenicity of P1 and P30 adhesins. Also, the data presented in this study localized, within their sequences, the immunodominant epitopes recognized by human immunoglobulins. Furthermore, library screening allowed the identification of four novel immunogenic polypeptides, respectively, encoded by fragments of the MPN152, MPN426, MPN456 and MPN-500 open reading frames, highlighting and further confirming the potential of lambda display technology in antigen and epitope discovery. PMID:18992837

  7. Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications

    Directory of Open Access Journals (Sweden)

    Asadollahi Mohammad A

    2010-12-01

    Full Text Available Abstract Background The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. Results In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c. Considering only metabolic genes (782 of 5,596 annotated genes, a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications. Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10 and ergosterol biosynthetic pathway (ERG8, ERG9. Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that

  8. Mining metagenomic whole genome sequences revealed subdominant but constant Lactobacillus population in the human gut microbiota.

    Science.gov (United States)

    Rossi, Maddalena; Martínez-Martínez, Daniel; Amaretti, Alberto; Ulrici, Alessandro; Raimondi, Stefano; Moya, Andrés

    2016-06-01

    The genus Lactobacillus includes over 215 species that colonize plants, foods, sewage and the gastrointestinal tract (GIT) of humans and animals. In the GIT, Lactobacillus population can be made by true inhabitants or by bacteria occasionally ingested with fermented or spoiled foods, or with probiotics. This study longitudinally surveyed Lactobacillus species and strains in the feces of a healthy subject through whole genome sequencing (WGS) data-mining, in order to identify members of the permanent or transient populations. In three time-points (0, 670 and 700 d), 58 different species were identified, 16 of them being retrieved for the first time in human feces. L. rhamnosus, L. ruminis, L. delbrueckii, L. plantarum, L. casei and L. acidophilus were the most represented, with estimated amounts ranging between 6 and 8 Log (cells g(-1) ), while the other were detected at 4 or 5 Log (cells g(-1) ). 86 Lactobacillus strains belonging to 52 species were identified. 43 seemingly occupied the GIT as true residents, since were detected in a time span of almost 2 years in all the three samples or in 2 samples separated by 670 or 700 d. As a whole, a stable community of lactobacilli was disclosed, with wide and understudied biodiversity. PMID:27043715

  9. Whole genome sequencing and complete genetic analysis reveals novel pathways to glycopeptide resistance in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Adriana Renzoni

    Full Text Available The precise mechanisms leading to the emergence of low-level glycopeptide resistance in Staphylococcus aureus are poorly understood. In this study, we used whole genome deep sequencing to detect differences between two isogenic strains: a parental strain and a stable derivative selected stepwise for survival on 4 µg/ml teicoplanin, but which grows at higher drug concentrations (MIC 8 µg/ml. We uncovered only three single nucleotide changes in the selected strain. Nonsense mutations occurred in stp1, encoding a serine/threonine phosphatase, and in yjbH, encoding a post-transcriptional negative regulator of the redox/thiol stress sensor and global transcriptional regulator, Spx. A missense mutation (G45R occurred in the histidine kinase sensor of cell wall stress, VraS. Using genetic methods, all single, pairwise combinations, and a fully reconstructed triple mutant were evaluated for their contribution to low-level glycopeptide resistance. We found a synergistic cooperation between dual phospho-signalling systems and a subtle contribution from YjbH, suggesting the activation of oxidative stress defences via Spx. To our knowledge, this is the first genetic demonstration of multiple sensor and stress pathways contributing simultaneously to glycopeptide resistance development. The multifactorial nature of glycopeptide resistance in this strain suggests a complex reprogramming of cell physiology to survive in the face of drug challenge.

  10. Whole-genome transcriptional analysis of heavy metal stresses inCaulobacter crescentus

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Ping; Brodie, Eoin L.; Suzuki, Yohey; McAdams, Harley H.; Andersen, Gary L.

    2005-09-21

    The bacterium Caulobacter crescentus and related stalkbacterial species are known for their distinctive ability to live in lownutrient environments, a characteristic of most heavy metal contaminatedsites. Caulobacter crescentus is a model organism for studying cell cycleregulation with well developed genetics. We have identified the pathwaysresponding to heavy metal toxicity in C. crescentus to provide insightsfor possible application of Caulobacter to environmental restoration. Weexposed C. crescentus cells to four heavy metals (chromium, cadmium,selenium and uranium) and analyzed genome wide transcriptional activitiespost exposure using a Affymetrix GeneChip microarray. C. crescentusshowed surprisingly high tolerance to uranium, a possible mechanism forwhich may be formation of extracellular calcium-uranium-phosphateprecipitates. The principal response to these metals was protectionagainst oxidative stress (up-regulation of manganese-dependent superoxidedismutase, sodA). Glutathione S-transferase, thioredoxin, glutaredoxinsand DNA repair enzymes responded most strongly to cadmium and chromate.The cadmium and chromium stress response also focused on reducing theintracellular metal concentration, with multiple efflux pumps employed toremove cadmium while a sulfate transporter was down-regulated to reducenon-specific uptake of chromium. Membrane proteins were also up-regulatedin response to most of the metals tested. A two-component signaltransduction system involved in the uranium response was identified.Several differentially regulated transcripts from regions previously notknown to encode proteins were identified, demonstrating the advantage ofevaluating the transcriptome using whole genome microarrays.

  11. The "most wanted" taxa from the human microbiome for whole genome sequencing.

    Directory of Open Access Journals (Sweden)

    Anthony A Fodor

    Full Text Available The goal of the Human Microbiome Project (HMP is to generate a comprehensive catalog of human-associated microorganisms including reference genomes representing the most common species. Toward this goal, the HMP has characterized the microbial communities at 18 body habitats in a cohort of over 200 healthy volunteers using 16S rRNA gene (16S sequencing and has generated nearly 1,000 reference genomes from human-associated microorganisms. To determine how well current reference genome collections capture the diversity observed among the healthy microbiome and to guide isolation and future sequencing of microbiome members, we compared the HMP's 16S data sets to several reference 16S collections to create a 'most wanted' list of taxa for sequencing. Our analysis revealed that the diversity of commonly occurring taxa within the HMP cohort microbiome is relatively modest, few novel taxa are represented by these OTUs and many common taxa among HMP volunteers recur across different populations of healthy humans. Taken together, these results suggest that it should be possible to perform whole-genome sequencing on a large fraction of the human microbiome, including the 'most wanted', and that these sequences should serve to support microbiome studies across multiple cohorts. Also, in stark contrast to other taxa, the 'most wanted' organisms are poorly represented among culture collections suggesting that novel culture- and single-cell-based methods will be required to isolate these organisms for sequencing.

  12. Enterobacter asburiae Strain L1: Complete Genome and Whole Genome Optical Mapping Analysis of a Quorum Sensing Bacterium

    Directory of Open Access Journals (Sweden)

    Yin Yin Lau

    2014-07-01

    Full Text Available Enterobacter asburiae L1 is a quorum sensing bacterium isolated from lettuce leaves. In this study, for the first time, the complete genome of E. asburiae L1 was sequenced using the single molecule real time sequencer (PacBio RSII and the whole genome sequence was verified by using optical genome mapping (OpGen technology. In our previous study, E. asburiae L1 has been reported to produce AHLs, suggesting the possibility of virulence factor regulation which is quorum sensing dependent. This evoked our interest to study the genome of this bacterium and here we present the complete genome of E. asburiae L1, which carries the virulence factor gene virK, the N-acyl homoserine lactone-based QS transcriptional regulator gene luxR and the N-acyl homoserine lactone synthase gene which we firstly named easI. The availability of the whole genome sequence of E. asburiae L1 will pave the way for the study of the QS-mediated gene expression in this bacterium. Hence, the importance and functions of these signaling molecules can be further studied in the hope of elucidating the mechanisms of QS-regulation in E. asburiae. To the best of our knowledge, this is the first documentation of both a complete genome sequence and the establishment of the molecular basis of QS properties of E. asburiae.

  13. Enterobacter asburiae strain L1: complete genome and whole genome optical mapping analysis of a quorum sensing bacterium.

    Science.gov (United States)

    Lau, Yin Yin; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    Enterobacter asburiae L1 is a quorum sensing bacterium isolated from lettuce leaves. In this study, for the first time, the complete genome of E. asburiae L1 was sequenced using the single molecule real time sequencer (PacBio RSII) and the whole genome sequence was verified by using optical genome mapping (OpGen) technology. In our previous study, E. asburiae L1 has been reported to produce AHLs, suggesting the possibility of virulence factor regulation which is quorum sensing dependent. This evoked our interest to study the genome of this bacterium and here we present the complete genome of E. asburiae L1, which carries the virulence factor gene virK, the N-acyl homoserine lactone-based QS transcriptional regulator gene luxR and the N-acyl homoserine lactone synthase gene which we firstly named easI. The availability of the whole genome sequence of E. asburiae L1 will pave the way for the study of the QS-mediated gene expression in this bacterium. Hence, the importance and functions of these signaling molecules can be further studied in the hope of elucidating the mechanisms of QS-regulation in E. asburiae. To the best of our knowledge, this is the first documentation of both a complete genome sequence and the establishment of the molecular basis of QS properties of E. asburiae. PMID:25196111

  14. Integrated analysis of whole genome and transcriptome sequencing reveals diverse transcriptomic aberrations driven by somatic genomic changes in liver cancers.

    Directory of Open Access Journals (Sweden)

    Yuichi Shiraishi

    Full Text Available Recent studies applying high-throughput sequencing technologies have identified several recurrently mutated genes and pathways in multiple cancer genomes. However, transcriptional consequences from these genomic alterations in cancer genome remain unclear. In this study, we performed integrated and comparative analyses of whole genomes and transcriptomes of 22 hepatitis B virus (HBV-related hepatocellular carcinomas (HCCs and their matched controls. Comparison of whole genome sequence (WGS and RNA-Seq revealed much evidence that various types of genomic mutations triggered diverse transcriptional changes. Not only splice-site mutations, but also silent mutations in coding regions, deep intronic mutations and structural changes caused splicing aberrations. HBV integrations generated diverse patterns of virus-human fusion transcripts depending on affected gene, such as TERT, CDK15, FN1 and MLL4. Structural variations could drive over-expression of genes such as WNT ligands, with/without creating gene fusions. Furthermore, by taking account of genomic mutations causing transcriptional aberrations, we could improve the sensitivity of deleterious mutation detection in known cancer driver genes (TP53, AXIN1, ARID2, RPS6KA3, and identified recurrent disruptions in putative cancer driver genes such as HNF4A, CPS1, TSC1 and THRAP3 in HCCs. These findings indicate genomic alterations in cancer genome have diverse transcriptomic effects, and integrated analysis of WGS and RNA-Seq can facilitate the interpretation of a large number of genomic alterations detected in cancer genome.

  15. Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Karen Elizabeth Kempsell

    2015-08-01

    Full Text Available A commercial Bacillus anthracis (Anthrax whole genome protein microarray has been used to identify immunogenic Anthrax proteins using sera from groups of donors with (a confirmed B. anthracis naturally acquired cutaneous infection, (b confirmed B. anthracis intravenous drug use-acquired infection (c occupational exposure in a wool-sorters factory (d humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups.Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However a number of other chromosomally-located and plasmid encoded open reading frames were also recognised by infected or exposed groups in comparison to controls. Some of these antigens e.g. BA4182 are not recognised by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo and are not currently found in the UK licensed Anthrax Vaccine (AVP. These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis ‘infectome’. These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesised, tested in mouse immunogenicity studies and validated in parallel using human sera from the

  16. Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4. 1 and its use for whole-genome shotgun sequence assembly

    Energy Technology Data Exchange (ETDEWEB)

    Shou, S. [Univ. Wisc.-Madison; Kvikstad, E. [Univ. Wisc.-Madison; Kile, A. [Univ. Wisc.-Madison; Severin, J. [Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4. 1 and its use for whole-genome shotgun sequence assembly; Forrest, D. [Univ. Wisc.-Madison; Runnheim, R. [Univ. Wisc.-Madison; Churas, C. [Univ. Wisc.-Madison; Hickman, J. W. [Univ. Wisc.-Madison; Mackenzie, C. [University of Texas–Houston Medical School; Choudhary, M. [University of Texas–Houston Medical School; Donohue, T. [Univ. Wisc.-Madison; Kaplan, S. [University of Texas–Houston Medical School; Schwartz, D. C. [Univ. Wisc.-Madison

    2003-09-01

    Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.

  17. Whole-Genome de novo Sequencing Of Quail And Grey Partridge

    DEFF Research Database (Denmark)

    Holm, Lars-Erik; Panitz, Frank; Burt, Dave;

    2011-01-01

    The development in sequencing methods has made it possible to perform whole genome de novo sequencing of species without large commercial interests. Within the EU-financed QUANTOMICS project (KBBE-2A-222664), we have performed de novo sequencing of quail (Coturnix coturnix) and grey partridge...... (Perdix perdix) on a Genome Analyzer GAII (Illumina) using paired-end sequencing. The amount of generated sequences amounts to 8 to 9 Gb for each species. The analysis and assembly of the generated sequences is ongoing. Access to the whole genome sequence from these two species will enable enhanced...... comparative studies towards the chicken genome and will aid in identifying evolutionarily conserved sequences within the Galliformes. The obtained sequences from quail and partridge represent a beginning of generating the whole genome sequence for these species. The continuation of establishing the genome...

  18. Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method.

    Science.gov (United States)

    Nakamura, Kosuke; Kondo, Kazunari; Akiyama, Hiroshi; Ishigaki, Takumi; Noguchi, Akio; Katsumata, Hiroshi; Takasaki, Kazuto; Futo, Satoshi; Sakata, Kozue; Fukuda, Nozomi; Mano, Junichi; Kitta, Kazumi; Tanaka, Hidenori; Akashi, Ryo; Nishimaki-Mogami, Tomoko

    2016-08-15

    Identification of transgenic sequences in an unknown genetically modified (GM) papaya (Carica papaya L.) by whole genome sequence analysis was demonstrated. Whole genome sequence data were generated for a GM-positive fresh papaya fruit commodity detected in monitoring using real-time polymerase chain reaction (PCR). The sequences obtained were mapped against an open database for papaya genome sequence. Transgenic construct- and event-specific sequences were identified as a GM papaya developed to resist infection from a Papaya ringspot virus. Based on the transgenic sequences, a specific real-time PCR detection method for GM papaya applicable to various food commodities was developed. Whole genome sequence analysis enabled identifying unknown transgenic construct- and event-specific sequences in GM papaya and development of a reliable method for detecting them in papaya food commodities. PMID:27006240

  19. Whole-Genome Saliva and Blood DNA Methylation Profiling in Individuals with a Respiratory Allergy

    Science.gov (United States)

    Declerck, Ken; Traen, Sophie; Koppen, Gudrun; Van Camp, Guy; Schoeters, Greet; Vanden Berghe, Wim; De Boever, Patrick

    2016-01-01

    The etiology of respiratory allergies (RA) can be partly explained by DNA methylation changes caused by adverse environmental and lifestyle factors experienced early in life. Longitudinal, prospective studies can aid in the unravelment of the epigenetic mechanisms involved in the disease development. High compliance rates can be expected in these studies when data is collected using non-invasive and convenient procedures. Saliva is an attractive biofluid to analyze changes in DNA methylation patterns. We investigated in a pilot study the differential methylation in saliva of RA (n = 5) compared to healthy controls (n = 5) using the Illumina Methylation 450K BeadChip platform. We evaluated the results against the results obtained in mononuclear blood cells from the same individuals. Differences in methylation patterns from saliva and mononuclear blood cells were clearly distinguishable (PAdj0.2), though the methylation status of about 96% of the cg-sites was comparable between peripheral blood mononuclear cells and saliva. When comparing RA cases with healthy controls, the number of differentially methylated sites (DMS) in saliva and blood were 485 and 437 (P0.1), respectively, of which 216 were in common. The methylation levels of these sites were significantly correlated between blood and saliva. The absolute levels of methylation in blood and saliva were confirmed for 3 selected DMS in the PM20D1, STK32C, and FGFR2 genes using pyrosequencing analysis. The differential methylation could only be confirmed for DMS in PM20D1 and STK32C genes in saliva. We show that saliva can be used for genome-wide methylation analysis and that it is possible to identify DMS when comparing RA cases and healthy controls. The results were replicated in blood cells of the same individuals and confirmed by pyrosequencing analysis. This study provides proof-of-concept for the applicability of saliva-based whole-genome methylation analysis in the field of respiratory allergy. PMID

  20. An efficient procedure for plant organellar genome assembly, based on whole genome data from the 454 GS FLX sequencing platform

    Directory of Open Access Journals (Sweden)

    Zhang Tongwu

    2011-11-01

    Full Text Available Abstract Motivation Complete organellar genome sequences (chloroplasts and mitochondria provide valuable resources and information for studying plant molecular ecology and evolution. As high-throughput sequencing technology advances, it becomes the norm that a shotgun approach is used to obtain complete genome sequences. Therefore, to assemble organellar sequences from the whole genome, shotgun reads are inevitable. However, associated techniques are often cumbersome, time-consuming, and difficult, because true organellar DNA is difficult to separate efficiently from nuclear copies, which have been transferred to the nucleus through the course of evolution. Results We report a new, rapid procedure for plant chloroplast and mitochondrial genome sequencing and assembly using the Roche/454 GS FLX platform. Plant cells can contain multiple copies of the organellar genomes, and there is a significant correlation between the depth of sequence reads in contigs and the number of copies of the genome. Without isolating organellar DNA from the mixture of nuclear and organellar DNA for sequencing, we retrospectively extracted assembled contigs of either chloroplast or mitochondrial sequences from the whole genome shotgun data. Moreover, the contig connection graph property of Newbler (a platform-specific sequence assembler ensures an efficient final assembly. Using this procedure, we assembled both chloroplast and mitochondrial genomes of a resurrection plant, Boea hygrometrica, with high fidelity. We also present information and a minimal sequence dataset as a reference for the assembly of other plant organellar genomes.

  1. Postzygotic single-nucleotide mosaicisms in whole-genome sequences of clinically unremarkable individuals

    OpenAIRE

    Huang, August Y.; Xu, Xiaojing; Ye, Adam Y.; Wu, Qixi; Yan, Linlin; Zhao, Boxun; Yang, Xiaoxu; He, Yao; Wang, Sheng; Zhang, Zheng; Gu, Bowen; Han-qing ZHAO; Wang, Meng; Gao, Hua; Gao, Ge

    2014-01-01

    Postzygotic single-nucleotide mutations (pSNMs) have been studied in cancer and a few other overgrowth human disorders at whole-genome scale and found to play critical roles. However, in clinically unremarkable individuals, pSNMs have never been identified at whole-genome scale largely due to technical difficulties and lack of matched control tissue samples, and thus the genome-wide characteristics of pSNMs remain unknown. We developed a new Bayesian-based mosaic genotyper and a series of eff...

  2. Whole-Genome Sequences of Two Borrelia afzelii and Two Borrelia garinii Lyme Disease Agent Isolates

    Energy Technology Data Exchange (ETDEWEB)

    Casjens, S.R.; Dunn, J.; Mongodin, E. F.; Qiu, W.-G.; Luft, B. J.; Fraser-Liggett, C. M.; Schutzer, S. E.

    2011-12-01

    Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B. burgdorferi, and B. bavariensis sp. nov. Whole-genome sequencing is an excellent tool for investigating and understanding the influence of bacterial diversity on the pathogenesis and etiology of Lyme disease. We report here the whole-genome sequences of four isolates from two of the Borrelia species that cause human Lyme disease, B. afzelii isolates ACA-1 and PKo and B. garinii isolates PBr and Far04.

  3. Identification of innate immunodeficiencies by whole genome sequencing

    DEFF Research Database (Denmark)

    Mogensen, Trine; Christiansen, Mette; Veirum, Jens Erik;

    2014-01-01

    , TBK1 and Unc93B) may contribute to the development of herpes encephalitis. Common to these genetic defects is that they lead to reduced antiviral interferon (IFN) responses. In this study whole exome sequencing (WES) was performed to identify mutations associated with susceptibility to herpes....... Exome sequencing coverage was on average 100. Any identified mutations were confirmed with Sanger sequencing. Next in vitro studies to evaluate the functional consequences of identified mutations will be done. We plan to express the mutated and wild type versions of these molecules by stable...

  4. Whole genome transcript profiling from fingerstick blood samples: a comparison and feasibility study

    Directory of Open Access Journals (Sweden)

    Williams Adam R

    2009-12-01

    Full Text Available Abstract Background Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5 mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small human samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 μl of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit. Results From two sets of fingerstick collections, about 70 uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6 ng total RNA with an average RIN of 9.3. The post-amplification yields for 50 ng of total RNA averaged at 7.0 ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with r2 values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r2 values ranging from 0.88 to 0

  5. Whole-Genome Sequencing Detection of Ongoing Listeria Contamination at a Restaurant, Rhode Island, USA, 2014

    Science.gov (United States)

    Gosciminski, Michael; Miller, Adam

    2016-01-01

    In November 2014, the Rhode Island Department of Health investigated a cluster of 3 listeriosis cases. Using whole-genome sequencing to support epidemiologic, laboratory, and environmental investigations, the department identified 1 restaurant as the likely source of the outbreak and also linked the establishment to a listeriosis case that occurred in 2013. PMID:27434089

  6. Whole-genome sequence of “Candidatus Liberibacter solanacearum” strain R1 from California

    Science.gov (United States)

    The draft whole-genome sequence of “Candidatus Liberibacter solanacearum” strain R1, isolated from a tomato plant in California, United States, is reported. The R1 strain genome is 1,204,257 bp in size (G+C content of 35.3%), encoding 1,101 open reading frames and 57 RNA genes....

  7. Whole Genome Selection Project Involving 2,000 Industry AI Sires

    Science.gov (United States)

    Whole genome selection (WGS) uses markers spanning the genome to predict genetic merit for economically important traits. WGS may increase the rate of genetic progress through improved accuracy and reduced generation interval especially for traits that cannot be measured on breeding animals. In cont...

  8. Draft Whole-Genome Sequence of the Type Strain Bacillus horikoshii DSM 8719

    Science.gov (United States)

    Hernández-González, Ismael L.

    2016-01-01

    Members of the Bacillus genus have been extensively studied because of their ability to produce enzymes with high biotechnological value. Here, we report the draft of the whole-genome sequence of the type strain Bacillus horikoshii DSM 8719, an alkali-tolerant strain. PMID:27417833

  9. Whole-Genome Sequencing Detection of Ongoing Listeria Contamination at a Restaurant, Rhode Island, USA, 2014.

    Science.gov (United States)

    Barkley, Jonathan S; Gosciminski, Michael; Miller, Adam

    2016-08-01

    In November 2014, the Rhode Island Department of Health investigated a cluster of 3 listeriosis cases. Using whole-genome sequencing to support epidemiologic, laboratory, and environmental investigations, the department identified 1 restaurant as the likely source of the outbreak and also linked the establishment to a listeriosis case that occurred in 2013. PMID:27434089

  10. Whole genome analysis of Klebsiella pneumoniae T2-1-1 from human oral cavity

    Directory of Open Access Journals (Sweden)

    Kok-Gan Chan

    2016-03-01

    Full Text Available Klebsiella pneumoniae T2-1-1 was isolated from the human tongue debris and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession JAQL00000000.

  11. Whole-Genome Shotgun Sequencing of Lactobacillus rhamnosus MTCC 5462, a Strain with Probiotic Potential

    OpenAIRE

    Prajapati, J. B.; Khedkar, C. D.; Chitra, J.; Suja, Senan; V. Mishra; Sreeja, V.; Patel, R. K.; Ahir, V. B.; Bhatt, V. D.; Sajnani, M. R.; Jakhesara, S. J.; Koringa, P. G.; Joshi, C. G.

    2012-01-01

    Lactobacillus rhamnosus MTCC 5462 was isolated from infant gastrointestinal flora. The strain exhibited an ability to reduce cholesterol and stimulate immunity. The strain has exhibited positive results in alleviating gastrointestinal discomfort and good potential as a probiotic. We sequenced the whole genome of the strain and compared it to the published genome sequence of Lactobacillus rhamnosus GG (ATCC 53103).

  12. Capsular Typing Method for Streptococcus agalactiae Using Whole-Genome Sequence Data

    OpenAIRE

    Sheppard, AE; Vaughan, A; Jones, N.; Turner, P; Turner, C.; Efstratiou, A.; Patel, D.; MMM Informatics Group; Walker, AS; Berkley, J.; Crook, DW; Seale, AC

    2016-01-01

    Group B streptococcus (GBS) capsular serotype is a major determ inant of virulence, and affects potential vaccine coverage. Here we report a whole genome sequencing-based method for GBS serotype assignment. This shows high agree ment (kappa=0.92) with conventional methods, and increased serotype assignment (100%) to all ten capsular types.

  13. Capsular Typing Method for Streptococcus agalactiae Using Whole-Genome Sequence Data.

    Science.gov (United States)

    Sheppard, Anna E; Vaughan, Alison; Jones, Nicola; Turner, Paul; Turner, Claudia; Efstratiou, Androulla; Patel, Darshana; Walker, A Sarah; Berkley, James A; Crook, Derrick W; Seale, Anna C

    2016-05-01

    Group B streptococcus (GBS) capsular serotypes are major determinants of virulence and affect potential vaccine coverage. Here we report a whole-genome-sequencing-based method for GBS serotype assignment. This method shows strong agreement (kappa of 0.92) with conventional methods and increased serotype assignment (100%) to all 10 capsular types. PMID:26962081

  14. Whole genome analysis of Klebsiella pneumoniae T2-1-1 from human oral cavity

    OpenAIRE

    Kok-Gan Chan; Wai-Fong Yin; Xin-Yue Chan

    2015-01-01

    Klebsiella pneumoniae T2-1-1 was isolated from the human tongue debris and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession JAQL00000000.

  15. Whole genome analysis of Klebsiella pneumoniae T2-1-1 from human oral cavity.

    Science.gov (United States)

    Chan, Kok-Gan; Yin, Wai-Fong; Chan, Xin-Yue

    2016-03-01

    Klebsiella pneumoniae T2-1-1 was isolated from the human tongue debris and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession JAQL00000000. PMID:26981378

  16. Whole-Genome Shotgun Sequence of Pseudomonas viridiflava, a Bacterium Species Pathogenic to Arabidopsis thaliana

    OpenAIRE

    Lefort, Francois; Calmin, Gautier; Crovadore, Julien; Osteras, Magne; Farinelli, Laurent

    2013-01-01

    We report here the first whole-genome shotgun sequence of Pseudomonas viridiflava strain UASWS38, a bacterium species pathogenic to the biological model plant Arabidopsis thaliana but also usable as a biological control agent and thus of great scientific interest for understanding the genetics of plant-microbe interactions.

  17. Whole-Genome Shotgun Sequence of Rhodococcus Species Strain JVH1

    OpenAIRE

    Brooks, Shannon L.; Van Hamme, Jonathan D.

    2012-01-01

    Here we present a whole-genome shotgun sequence of Rhodococcus species strain JVH1, an organism capable of degrading a variety of organosulfur compounds. In particular, JVH1 is able to selectively cleave carbon-sulfur bonds within alkyl chains. A large number of oxygenases were identified, consistent with other members of the genus.

  18. WIDE-CROSS WHOLE-GENOME RADIATION HYBIRD MAPPING OF THE COTTON (GOSSYPIUM BARBADENSE L.) GENOME

    Science.gov (United States)

    Whole-genome radiation hybrid mapping has been applied extensively to human and certain animal species but little to plants. We recently demonstrated an alternative mapping approach in cotton (Gossypium hirsutum L.) based on segmentation by 5-krad gamma-irradiation and derivation of wild-cross whol...

  19. A whole-genome assembly of the domestic cow, Bos taurus

    Science.gov (United States)

    Background: The genome of the domestic cow, Bos taurus, was sequenced using a mixture of hierarchical and whole-genome shotgun sequencing methods. Results: We have assembled the 35 million sequence reads and applied a variety of assembly improvement techniques, creating an assembly of 2.86 billion b...

  20. Whole genome analysis of Klebsiella pneumoniae T2-1-1 from human oral cavity

    OpenAIRE

    Kok-Gan Chan; Wai-Fong Yin; Xin-Yue Chan

    2016-01-01

    Klebsiella pneumoniae T2-1-1 was isolated from the human tongue debris and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession JAQL00000000.

  1. Whole-genome analyses resolve early branches in the tree of life of modern birds

    DEFF Research Database (Denmark)

    Sicheritz-Pontén, Thomas; Li, Cai; Li, Bo;

    2014-01-01

    and confirm independent gains of vocal learning. Among Columbea, we identify pigeons and flamingoes as belonging to sister clades. Even with whole genomes, some of the earliest branches in Neoaves proved challenging to resolve, which was best explained by massive protein-coding sequence convergence...

  2. Whole-Genome Sequence of Aeromonas hydrophila Strain AH-1 (Serotype O11).

    Science.gov (United States)

    Forn-Cuní, Gabriel; Tomás, Juan M; Merino, Susana

    2016-01-01

    Aeromonas hydrophila is an emerging pathogen of aquatic and terrestrial animals, including humans. Here, we report the whole-genome sequence of the septicemic A. hydrophila AH-1 strain, belonging to the serotype O11, and the first mesophilic Aeromonas with surface layer (S-layer) to be sequenced. PMID:27587829

  3. Whole-Genome Shotgun Sequencing of a Colonizing Multilocus Sequence Type 17 Streptococcus agalactiae Strain

    Science.gov (United States)

    Singh, Pallavi; Springman, A. Cody; Davies, H. Dele

    2012-01-01

    This report highlights the whole-genome shotgun draft sequence for a Streptococcus agalactiae strain representing multilocus sequence type (ST) 17, isolated from a colonized woman at 8 weeks postpartum. This sequence represents an important addition to the published genomes and will promote comparative genomic studies of S. agalactiae recovered from diverse sources. PMID:23045509

  4. Whole-genome sequencing of giant pandas provides insights into demographic history and local adaptation

    DEFF Research Database (Denmark)

    Zhao, Shancen; Zheng, Pingping; Dong, Shanshan;

    2013-01-01

    dynamics remains largely unknown. We sequenced the whole genomes of 34 pandas at an average 4.7-fold coverage and used this data set together with the previously deep-sequenced panda genome to reconstruct a continuous demographic history of pandas from their origin to the present. We identify two...

  5. Whole-Genome Scans Provide Evidence of Adaptive Evolution in Malawian Plasmodium falciparum Isolates

    DEFF Research Database (Denmark)

    Ocholla, Harold; Preston, Mark D; Mipando, Mwapatsa;

    2014-01-01

    BACKGROUND:  Selection by host immunity and antimalarial drugs has driven extensive adaptive evolution in Plasmodium falciparum and continues to produce ever-changing landscapes of genetic variation. METHODS:  We performed whole-genome sequencing of 69 P. falciparum isolates from Malawi and used ...

  6. Whole-Genome Shotgun Sequencing of a Colonizing Multilocus Sequence Type 17 Streptococcus agalactiae Strain

    OpenAIRE

    Singh, Pallavi; Springman, A. Cody; Davies, H Dele; Manning, Shannon D.

    2012-01-01

    This report highlights the whole-genome shotgun draft sequence for a Streptococcus agalactiae strain representing multilocus sequence type (ST) 17, isolated from a colonized woman at 8 weeks postpartum. This sequence represents an important addition to the published genomes and will promote comparative genomic studies of S. agalactiae recovered from diverse sources.

  7. Generation of physical map contig-specific sequences useful for whole genome sequence scaffolding.

    Directory of Open Access Journals (Sweden)

    Yanliang Jiang

    Full Text Available Along with the rapid advances of the nextgen sequencing technologies, more and more species are added to the list of organisms whose whole genomes are sequenced. However, the assembled draft genome of many organisms consists of numerous small contigs, due to the short length of the reads generated by nextgen sequencing platforms. In order to improve the assembly and bring the genome contigs together, more genome resources are needed. In this study, we developed a strategy to generate a valuable genome resource, physical map contig-specific sequences, which are randomly distributed genome sequences in each physical contig. Two-dimensional tagging method was used to create specific tags for 1,824 physical contigs, in which the cost was dramatically reduced. A total of 94,111,841 100-bp reads and 315,277 assembled contigs are identified containing physical map contig-specific tags. The physical map contig-specific sequences along with the currently available BAC end sequences were then used to anchor the catfish draft genome contigs. A total of 156,457 genome contigs (~79% of whole genome sequencing assembly were anchored and grouped into 1,824 pools, in which 16,680 unique genes were annotated. The physical map contig-specific sequences are valuable resources to link physical map, genetic linkage map and draft whole genome sequences, consequently have the capability to improve the whole genome sequences assembly and scaffolding, and improve the genome-wide comparative analysis as well. The strategy developed in this study could also be adopted in other species whose whole genome assembly is still facing a challenge.

  8. Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates.

    Science.gov (United States)

    Brunet, Frédéric G; Volff, Jean-Nicolas; Schartl, Manfred

    2016-01-01

    The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75% after 1R/2R, 64.4% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling. PMID:27260203

  9. Increased frequency of single base substitutions in a population of transcripts expressed in cancer cells

    International Nuclear Information System (INIS)

    Single Base Substitutions (SBS) that alter transcripts expressed in cancer originate from somatic mutations. However, recent studies report SBS in transcripts that are not supported by the genomic DNA of tumor cells. We used sequence based whole genome expression profiling, namely Long-SAGE (L-SAGE) and Tag-seq (a combination of L-SAGE and deep sequencing), and computational methods to identify transcripts with greater SBS frequencies in cancer. Millions of tags produced by 40 healthy and 47 cancer L-SAGE experiments were compared to 1,959 Reference Tags (RT), i.e. tags matching the human genome exactly once. Similarly, tens of millions of tags produced by 7 healthy and 8 cancer Tag-seq experiments were compared to 8,572 RT. For each transcript, SBS frequencies in healthy and cancer cells were statistically tested for equality. In the L-SAGE and Tag-seq experiments, 372 and 4,289 transcripts respectively, showed greater SBS frequencies in cancer. Increased SBS frequencies could not be attributed to known Single Nucleotide Polymorphisms (SNP), catalogued somatic mutations or RNA-editing enzymes. Hypothesizing that Single Tags (ST), i.e. tags sequenced only once, were indicators of SBS, we observed that ST proportions were heterogeneously distributed across Embryonic Stem Cells (ESC), healthy differentiated and cancer cells. ESC had the lowest ST proportions, whereas cancer cells had the greatest. Finally, in a series of experiments carried out on a single patient at 1 healthy and 3 consecutive tumor stages, we could show that SBS frequencies increased during cancer progression. If the mechanisms generating the base substitutions could be known, increased SBS frequency in transcripts would be a new useful biomarker of cancer. With the reduction of sequencing cost, sequence based whole genome expression profiling could be used to characterize increased SBS frequency in patient’s tumor and aid diagnostic

  10. Increased frequency of single base substitutions in a population of transcripts expressed in cancer cells

    Directory of Open Access Journals (Sweden)

    Bianchetti Laurent

    2012-11-01

    Full Text Available Abstract Background Single Base Substitutions (SBS that alter transcripts expressed in cancer originate from somatic mutations. However, recent studies report SBS in transcripts that are not supported by the genomic DNA of tumor cells. Methods We used sequence based whole genome expression profiling, namely Long-SAGE (L-SAGE and Tag-seq (a combination of L-SAGE and deep sequencing, and computational methods to identify transcripts with greater SBS frequencies in cancer. Millions of tags produced by 40 healthy and 47 cancer L-SAGE experiments were compared to 1,959 Reference Tags (RT, i.e. tags matching the human genome exactly once. Similarly, tens of millions of tags produced by 7 healthy and 8 cancer Tag-seq experiments were compared to 8,572 RT. For each transcript, SBS frequencies in healthy and cancer cells were statistically tested for equality. Results In the L-SAGE and Tag-seq experiments, 372 and 4,289 transcripts respectively, showed greater SBS frequencies in cancer. Increased SBS frequencies could not be attributed to known Single Nucleotide Polymorphisms (SNP, catalogued somatic mutations or RNA-editing enzymes. Hypothesizing that Single Tags (ST, i.e. tags sequenced only once, were indicators of SBS, we observed that ST proportions were heterogeneously distributed across Embryonic Stem Cells (ESC, healthy differentiated and cancer cells. ESC had the lowest ST proportions, whereas cancer cells had the greatest. Finally, in a series of experiments carried out on a single patient at 1 healthy and 3 consecutive tumor stages, we could show that SBS frequencies increased during cancer progression. Conclusion If the mechanisms generating the base substitutions could be known, increased SBS frequency in transcripts would be a new useful biomarker of cancer. With the reduction of sequencing cost, sequence based whole genome expression profiling could be used to characterize increased SBS frequency in patient’s tumor and aid diagnostic.

  11. Whole genome sequencing of mutation accumulation lines reveals a low mutation rate in the social amoeba Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Gerda Saxer

    Full Text Available Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10(-9, with a Poisson confidence interval of 4.1×10(-9 - 9.5×10(-9, per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10(-11, with a Poisson confidence interval ranging from 7.4×10(-13 to 1.6×10(-10, is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.

  12. Downsizing genomic medicine: approaching the ethical complexity of whole-genome sequencing by starting small.

    Science.gov (United States)

    Sharp, Richard R

    2011-03-01

    As we look to a time when whole-genome sequencing is integrated into patient care, it is possible to anticipate a number of ethical challenges that will need to be addressed. The most intractable of these concern informed consent and the responsible management of very large amounts of genetic information. Given the range of possible findings, it remains unclear to what extent it will be possible to obtain meaningful patient consent to genomic testing. Equally unclear is how clinicians will disseminate the enormous volume of genetic information produced by whole-genome sequencing. Toward developing practical strategies for managing these ethical challenges, we propose a research agenda that approaches multiplexed forms of clinical genetic testing as natural laboratories in which to develop best practices for managing the ethical complexities of genomic medicine. PMID:21311340

  13. Genomic Prediction from Whole Genome Sequence in Livestock: The 1000 Bull Genomes Project

    DEFF Research Database (Denmark)

    Hayes, Benjamin J; MacLeod, Iona M; Daetwyler, Hans D;

    Advantages of using whole genome sequence data to predict genomic estimated breeding values (GEBV) include better persistence of accuracy of GEBV across generations and more accurate GEBV across breeds. The 1000 Bull Genomes Project provides a database of whole genome sequenced key ancestor bulls......, for imputing sequence variant genotypes into reference sets for genomic prediction. Run 3.0 included 429 sequences, with 31.8 million variants detected. BayesRC, a new method for genomic prediction, addresses some challenges associated with using the sequence data, and takes advantage of biological...... information. In a dairy data set, predictions using BayesRC and imputed sequence data from 1000 Bull Genomes were 2% more accurate than with 800k data. We could demonstrate the method identified causal mutations in some cases. Further improvements will come from more accurate imputation of sequence variant...

  14. Whole genome sequencing of Mycobacterium tuberculosis SB24 isolated from Sabah, Malaysia.

    Science.gov (United States)

    Philip, Noraini; Rodrigues, Kenneth Francis; William, Timothy; John, Daisy Vanitha

    2016-09-01

    Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB) that causes millions of death every year. We have sequenced the genome of M. tuberculosis isolated from cerebrospinal fluid (CSF) of a patient diagnosed with tuberculous meningitis (TBM). The isolated strain was referred as M. tuberculosis SB24. Genomic DNA of the M. tuberculosis SB24 was extracted and subjected to whole genome sequencing using PacBio platform. The draft genome size of M. tuberculosis SB24 was determined to be 4,452,489 bp with a G + C content of 65.6%. The whole genome shotgun project has been deposited in NCBI SRA under the accession number SRP076503. PMID:27556011

  15. Applications of the double-barreled data in whole-genome shotgun sequence assembly and analysis

    Institute of Scientific and Technical Information of China (English)

    HAN Yujun; WANG Jing; GU Xiaocheng; YU Jun; LI Songgang; NI Peixiang; L(U) Hong; YE Jia; HU Jianfei; CHEN Chen; HUANG Xiangang; CONG Lijuan; LI Guangyuan

    2005-01-01

    Double-barreled (DB) data have been widely used for the assembly of large genomes. Based on the experience of building the whole-genome working draft of Oryza sativa L.ssp. Indica, we present here the prevailing and improved uses of DB data in the assembly procedure and report on novel applications during the following data-mining processes such as acquiring precise insert fragment information of each clone across the genome, and a new kind of Iow-cost whole-genome microarray. With the increasing number of organisms being sequenced,we believe that DB data will play an important role both in other assembly procedures and infuture genomic studies.

  16. Unique Features of a Japanese ‘Candidatus Liberibacter asiaticus’ Strain Revealed by Whole Genome Sequencing

    OpenAIRE

    Hiroshi Katoh; Shin-Ichi Miyata; Hiromitsu Inoue; Toru Iwanami

    2014-01-01

    Citrus greening (huanglongbing) is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with phloem-limited bacteria of three species of α-Proteobacteria, namely, 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus', and 'Ca. L. africanus'. Recent findings suggested that some Japanese strains lack the bacteriophage-type DNA polymerase region (DNA pol), in contrast to the Floridian psy62 strain. The whole genome sequence of the pol-negative 'C...

  17. A Model for Carbohydrate Metabolism in the Diatom Phaeodactylum tricornutum Deduced from Comparative Whole Genome Analysis

    OpenAIRE

    Kroth, Peter G.; Chiovitti, Anthony; Gruber, Ansgar; Martin-jezequel, Veronique; Mock, Thomas; Schnitzler Parker, Micaela; Michele S. Stanley; Kaplan, Aaron; Caron, Lise; Weber, Till; Maheswari, Uma; Armbrust, Elisabeth Virginia; Bowler, Chris

    2008-01-01

    Background:Diatoms are unicellular algae responsible for approximately 20% of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids.Methodology/Principal Findings:The whole genome sequence of the diatom Phaeodactylum tricornutum has recently been completed. We identified and annotated genes for enzymes involved in carbohydrate pathways based on extensive EST support and comparison to ...

  18. Effective Normalization for Copy Number Variation Detection from Whole Genome Sequencing

    OpenAIRE

    Janevski Angel; Varadan Vinay; Kamalakaran Sitharthan; Banerjee Nilanjana; Dimitrova Nevenka

    2012-01-01

    Abstract Background Whole genome sequencing enables a high resolution view of the human genome and provides unique insights into genome structure at an unprecedented scale. There have been a number of tools to infer copy number variation in the genome. These tools, while validated, also include a number of parameters that are configurable to genome data being analyzed. These algorithms allow for normalization to account for individual and population-specific effects on individual genome CNV e...

  19. Using Mendelian inheritance errors as quality control criteria in whole genome sequencing data set

    OpenAIRE

    Pilipenko, Valentina V; He, Hua; Kurowski, Brad G.; Alexander, Eileen S.; Zhang, Xue; Ding, Lili; Mersha, Tesfaye B.; Kottyan, Leah; Fardo, David W.; Martin, Lisa J.

    2014-01-01

    Although the technical and analytic complexity of whole genome sequencing is generally appreciated, best practices for data cleaning and quality control have not been defined. Family based data can be used to guide the standardization of specific quality control metrics in nonfamily based data. Given the low mutation rate, Mendelian inheritance errors are likely as a result of erroneous genotype calls. Thus, our goal was to identify the characteristics that determine Mendelian inheritance err...

  20. Whole-Genome Sequence of Rummeliibacillus stabekisii Strain PP9 Isolated from Antarctic Soil

    Science.gov (United States)

    da Mota, Fábio Faria; Vollú, Renata Estebanez; Jurelevicius, Diogo

    2016-01-01

    The whole genome of Rummeliibacillus stabekisii PP9, isolated from a soil sample from Antarctica, consists of a circular chromosome of 3,412,092 bp and a circular plasmid of 8,647 bp, with 3,244 protein-coding genes, 12 copies of the 16S-23S-5S rRNA operon, 101 tRNA genes, and 6 noncoding RNAs (ncRNAs). PMID:27231360

  1. Self-organizing Approach for Automated Gene Identification in Whole Genomes

    CERN Document Server

    Gorban, A N; Popova, T G; Gorban, Alexander N.; Zinovyev, Andrey Yu.; Popova, Tatyana G.

    2001-01-01

    An approach based on using the idea of distinguished coding phase in explicit form for identification of protein-coding regions (exons) in whole genome has been proposed. For several genomes an optimal window length for averaging GC-content function and calculating codon frequencies has been found. Self-training procedure based on clustering in multidimensional space of triplet frequencies is proposed. For visualization of data in the space of triplet requiencies method of elastic maps was applied.

  2. Clinical Application of Whole-Genome Sequencing To Inform Treatment for Multidrug-Resistant Tuberculosis Cases

    OpenAIRE

    Witney, Adam A.; Gould, Katherine A.; Arnold, Amber; Coleman, David; Delgado, Rachel; Dhillon, Jasvir; Pond, Marcus J.; Pope, Cassie F.; Planche, Tim D.; Stoker, Neil G.; Cosgrove, Catherine A.; Butcher, Philip D.; Harrison, Thomas S; Hinds, Jason

    2015-01-01

    The treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time fram...

  3. Prospective Whole-Genome Sequencing Enhances National Surveillance of Listeria monocytogenes

    OpenAIRE

    Kwong, Jason C.; Mercoulia, Karolina; Tomita, Takehiro; Easton, Marion; Li, Hua Y.; Bulach, Dieter M.; Stinear, Timothy P.; Seemann, Torsten; Benjamin P Howden

    2016-01-01

    Whole-genome sequencing (WGS) has emerged as a powerful tool for comparing bacterial isolates in outbreak detection and investigation. Here we demonstrate that WGS performed prospectively for national epidemiologic surveillance of Listeria monocytogenes has the capacity to be superior to our current approaches using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), binary typing, and serotyping. Initially 423 ...

  4. Whole genome nucleosome sequencing identifies novel types of forensic markers in degraded DNA samples

    OpenAIRE

    Chun-nan Dong; Ya-dong Yang; Shu-jin Li; Ya-ran Yang; Xiao-jing Zhang; Xiang-dong Fang; Jiang-wei Yan; Bin Cong

    2016-01-01

    In the case of mass disasters, missing persons and forensic caseworks, highly degraded biological samples are often encountered. It can be a challenge to analyze and interpret the DNA profiles from these samples. Here we provide a new strategy to solve the problem by taking advantage of the intrinsic structural properties of DNA. We have assessed the in vivo positions of more than 35 million putative nucleosome cores in human leukocytes using high-throughput whole genome sequencing, and ident...

  5. Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples

    OpenAIRE

    Minsoung Rhee; Yooli K Light; Meagher, Robert J.; Anup K. Singh

    2016-01-01

    Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template D...

  6. Whole-Genome Sequence of Chlamydia gallinacea Type Strain 08-1274/3

    Science.gov (United States)

    Hölzer, Martin; Laroucau, Karine; Creasy, Heather Huot; Ott, Sandra; Vorimore, Fabien; Bavoil, Patrik M.; Marz, Manja

    2016-01-01

    The recently introduced bacterial species Chlamydia gallinacea is known to occur in domestic poultry and other birds. Its potential as an avian pathogen and zoonotic agent is under investigation. The whole-genome sequence of its type strain, 08-1274/3, consists of a 1,059,583-bp chromosome with 914 protein-coding sequences (CDSs) and a plasmid (p1274) comprising 7,619 bp with 9 CDSs. PMID:27445388

  7. Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity

    OpenAIRE

    Dulak, Austin M.; Stojanov, Petar; Peng, Shouyong; Lawrence, Michael S; Fox, Cameron; Stewart, Chip; Bandla, Santhoshi; Imamura, Yu; Schumacher, Steven E; Shefler, Erica; McKenna, Aaron; Cibulskis, Kristian; Sivachenko, Andrey; Carter, Scott L.; Saksena, Gordon

    2012-01-01

    The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a five-year survival rate of 15%, identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole exome sequencing of 149 EAC tumors/normal pairs, 15 of which have also been subjected to whole genome sequencing. We identify a mutational signature defined by a high prevalence of A to C transversions at AA dinucleotides. Statistical analysis of exome da...

  8. Systematic Pharmacogenomics Analysis of a Malay Whole Genome: Proof of Concept for Personalized Medicine

    OpenAIRE

    Salleh, Mohd Zaki; Teh, Lay Kek; Lee, Lian Shien; Ismet, Rose Iszati; Patowary, Ashok; Joshi, Kandarp; Pasha, Ayesha; Ahmed, Azni Zain; Janor, Roziah Mohd; Hamzah, Ahmad Sazali; Adam, Aishah; Yusoff, Khalid; Hoh, Boon Peng; Hatta, Fazleen Haslinda Mohd; Ismail, Mohamad Izwan

    2013-01-01

    Background With a higher throughput and lower cost in sequencing, second generation sequencing technology has immense potential for translation into clinical practice and in the realization of pharmacogenomics based patient care. The systematic analysis of whole genome sequences to assess patient to patient variability in pharmacokinetics and pharmacodynamics responses towards drugs would be the next step in future medicine in line with the vision of personalizing medicine. Methods Genomic DN...

  9. Whole-genome sequencing and analysis of the Malaysian cynomolgus macaque (Macaca fascicularis) genome

    OpenAIRE

    Higashino, Atsunori; Sakate, Ryuichi; Kameoka, Yosuke; Takahashi, Ichiro; Hirata, Makoto; Tanuma, Reiko; Masui, Tohru; Yasutomi, Yasuhiro; Osada, Naoki

    2012-01-01

    Background The genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome. Results We identified approximately 9.7 million single nuc...

  10. Analytical validation of whole exome and whole genome sequencing for clinical applications

    OpenAIRE

    Linderman, Michael D.; Brandt, Tracy; Edelmann, Lisa; Jabado, Omar; Kasai, Yumi; Kornreich, Ruth; Mahajan, Milind; Shah, Hardik; Kasarskis, Andrew; Eric E Schadt

    2014-01-01

    Background Whole exome and genome sequencing (WES/WGS) is now routinely offered as a clinical test by a growing number of laboratories. As part of the test design process each laboratory must determine the performance characteristics of the platform, test and informatics pipeline. This report documents one such characterization of WES/WGS. Methods Whole exome and whole genome sequencing was performed on multiple technical replicates of five reference samples using the Illumina HiSeq 2000/2500...

  11. Coverage Bias and Sensitivity of Variant Calling for Four Whole-genome Sequencing Technologies

    OpenAIRE

    Nora Rieber; Marc Zapatka; Bärbel Lasitschka; David Jones1; Paul Northcott; Barbara Hutter; Natalie Jäger; Marcel Kool; Michael Taylor; Peter Lichter; Stefan Pfister; Stephan Wolf; Benedikt Brors; Roland Eils

    2013-01-01

    The emergence of high-throughput, next-generation sequencing technologies has dramatically altered the way we assess genomes in population genetics and in cancer genomics. Currently, there are four commonly used whole-genome sequencing platforms on the market: Illumina's HiSeq2000, Life Technologies' SOLiD 4 and its completely redesigned 5500xl SOLiD, and Complete Genomics' technology. A number of earlier studies have compared a subset of those sequencing platforms or compared those platforms...

  12. Rainbow: a tool for large-scale whole-genome sequencing data analysis using cloud computing

    OpenAIRE

    Zhao, Shanrong; Prenger, Kurt; Smith, Lance; Messina, Thomas; Fan, Hongtao; Jaeger, Edward; Stephens, Susan

    2013-01-01

    Background Technical improvements have decreased sequencing costs and, as a result, the size and number of genomic datasets have increased rapidly. Because of the lower cost, large amounts of sequence data are now being produced by small to midsize research groups. Crossbow is a software tool that can detect single nucleotide polymorphisms (SNPs) in whole-genome sequencing (WGS) data from a single subject; however, Crossbow has a number of limitations when applied to multiple subjects from la...

  13. Coverage Bias and Sensitivity of Variant Calling for Four Whole-genome Sequencing Technologies

    OpenAIRE

    Rieber, Nora; Zapatka, Marc; Lasitschka, Bärbel; Jones, David,; Northcott, Paul; Hutter, Barbara; Jäger, Natalie; Kool, Marcel; Taylor, Michael; Lichter, Peter; Pfister, Stefan; Wolf, Stephan; Brors, Benedikt; Eils, Roland

    2013-01-01

    The emergence of high-throughput, next-generation sequencing technologies has dramatically altered the way we assess genomes in population genetics and in cancer genomics. Currently, there are four commonly used whole-genome sequencing platforms on the market: Illumina’s HiSeq2000, Life Technologies’ SOLiD 4 and its completely redesigned 5500xl SOLiD, and Complete Genomics’ technology. A number of earlier studies have compared a subset of those sequencing platforms or compared those platforms...

  14. Identification of emergent blaCMY-2-carrying Proteus mirabilis lineages by whole-genome sequencing

    OpenAIRE

    Mac Aogáin, M.; Rogers, T.R.; Crowley, B.

    2016-01-01

    Whole-genome sequencing of 24 Proteus mirabilis isolates revealed the clonal expansion of two cefoxitin-resistant strains among patients with community-onset infection. These strains harboured bla CMY-2 within a chromosomally located integrative and conjugative element and exhibited multidrug resistance phenotypes. A predominant strain, identified in 18 patients, also harboured the PGI-1 genomic island and associated resistance genes, accounting for its broader antibiotic resistance profile. ...

  15. Organization and Evolution of Primate Centromeric DNA from Whole-Genome Shotgun Sequence Data

    OpenAIRE

    Alkan, Can; Eichler, Evan E.; Ventura, Mario; Archidiacono, Nicoletta; Rocchi, Mariano; Sahinalp, S Cenk

    2007-01-01

    Author Summary Centromeric DNA has been described as the last frontier of genomic sequencing; such regions are typically poorly assembled during the whole-genome shotgun sequence assembly process due to their repetitive complexity. This paper develops a computational algorithm to systematically extract data regarding primate centromeric DNA structure and organization from that ∼5% of sequence that is not included as part of standard genome sequence assemblies. Using this computational approac...

  16. Whole-genome resequencing of Hanwoo (Korean cattle) and insight into regions of homozygosity

    OpenAIRE

    Lee, Kyung-Tai; Chung, Won-Hyong; Lee, Sung-Yeoun; Choi, Jung-Woo; Kim, Jiwoong; Lim, Dajeong; Lee, Seunghwan; Jang, Gul-Won; Kim, Bumsoo; Choy, Yun Ho; Liao, Xiaoping; Stothard, Paul; Moore, Stephen S; Lee, Sang-Heon; Ahn, Sungmin

    2013-01-01

    Background Hanwoo (Korean cattle), which originated from natural crossbreeding between taurine and zebu cattle, migrated to the Korean peninsula through North China. Hanwoo were raised as draft animals until the 1970s without the introduction of foreign germplasm. Since 1979, Hanwoo has been bred as beef cattle. Genetic variation was analyzed by whole-genome deep resequencing of a Hanwoo bull. The Hanwoo genome was compared to that of two other breeds, Black Angus and Holstein, and genes with...

  17. How accurately is ncRNA aligned within whole-genome multiple alignments?

    OpenAIRE

    Ruzzo Walter L; Wang Adrienne X; Tompa Martin

    2007-01-01

    Abstract Background Multiple alignment of homologous DNA sequences is of great interest to biologists since it provides a window into evolutionary processes. At present, the accuracy of whole-genome multiple alignments, particularly in noncoding regions, has not been thoroughly evaluated. Results We evaluate the alignment accuracy of certain noncoding regions using noncoding RNA alignments from Rfam as a reference. We inspect the MULTIZ 17-vertebrate alignment from the UCSC Genome Browser for...

  18. Screening of whole genome sequences identified high-impact variants for stallion fertility

    OpenAIRE

    Schrimpf, Rahel; Gottschalk, Maren; Metzger, Julia; Martinsson, Gunilla; Sieme, Harald; Distl, Ottmar

    2016-01-01

    Background Stallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions. Methods Gene ontology (GO) ter...

  19. De novo mutations discovered in 8 Mexican American families through whole genome sequencing

    OpenAIRE

    Wang, Heming; Zhu, Xiaofeng

    2014-01-01

    De novo mutations enrich the sequence diversity and carry the clue of evolutional selection. Recent studies suggest the de novo mutations could be one of the risk factors for complex diseases. We conducted a survey of de novo mutations using the whole genome sequence data but only available on the odd autosomes of Mexican American families provided by Genetic Analysis Workshop 18. We extracted 8 three-generation families who have sequencing data available from 20 large pedigrees. By comparing...

  20. Rapid Whole-Genome Sequencing for Surveillance of Salmonella enterica Serovar Enteritidis

    OpenAIRE

    den Bakker, Henk C.; Allard, Marc W.; Bopp, Dianna; Brown, Eric W.; Fontana, John; Iqbal, Zamin; Kinney, Aristea; Limberger, Ronald; Musser, Kimberlee A.; Shudt, Matthew; Strain, Errol; Wiedmann, Martin; Wolfgang, William J.

    2014-01-01

    For Salmonella enterica serovar Enteritidis, 85% of isolates can be classified into 5 pulsed-field gel electrophoresis (PFGE) types. However, PFGE has limited discriminatory power for outbreak detection. Although whole-genome sequencing has been found to improve discrimination of outbreak clusters, whether this procedure can be used in real-time in a public health laboratory is not known. Therefore, we conducted a retrospective and prospective analysis. The retrospective study investigated is...

  1. Microarray-based whole-genome hybridization as a tool for determining procaryotic species relatedness

    Energy Technology Data Exchange (ETDEWEB)

    Wu, L.; Liu, X.; Fields, M.W.; Thompson, D.K.; Bagwell, C.E.; Tiedje, J. M.; Hazen, T.C.; Zhou, J.

    2008-01-15

    The definition and delineation of microbial species are of great importance and challenge due to the extent of evolution and diversity. Whole-genome DNA-DNA hybridization is the cornerstone for defining procaryotic species relatedness, but obtaining pairwise DNA-DNA reassociation values for a comprehensive phylogenetic analysis of procaryotes is tedious and time consuming. A previously described microarray format containing whole-genomic DNA (the community genome array or CGA) was rigorously evaluated as a high-throughput alternative to the traditional DNA-DNA reassociation approach for delineating procaryotic species relationships. DNA similarities for multiple bacterial strains obtained with the CGA-based hybridization were comparable to those obtained with various traditional whole-genome hybridization methods (r=0.87, P<0.01). Significant linear relationships were also observed between the CGA-based genome similarities and those derived from small subunit (SSU) rRNA gene sequences (r=0.79, P<0.0001), gyrB sequences (r=0.95, P<0.0001) or REP- and BOX-PCR fingerprinting profiles (r=0.82, P<0.0001). The CGA hybridization-revealed species relationships in several representative genera, including Pseudomonas, Azoarcus and Shewanella, were largely congruent with previous classifications based on various conventional whole-genome DNA-DNA reassociation, SSU rRNA and/or gyrB analyses. These results suggest that CGA-based DNA-DNA hybridization could serve as a powerful, high-throughput format for determining species relatedness among microorganisms.

  2. Heritability of pulmonary function estimated from pedigree and whole-genome markers

    OpenAIRE

    Klimentidis, Yann C.; Vazquez, Ana I; de los Campos, Gustavo; Allison, David B.; Dransfield, Mark T.; Thannickal, Victor J.

    2013-01-01

    Asthma and chronic obstructive pulmonary disease (COPD) are major worldwide health problems. Pulmonary function testing is a useful diagnostic tool for these diseases, and is known to be influenced by genetic and environmental factors. Previous studies have demonstrated that a substantial proportion of the variation in pulmonary function phenotypes can be explained by familial relationships. The availability of whole-genome single nucleotide polymorphism (SNP) data enables us to further evalu...

  3. A whole-genome, radiation hybrid mapping resource of hexaploid wheat.

    Science.gov (United States)

    Tiwari, Vijay K; Heesacker, Adam; Riera-Lizarazu, Oscar; Gunn, Hilary; Wang, Shichen; Wang, Yi; Gu, Young Q; Paux, Etienne; Koo, Dal-Hoe; Kumar, Ajay; Luo, Ming-Cheng; Lazo, Gerard; Zemetra, Robert; Akhunov, Eduard; Friebe, Bernd; Poland, Jesse; Gill, Bikram S; Kianian, Shahryar; Leonard, Jeffrey M

    2016-04-01

    Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n = 6x = 42; approximately 17 GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole-genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics-based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the pericentromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole-genome using these approaches is nearly impossible. We developed a whole-genome radiation hybrid (WGRH) resource and tested it by genotyping a set of 115 randomly selected lines on a high-density single nucleotide polymorphism (SNP) array. At the whole-genome level, 26 299 SNP markers were mapped on the RH panel and provided an average mapping resolution of approximately 248 Kb/cR1500 with a total map length of 6866 cR1500 . The 7296 unique mapping bins provided a five- to eight-fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including pericentromeric regions. Our research provides a valuable and low-cost resource for anchoring and ordering sequenced BAC and next generation sequencing (NGS) contigs. The WGRH developed for reference wheat line Chinese Spring (CS-WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high-quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species. PMID:26945524

  4. Comparison of Whole-Genome Sequencing and Molecular-Epidemiological Techniques for Clostridium difficile Strain Typing.

    Science.gov (United States)

    Dominguez, Samuel R; Anderson, Lydia J; Kotter, Cassandra V; Littlehorn, Cynthia A; Arms, Lesley E; Dowell, Elaine; Todd, James K; Frank, Daniel N

    2016-09-01

    We analyzed in parallel 27 pediatric Clostridium difficile isolates by repetitive sequence-based polymerase chain reaction (RepPCR), pulsed-field gel electrophoresis (PFGE), and whole-genome next-generation sequencing. Next-generation sequencing distinguished 3 groups of isolates that were indistinguishable by RepPCR and 1 isolate that clustered in the same PFGE group as other isolates. PMID:26407257

  5. Inference of Gorilla Demographic and Selective History from Whole-Genome Sequence Data

    OpenAIRE

    McManus, Kimberly F; Kelley, Joanna L.; Song, Shiya; Veeramah, Krishna R; Woerner, August E.; Stevison, Laurie S.; Ryder, Oliver A.; Ape Genome Project, Great; Kidd, Jeffrey M.; Wall, Jeffrey D.; Bustamante, Carlos D.; Hammer, Michael F.

    2015-01-01

    Although population-level genomic sequence data have been gathered extensively for humans, similar data from our closest living relatives are just beginning to emerge. Examination of genomic variation within great apes offers many opportunities to increase our understanding of the forces that have differentially shaped the evolutionary history of hominid taxa. Here, we expand upon the work of the Great Ape Genome Project by analyzing medium to high coverage whole-genome sequences from 14 west...

  6. Reducing INDEL calling errors in whole genome and exome sequencing data

    OpenAIRE

    Fang, Han; Wu, Yiyang; Narzisi, Giuseppe; O’Rawe, Jason A; Barrón, Laura T Jimenez; Rosenbaum, Julie; Ronemus, Michael; Iossifov, Ivan; Schatz, Michael C.; Lyon, Gholson J

    2014-01-01

    Background INDELs, especially those disrupting protein-coding regions of the genome, have been strongly associated with human diseases. However, there are still many errors with INDEL variant calling, driven by library preparation, sequencing biases, and algorithm artifacts. Methods We characterized whole genome sequencing (WGS), whole exome sequencing (WES), and PCR-free sequencing data from the same samples to investigate the sources of INDEL errors. We also developed a classification schem...

  7. Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

    OpenAIRE

    Adriano C Coelho; Sébastien Boisvert; Angana Mukherjee; Philippe Leprohon; Jacques Corbeil; Marc Ouellette

    2012-01-01

    BACKGROUND: Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance. METHODS/PRINCIPAL FINDINGS: Analysis of the genome assemblies re...

  8. Colorectal cancer and the human gut microbiome: reproducibility with whole-genome shotgun sequencing

    OpenAIRE

    Emily Vogtmann; Xing Hua; Georg Zeller; Shinichi Sunagawa; Voigt, Anita Y.; Rajna Hercog; Goedert, James J.; Jianxin Shi; Peer Bork; Rashmi Sinha

    2016-01-01

    Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously pub...

  9. Self-organizing Approach for Automated Gene Identification in Whole Genomes

    OpenAIRE

    Gorban, Alexander N; Zinovyev, Andrey Yu.; Popova, Tatyana G.

    2001-01-01

    An approach based on using the idea of distinguished coding phase in explicit form for identification of protein-coding regions (exons) in whole genome has been proposed. For several genomes an optimal window length for averaging GC-content function and calculating codon frequencies has been found. Self-training procedure based on clustering in multidimensional space of triplet frequencies is proposed. For visualization of data in the space of triplet requiencies method of elastic maps was ap...

  10. Analysis on n-gram statistics and linguistic features of whole genome protein sequences

    Institute of Scientific and Technical Information of China (English)

    DONG Qi-wen; WANG Xiao-long; LIN Lei

    2008-01-01

    To obtain the statistical sequence analysis on a large number of genomic and proteomie sequences available for different organisms,the n-grams of whole genome protein sequences from 20 organisms were extracted.Their linguistic features were analyzed by two tests:Zipf power law and Shannon entropy,developed for analysis of natural languages and symbolic sequences.The natural genome proteins and the artificial genome proteins were compared with each other and some statistical features of n-grams were discovered.The results show that:the n-grams of whole genome protein sequences approximately follow the Zipf law when n is larger than 4;the Shannon n-gram entropy of natural genome proteins is lower than that of artificial proteins;a simple unigram model can distinguish different organisms;there exist organism-specific usages of "phrases" in protein sequences.It is suggested that further detailed analysis on n-gram of whole genome protein sequences will result in a powerful model for mapping the relationship of protein sequence,structure and function.

  11. Whole Genome Mapping with Feature Sets from High-Throughput Sequencing Data.

    Science.gov (United States)

    Pan, Yonglong; Wang, Xiaoming; Liu, Lin; Wang, Hao; Luo, Meizhong

    2016-01-01

    A good physical map is essential to guide sequence assembly in de novo whole genome sequencing, especially when sequences are produced by high-throughput sequencing such as next-generation-sequencing (NGS) technology. We here present a novel method, Feature sets-based Genome Mapping (FGM). With FGM, physical map and draft whole genome sequences can be generated, anchored and integrated using the same data set of NGS sequences, independent of restriction digestion. Method model was created and parameters were inspected by simulations using the Arabidopsis genome sequence. In the simulations, when ~4.8X genome BAC library including 4,096 clones was used to sequence the whole genome, ~90% of clones were successfully connected to physical contigs, and 91.58% of genome sequences were mapped and connected to chromosomes. This method was experimentally verified using the existing physical map and genome sequence of rice. Of 4,064 clones covering 115 Mb sequence selected from ~3 tiles of 3 chromosomes of a rice draft physical map, 3,364 clones were reconstructed into physical contigs and 98 Mb sequences were integrated into the 3 chromosomes. The physical map-integrated draft genome sequences can provide permanent frameworks for eventually obtaining high-quality reference sequences by targeted sequencing, gap filling and combining other sequences. PMID:27611682

  12. Divergent whole-genome methylation maps of human and chimpanzee brains reveal epigenetic basis of human regulatory evolution.

    Science.gov (United States)

    Zeng, Jia; Konopka, Genevieve; Hunt, Brendan G; Preuss, Todd M; Geschwind, Dan; Yi, Soojin V

    2012-09-01

    DNA methylation is a pervasive epigenetic DNA modification that strongly affects chromatin regulation and gene expression. To date, it remains largely unknown how patterns of DNA methylation differ between closely related species and whether such differences contribute to species-specific phenotypes. To investigate these questions, we generated nucleotide-resolution whole-genome methylation maps of the prefrontal cortex of multiple humans and chimpanzees. Levels and patterns of DNA methylation vary across individuals within species according to the age and the sex of the individuals. We also found extensive species-level divergence in patterns of DNA methylation and that hundreds of genes exhibit significantly lower levels of promoter methylation in the human brain than in the chimpanzee brain. Furthermore, we investigated the functional consequences of methylation differences in humans and chimpanzees by integrating data on gene expression generated with next-generation sequencing methods, and we found a strong relationship between differential methylation and gene expression. Finally, we found that differentially methylated genes are strikingly enriched with loci associated with neurological disorders, psychological disorders, and cancers. Our results demonstrate that differential DNA methylation might be an important molecular mechanism driving gene-expression divergence between human and chimpanzee brains and might potentially contribute to the evolution of disease vulnerabilities. Thus, comparative studies of humans and chimpanzees stand to identify key epigenomic modifications underlying the evolution of human-specific traits. PMID:22922032

  13. Increased Proportion of Variance Explained and Prediction Accuracy of Survival of Breast Cancer Patients with Use of Whole-Genome Multiomic Profiles.

    Science.gov (United States)

    Vazquez, Ana I; Veturi, Yogasudha; Behring, Michael; Shrestha, Sadeep; Kirst, Matias; Resende, Marcio F R; de Los Campos, Gustavo

    2016-07-01

    Whole-genome multiomic profiles hold valuable information for the analysis and prediction of disease risk and progression. However, integrating high-dimensional multilayer omic data into risk-assessment models is statistically and computationally challenging. We describe a statistical framework, the Bayesian generalized additive model ((BGAM), and present software for integrating multilayer high-dimensional inputs into risk-assessment models. We used BGAM and data from The Cancer Genome Atlas for the analysis and prediction of survival after diagnosis of breast cancer. We developed a sequence of studies to (1) compare predictions based on single omics with those based on clinical covariates commonly used for the assessment of breast cancer patients (COV), (2) evaluate the benefits of combining COV and omics, (3) compare models based on (a) COV and gene expression profiles from oncogenes with (b) COV and whole-genome gene expression (WGGE) profiles, and (4) evaluate the impacts of combining multiple omics and their interactions. We report that (1) WGGE profiles and whole-genome methylation (METH) profiles offer more predictive power than any of the COV commonly used in clinical practice (e.g., subtype and stage), (2) adding WGGE or METH profiles to COV increases prediction accuracy, (3) the predictive power of WGGE profiles is considerably higher than that based on expression from large-effect oncogenes, and (4) the gain in prediction accuracy when combining multiple omics is consistent. Our results show the feasibility of omic integration and highlight the importance of WGGE and METH profiles in breast cancer, achieving gains of up to 7 points area under the curve (AUC) over the COV in some cases. PMID:27129736

  14. Whole-Genome Sequencing Allows for Improved Identification of Persistent Listeria monocytogenes in Food-Associated Environments

    OpenAIRE

    Stasiewicz, Matthew J.; Oliver, Haley F; Wiedmann, Martin; den Bakker, Henk C

    2015-01-01

    While the food-borne pathogen Listeria monocytogenes can persist in food associated environments, there are no whole-genome sequence (WGS) based methods to differentiate persistent from sporadic strains. Whole-genome sequencing of 188 isolates from a longitudinal study of L. monocytogenes in retail delis was used to (i) apply single-nucleotide polymorphism (SNP)-based phylogenetics for subtyping of L. monocytogenes, (ii) use SNP counts to differentiate persistent from repeatedly reintroduced ...

  15. Accuracy of genomic prediction using imputed whole-genome sequence data in white layers.

    Science.gov (United States)

    Heidaritabar, M; Calus, M P L; Megens, H-J; Vereijken, A; Groenen, M A M; Bastiaansen, J W M

    2016-06-01

    There is an increasing interest in using whole-genome sequence data in genomic selection breeding programmes. Prediction of breeding values is expected to be more accurate when whole-genome sequence is used, because the causal mutations are assumed to be in the data. We performed genomic prediction for the number of eggs in white layers using imputed whole-genome resequence data including ~4.6 million SNPs. The prediction accuracies based on sequence data were compared with the accuracies from the 60 K SNP panel. Predictions were based on genomic best linear unbiased prediction (GBLUP) as well as a Bayesian variable selection model (BayesC). Moreover, the prediction accuracy from using different types of variants (synonymous, non-synonymous and non-coding SNPs) was evaluated. Genomic prediction using the 60 K SNP panel resulted in a prediction accuracy of 0.74 when GBLUP was applied. With sequence data, there was a small increase (~1%) in prediction accuracy over the 60 K genotypes. With both 60 K SNP panel and sequence data, GBLUP slightly outperformed BayesC in predicting the breeding values. Selection of SNPs more likely to affect the phenotype (i.e. non-synonymous SNPs) did not improve the accuracy of genomic prediction. The fact that sequence data were based on imputation from a small number of sequenced animals may have limited the potential to improve the prediction accuracy. A small reference population (n = 1004) and possible exclusion of many causal SNPs during quality control can be other possible reasons for limited benefit of sequence data. We expect, however, that the limited improvement is because the 60 K SNP panel was already sufficiently dense to accurately determine the relationships between animals in our data. PMID:26776363

  16. High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic

    Directory of Open Access Journals (Sweden)

    Sealfon Rachel

    2012-09-01

    Full Text Available Abstract Background Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x; four of the seven isolates were previously sequenced. Results Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961, 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Conclusions Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

  17. Gene duplication and paleopolyploidy in soybean and the implications for whole genome sequencing

    Directory of Open Access Journals (Sweden)

    Nelson Rex T

    2007-09-01

    Full Text Available Abstract Background Soybean, Glycine max (L. Merr., is a well documented paleopolyploid. What remains relatively under characterized is the level of sequence identity in retained homeologous regions of the genome. Recently, the Department of Energy Joint Genome Institute and United States Department of Agriculture jointly announced the sequencing of the soybean genome. One of the initial concerns is to what extent sequence identity in homeologous regions would have on whole genome shotgun sequence assembly. Results Seventeen BACs representing ~2.03 Mb were sequenced as representative potential homeologous regions from the soybean genome. Genetic mapping of each BAC shows that 11 of the 20 chromosomes are represented. Sequence comparisons between homeologous BACs shows that the soybean genome is a mosaic of retained paleopolyploid regions. Some regions appear to be highly conserved while other regions have diverged significantly. Large-scale "batch" reassembly of all 17 BACs combined showed that even the most homeologous BACs with upwards of 95% sequence identity resolve into their respective homeologous sequences. Potential assembly errors were generated by tandemly duplicated pentatricopeptide repeat containing genes and long simple sequence repeats. Analysis of a whole-genome shotgun assembly of 80,000 randomly chosen JGI-DOE sequence traces reveals some new soybean-specific repeat sequences. Conclusion This analysis investigated both the structure of the paleopolyploid soybean genome and the potential effects retained homeology will have on assembling the whole genome shotgun sequence. Based upon these results, homeologous regions similar to those characterized here will not cause major assembly issues.

  18. Genomic Epidemiology: Whole-Genome-Sequencing–Powered Surveillance and Outbreak Investigation of Foodborne Bacterial Pathogens

    DEFF Research Database (Denmark)

    Deng, Xiangyu; den Bakker, Henk C.; Hendriksen, Rene S.

    2016-01-01

    As we are approaching the twentieth anniversary of PulseNet, a network of public health and regulatory laboratories that has changed the landscape of foodborne illness surveillance through molecular subtyping, public health microbiology is undergoing another transformation brought about by so......-called next-generation sequencing (NGS) technologies that have made whole-genome sequencing (WGS) of foodborne bacterial pathogens a realistic and superior alternative to traditional subtyping methods. Routine, real-time, and widespread application of WGS in food safety and public health is on the horizon...

  19. Whole-genome sequencing identifies a recurrent functional synonymous mutation in melanoma

    OpenAIRE

    Gartner, Jared J; Stephen C. J. Parker; Prickett, Todd D.; Dutton-Regester, Ken; Stitzel, Michael L.; Lin, Jimmy C.; Davis, Sean; Simhadri, Vijaya L.; Jha, Sujata; Katagiri, Nobuko; Gotea, Valer; Jamie K. Teer; Wei, Xiaomu; Morken, Mario A; Umesh K Bhanot

    2013-01-01

    Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683–691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This muta...

  20. Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

    OpenAIRE

    Votintseva, Antonina A.; Pankhurst, Louise J.; Anson, Luke W.; Morgan, Marcus R.; Gascoyne-Binzi, Deborah; Walker, Timothy M; Quan, T. Phuong; Wyllie, David H; Del Ojo Elias, Carlos; Wilcox, Mark; Walker, A. Sarah; Peto, Tim E A; Crook, Derrick W.

    2015-01-01

    We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reve...

  1. Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures

    OpenAIRE

    Votintseva, AA; Pankhurst, LJ; Anson, LW; Morgan, *; Gascoyne-Binzi, D.; Walker, TM; quan, TP; Wyllie, DH; Del Ojo Elias, C; Wilcox, M; Walker, AS; Peto, TE; Crook, DW

    2015-01-01

    We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reve...

  2. Unique Features of a Japanese ‘Candidatus Liberibacter asiaticus’ Strain Revealed by Whole Genome Sequencing

    OpenAIRE

    Katoh, Hiroshi; Miyata, Shin-ichi; Inoue, Hiromitsu; Iwanami, Toru

    2014-01-01

    Citrus greening (huanglongbing) is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with phloem-limited bacteria of three species of α-Proteobacteria, namely, ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. americanus’, and ‘Ca. L. africanus’. Recent findings suggested that some Japanese strains lack the bacteriophage-type DNA polymerase region (DNA pol), in contrast to the Floridian psy62 strain. The whole genome sequence of the pol-negative ‘C...

  3. Refining QTL with high-density SNP genotyping and whole genome sequence in three cattle breeds

    DEFF Research Database (Denmark)

    Sahana, Goutam; Guldbrandtsen, Bernt; Lund, Mogens Sandø

    2012-01-01

    Genome-wide association study was carried out in Nordic Holsteins, Nordic Red and Jersey breeds for functional traits using BovineHD Genotyping BreadChip (Illumina, San Diego, CA). The association analyses were carried out using both linear mixed model approach and a Bayesian variable selection...... method. Principal components were used to account for population structure. The QTL segregating in all three breeds were selected and a few of the most significant ones were followed in further analyses. The polymorphisms in the identified QTL regions were imputed using 90 whole genome sequences...

  4. Two Methods of Whole-Genome Amplification Enable Accurate Genotyping Across a 2320-SNP Linkage Panel

    OpenAIRE

    Barker, David L.; Hansen, Mark S. T.; Faruqi, A. Fawad; Giannola, Diane; Irsula, Orlando R.; Lasken, Roger S; Latterich, Martin; Makarov, Vladimir; Oliphant, Arnold; Pinter, Jonathon H.; Shen, Richard; Sleptsova, Irina; Ziehler, William; Lai, Eric

    2004-01-01

    Comprehensive genome scans involving many thousands of SNP assays will require significant amounts of genomic DNA from each sample. We report two successful methods for amplifying whole-genomic DNA prior to SNP analysis, multiple displacement amplification, and OmniPlex technology. We determined the coverage of amplification by analyzing a SNP linkage marker set that contained 2320 SNP markers spread across the genome at an average distance of 2.5 cM. We observed a concordance of >99.8% in ge...

  5. Molecular footprints of domestication and improvement in soybean revealed by whole genome re-sequencing

    DEFF Research Database (Denmark)

    Li, Ying-hui; Zhao, Shan-cen; Ma, Jian-xin; Li, Dong; Yan, Long; Li, Jun; Qi, Xiao-tian; Guo, Xiao-sen; Zhang, Le; He, Wei-ming; Chang, Ru-zhen; Liang, Qin-si; Guo, Yong; Ye, Chen; Wang, Xiao-bo; Tao, Yong; Guan, Rong-xia; Wang, Jun-yi; Liu, Yu-lin; Jin, Long-guo; Zhang, Xiu-qing; Liu, Zhang-xiong; Zhang, Li-juan; Chen, Jie; Wang, Ke-jing; Nielsen, Rasmus; Li, Rui-qiang; Chen, Peng-yin; Li, Wen-bin; Reif, Jochen C.; Purugganan, Michael; Wang, Jian; Zhang, Meng-chen; Wang, Jun; Qiu, Li-juan

    2013-01-01

    artificial selection during domestication led to more pronounced reduction in the genetic diversity of soybean than the switch from landraces to elite cultivars. Only a small proportion (2.99%) of the whole genomic regions appear to be affected by artificial selection for preferred agricultural traits. The...... and genetic improvement were identified.CONCLUSIONS:Given the uniqueness of the soybean germplasm sequenced, this study drew a clear picture of human-mediated evolution of the soybean genomes. The genomic resources and information provided by this study would also facilitate the discovery of genes...

  6. Whole genome sequences and annotation of Micrococcus luteus SUBG006, a novel phytopathogen of mango

    Directory of Open Access Journals (Sweden)

    Purvi M. Rakhashiya

    2015-12-01

    Full Text Available Actinobaceria, Micrococcus luteus SUBG006 was isolated from infected leaves of Mangifera indica L. vr. Nylon in Rajkot, (22.30°N, 70.78°E, Gujarat, India. The genome size is 3.86 Mb with G + C content of 69.80% and contains 112 rRNA sequences (5S, 16S and 23S. The whole genome sequencing has been deposited in DDBJ/EMBL/GenBank under the accession number JOKP00000000.

  7. Whole genome sequencing in drug discovery research: a one fits all solution?

    OpenAIRE

    Marc Sultan

    2015-01-01

    With the recent availability of Illumina's HiSeq X ten sequencing platform, the cost of whole genome sequencing (WGS) has dropped to nearly $1,000 per genome. The affordability of WGS has now the potential of replacing other genotyping platforms such as whole exome sequencing (WES) and array based genotyping for (smaller) clinical study cohorts. In a recent pilot study, we compared the performance and genotyping quality of the HiSeq X WGS approach against WES and array based genotyping with r...

  8. Identification of emergent bla CMY-2 -carrying Proteus mirabilis lineages by whole-genome sequencing.

    Science.gov (United States)

    Mac Aogáin, M; Rogers, T R; Crowley, B

    2016-01-01

    Whole-genome sequencing of 24 Proteus mirabilis isolates revealed the clonal expansion of two cefoxitin-resistant strains among patients with community-onset infection. These strains harboured bla CMY-2 within a chromosomally located integrative and conjugative element and exhibited multidrug resistance phenotypes. A predominant strain, identified in 18 patients, also harboured the PGI-1 genomic island and associated resistance genes, accounting for its broader antibiotic resistance profile. The identification of these novel multidrug-resistant strains among community-onset infections suggests that they are endemic to this region and represent emergent P. mirabilis lineages of clinical significance. PMID:26865983

  9. Sequence Determination from Overlapping Fragments: A Simple Model of Whole-Genome Shotgun Sequencing

    Science.gov (United States)

    Derrida, Bernard; Fink, Thomas M.

    2002-02-01

    Assembling fragments randomly sampled from along a sequence is the basis of whole-genome shotgun sequencing, a technique used to map the DNA of the human and other genomes. We calculate the probability that a random sequence can be recovered from a collection of overlapping fragments. We provide an exact solution for an infinite alphabet and in the case of constant overlaps. For the general problem we apply two assembly strategies and give the probability that the assembly puzzle can be solved in the limit of infinitely many fragments.

  10. Kuwaiti population subgroup of nomadic Bedouin ancestry—Whole genome sequence and analysis

    OpenAIRE

    Sumi Elsa John; Gaurav Thareja; Prashantha Hebbar; Kazem Behbehani; Thangavel Alphonse Thanaraj; Osama Alsmadi

    2015-01-01

    Kuwaiti native population comprises three distinct genetic subgroups of Persian, “city-dwelling” Saudi Arabian tribe, and nomadic “tent-dwelling” Bedouin ancestry. Bedouin subgroup is characterized by presence of 17% African ancestry; it owes it origin to nomadic tribes of the deserts of Arabian Peninsula and North Africa. By sequencing whole genome of a Kuwaiti male from this subgroup at 41X coverage, we report 3,752,878 SNPs, 411,839 indels, and 8451 structural variations. Neighbor-joining ...

  11. Comparing Platforms for C. elegans Mutant Identification Using High-Throughput Whole-Genome Sequencing

    OpenAIRE

    Shen, Yufeng; Sarin, Sumeet; Liu, Ye; Hobert, Oliver; Pe'er, Itsik

    2008-01-01

    Background Whole-genome sequencing represents a promising approach to pinpoint chemically induced mutations in genetic model organisms, thereby short-cutting time-consuming genetic mapping efforts. Principal Findings We compare here the ability of two leading high-throughput platforms for paired-end deep sequencing, SOLiD (ABI) and Genome Analyzer (Illumina; “Solexa”), to achieve the goal of mutant detection. As a test case we used a mutant C. elegans strain that harbors a mutation in the lsy...

  12. A Bivariate Whole Genome Linkage Study Identified Genomic Regions Influencing Both BMD and Bone Structure

    OpenAIRE

    Liu, Xiao-Gang; Liu, Yong-Jun; Liu, Jianfeng; Pei, Yufang; Xiong, Dong-Hai; Shen, Hui; Deng, Hong-Yi; Papasian, Christopher J.; Drees, Betty M.; Hamilton, James J.; Recker, Robert R.; Deng, Hong-Wen

    2008-01-01

    Areal BMD (aBMD) and areal bone size (ABS) are biologically correlated traits and are each important determinants of bone strength and risk of fractures. Studies showed that aBMD and ABS are genetically correlated, indicating that they may share some common genetic factors, which, however, are largely unknown. To study the genetic factors influencing both aBMD and ABS, bivariate whole genome linkage analyses were conducted for aBMD-ABS at the femoral neck (FN), lumbar spine (LS), and ultradis...

  13. The First Kazakh Whole Genomes: The First Report of NGS Data

    Directory of Open Access Journals (Sweden)

    Ainur Akilzhanova

    2014-12-01

    Full Text Available Introduction: The human genome sequence will underpin human biology and medicine in the next century, providing a single, essential reference to all genetic information. Extraordinary technological advances and decreases in the cost of DNA sequencing have made the possibility of whole genome sequencing (WGS feasible as a highly accessible test for numerous indications. The international project “Genetic architecture of Kazakh population” is well underway to determine the complete DNA. Next generation sequencing is a powerful tool for genetic analysis, which will enable us to uncover the association of loci at specific sites in the genome associated with disease. The aim of this study was to introduce first data on WGS of 6 Kazakh individuals.Methods: This pilot study is among the first WGS performed on 6 healthy Kazakh individuals, using next generation sequencing platform HiSeq2000, Illumina by manufacturer’s protocols. All generated *.bcl files were simultaneously converted and demultiplexed using bcl2fasta application. Alignment of sequence reads performed using bwa-mem against human b19 reference genome. Sorting, removing of intermediate files, *.bam files assembling, and marking duplicates were performed using PicardTools package. GATK haplotype caller tool was used for variant calling. ClinVar, SNPedia, and Cosmic databases were processed to identify clinical genomic variants in 6 Kazakh whole genomes. Java Runtime Environment and R. Bioconductor packages were installed to perform raw data processing and run program scripts.Results: The sequence alignment and mapping procedures on reference genome hg19 of each 6 healthy Kazakh individual were completed. Between 87,308,581,400 and 107,526,741,301 total base pairs were sequenced with average coverage x29.85. Between 98.85% and 99.58% base pairs were totally mapped and on average 96.07% were properly paired. Het/Hom and Ti/Tv ratios for each whole genome ranged from 1.35 to 1.52 and

  14. Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture

    DEFF Research Database (Denmark)

    Zheng, Hou-Feng; Forgetta, Vincenzo; Hsu, Yi-Hsiang;

    2015-01-01

    The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is mainly unknown. Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been...... provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population....

  15. Whole genome sequences and annotation of Micrococcus luteus SUBG006, a novel phytopathogen of mango.

    Science.gov (United States)

    Rakhashiya, Purvi M; Patel, Pooja P; Thaker, Vrinda S

    2015-12-01

    Actinobaceria, Micrococcus luteus SUBG006 was isolated from infected leaves of Mangifera indica L. vr. Nylon in Rajkot, (22.30°N, 70.78°E), Gujarat, India. The genome size is 3.86 Mb with G + C content of 69.80% and contains 112 rRNA sequences (5S, 16S and 23S). The whole genome sequencing has been deposited in DDBJ/EMBL/GenBank under the accession number JOKP00000000. PMID:26697318

  16. Whole genome sequences and annotation of Micrococcus luteus SUBG006, a novel phytopathogen of mango

    OpenAIRE

    Rakhashiya, Purvi M.; Patel, Pooja P.; Thaker, Vrinda S.

    2015-01-01

    Actinobaceria, Micrococcus luteus SUBG006 was isolated from infected leaves of Mangifera indica L. vr. Nylon in Rajkot, (22.30°N, 70.78°E), Gujarat, India. The genome size is 3.86 Mb with G + C content of 69.80% and contains 112 rRNA sequences (5S, 16S and 23S). The whole genome sequencing has been deposited in DDBJ/EMBL/GenBank under the accession number JOKP00000000.

  17. Global gene expression profiling data analysis reveals key gene families and biological processes inhibited by Mithramycin in sarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Kirti K. Kulkarni

    2015-03-01

    Full Text Available The role of Mithramycin as an anticancer drug has been well studied. Sarcoma is a type of cancer arising from cells of mesenchymal origin. Though incidence of sarcoma is not of significant percentage, it becomes vital to understand the role of Mithramycin in controlling tumor progression of sarcoma. In this article, we have analyzed the global gene expression profile changes induced by Mithramycin in two different sarcoma lines from whole genome gene expression profiling microarray data. We have found that the primary mode of action of Mithramycin is by global repression of key cellular processes and gene families like phosphoproteins, kinases, alternative splicing, regulation of transcription, DNA binding, regulation of histone acetylation, negative regulation of gene expression, chromosome organization or chromatin assembly and cytoskeleton.

  18. Estrogen Receptor-Mediated Effects of Isoflavone Supplementation Were Not Observed in Whole-Genome Gene Expression Profiles of Peripheral Blood Mononuclear Cells in Postmenopausal, Equol-Producing Women

    NARCIS (Netherlands)

    Velpen, van der V.; Geelen, A.; Schouten, E.G.; Hollman, P.C.H.; Afman, L.A.; Veer, van 't P.

    2013-01-01

    Isoflavones (genistein, daidzein, and glycitein) are suggested to have benefits as well as risks for human health. Approximately one-third of the Western population is able to metabolize daidzein into the more potent metabolite equol. Having little endogenous estradiol, equol-producing postmenopausa

  19. Human embryonic stem cells as a model for cardiac gene discovery : from chip to chap

    NARCIS (Netherlands)

    Beqqali, A.

    2008-01-01

    Here we described the use of human embryonic stem cells (hESCs) as a model to obtain insights into commitment to the mesoderm and endoderm lineages and the early steps in human cardiac cell differentiation by means of whole-genome temporal expression profiling. Furthermore, we used it as an approach

  20. Reconstruction of thermotolerant yeast by one-point mutation identified through whole-genome analyses of adaptively-evolved strains.

    Science.gov (United States)

    Satomura, Atsushi; Miura, Natsuko; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-01-01

    Saccharomyces cerevisiae is used as a host strain in bioproduction, because of its rapid growth, ease of genetic manipulation, and high reducing capacity. However, the heat produced during the fermentation processes inhibits the biological activities and growth of the yeast cells. We performed whole-genome sequencing of 19 intermediate strains previously obtained during adaptation experiments under heat stress; 49 mutations were found in the adaptation steps. Phylogenetic tree revealed at least five events in which these strains had acquired mutations in the CDC25 gene. Reconstructed CDC25 point mutants based on a parental strain had acquired thermotolerance without any growth defects. These mutations led to the downregulation of the cAMP-dependent protein kinase (PKA) signaling pathway, which controls a variety of processes such as cell-cycle progression and stress tolerance. The one-point mutations in CDC25 were involved in the global transcriptional regulation through the cAMP/PKA pathway. Additionally, the mutations enabled efficient ethanol fermentation at 39 °C, suggesting that the one-point mutations in CDC25 may contribute to bioproduction. PMID:26984760

  1. Whole genome duplication affects evolvability of flowering time in an autotetraploid plant.

    Directory of Open Access Journals (Sweden)

    Sara L Martin

    Full Text Available Whole genome duplications have occurred recurrently throughout the evolutionary history of eukaryotes. The resulting genetic and phenotypic changes can influence physiological and ecological responses to the environment; however, the impact of genome copy number on evolvability has rarely been examined experimentally. Here, we evaluate the effect of genome duplication on the ability to respond to selection for early flowering time in lines drawn from naturally occurring diploid and autotetraploid populations of the plant Chamerion angustifolium (fireweed. We contrast this with the result of four generations of selection on synthesized neoautotetraploids, whose genic variability is similar to diploids but genome copy number is similar to autotetraploids. In addition, we examine correlated responses to selection in all three groups. Diploid and both extant tetraploid and neoautotetraploid lines responded to selection with significant reductions in time to flowering. Evolvability, measured as realized heritability, was significantly lower in extant tetraploids (^b(T =  0.31 than diploids (^b(T =  0.40. Neotetraploids exhibited the highest evolutionary response (^b(T  =  0.55. The rapid shift in flowering time in neotetraploids was associated with an increase in phenotypic variability across generations, but not with change in genome size or phenotypic correlations among traits. Our results suggest that whole genome duplications, without hybridization, may initially alter evolutionary rate, and that the dynamic nature of neoautopolyploids may contribute to the prevalence of polyploidy throughout eukaryotes.

  2. Independent Evolution of Winner Traits without Whole Genome Duplication in Dekkera Yeasts

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    Dai, Shao-Xing; Li, Wen-Xing; Zheng, Jun-Juan; Li, Gong-Hua; Huang, Jing-Fei

    2016-01-01

    Dekkera yeasts have often been considered as alternative sources of ethanol production that could compete with S. cerevisiae. The two lineages of yeasts independently evolved traits that include high glucose and ethanol tolerance, aerobic fermentation, and a rapid ethanol fermentation rate. The Saccharomyces yeasts attained these traits mainly through whole genome duplication approximately 100 million years ago (Mya). However, the Dekkera yeasts, which were separated from S. cerevisiae approximately 200 Mya, did not undergo whole genome duplication (WGD) but still occupy a niche similar to S. cerevisiae. Upon analysis of two Dekkera yeasts and five closely related non-WGD yeasts, we found that a massive loss of cis-regulatory elements occurred in an ancestor of the Dekkera yeasts, which led to improved mitochondrial functions similar to the S. cerevisiae yeasts. The evolutionary analysis indicated that genes involved in the transcription and translation process exhibited faster evolution in the Dekkera yeasts. We detected 90 positively selected genes, suggesting that the Dekkera yeasts evolved an efficient translation system to facilitate adaptive evolution. Moreover, we identified that 12 vacuolar H+-ATPase (V-ATPase) function genes that were under positive selection, which assists in developing tolerance to high alcohol and high sugar stress. We also revealed that the enzyme PGK1 is responsible for the increased rate of glycolysis in the Dekkera yeasts. These results provide important insights to understand the independent adaptive evolution of the Dekkera yeasts and provide tools for genetic modification promoting industrial usage. PMID:27152421

  3. Kernel-based whole-genome prediction of complex traits: a review

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    Gota eMorota

    2014-10-01

    Full Text Available Prediction of genetic values has been a focus of applied quantitative genetics since the beginning of the 20th century, with renewed interest following the advent of the era of whole genome-enabled prediction. Opportunities offered by the emergence of high-dimensional genomic data fueled by post-Sanger sequencing technologies, especially molecular markers, have driven researchers to extend Ronald Fisher and Sewall Wright's models to confront new challenges. In particular, kernel methods are gaining consideration as a regression method of choice for genome-enabled prediction. Complex traits are presumably influenced by many genomic regions working in concert with others (clearly so when considering pathways, thus generating interactions. Motivated by this view, a growing number of statistical approaches based on kernels attempt to capture non-additive effects, either parametrically or non-parametrically. This review centers on whole-genome regression using kernel methods applied to a wide range of quantitative traits of agricultural importance in animals and plants. We discuss various kernel-based approaches tailored to capturing total genetic variation, with the aim of arriving at an enhanced predictive performance in the light of available genome annotation information. Connections between prediction machines born in animal breeding, statistics, and machine learning are revisited, and their empirical prediction performance is discussed. Overall, while some encouraging results have been obtained with non-parametric kernels, recovering non-additive genetic variation in a validation dataset remains a challenge in quantitative genetics.

  4. Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.

    Science.gov (United States)

    Thoendel, Matthew; Jeraldo, Patricio R; Greenwood-Quaintance, Kerryl E; Yao, Janet Z; Chia, Nicholas; Hanssen, Arlen D; Abdel, Matthew P; Patel, Robin

    2016-08-01

    Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing. PMID:27237775

  5. Construction of a phylogenetic tree of photosynthetic prokaryotes based on average similarities of whole genome sequences.

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    Soichirou Satoh

    Full Text Available Phylogenetic trees have been constructed for a wide range of organisms using gene sequence information, especially through the identification of orthologous genes that have been vertically inherited. The number of available complete genome sequences is rapidly increasing, and many tools for construction of genome trees based on whole genome sequences have been proposed. However, development of a reasonable method of using complete genome sequences for construction of phylogenetic trees has not been established. We have developed a method for construction of phylogenetic trees based on the average sequence similarities of whole genome sequences. We used this method to examine the phylogeny of 115 photosynthetic prokaryotes, i.e., cyanobacteria, Chlorobi, proteobacteria, Chloroflexi, Firmicutes and nonphotosynthetic organisms including Archaea. Although the bootstrap values for the branching order of phyla were low, probably due to lateral gene transfer and saturated mutation, the obtained tree was largely consistent with the previously reported phylogenetic trees, indicating that this method is a robust alternative to traditional phylogenetic methods.

  6. Organization and evolution of primate centromeric DNA from whole-genome shotgun sequence data.

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    Can Alkan

    2007-09-01

    Full Text Available The major DNA constituent of primate centromeres is alpha satellite DNA. As much as 2%-5% of sequence generated as part of primate genome sequencing projects consists of this material, which is fragmented or not assembled as part of published genome sequences due to its highly repetitive nature. Here, we develop computational methods to rapidly recover and categorize alpha-satellite sequences from previously uncharacterized whole-genome shotgun sequence data. We present an algorithm to computationally predict potential higher-order array structure based on paired-end sequence data and then experimentally validate its organization and distribution by experimental analyses. Using whole-genome shotgun data from the human, chimpanzee, and macaque genomes, we examine the phylogenetic relationship of these sequences and provide further support for a model for their evolution and mutation over the last 25 million years. Our results confirm fundamental differences in the dispersal and evolution of centromeric satellites in the Old World monkey and ape lineages of evolution.

  7. Rediscovery by Whole Genome Sequencing: Classical Mutations and Genome Polymorphisms in Neurospora crassa

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    McCluskey, Kevin; Wiest, Aric E.; Grigoriev, Igor V.; Lipzen, Anna; Martin, Joel; Schackwitz, Wendy; Baker, Scott E.

    2011-06-02

    Classical forward genetics has been foundational to modern biology, and has been the paradigm for characterizing the role of genes in shaping phenotypes for decades. In recent years, reverse genetics has been used to identify the functions of genes, via the intentional introduction of variation and subsequent evaluation in physiological, molecular, and even population contexts. These approaches are complementary and whole genome analysis serves as a bridge between the two. We report in this article the whole genome sequencing of eighteen classical mutant strains of Neurospora crassa and the putative identification of the mutations associated with corresponding mutant phenotypes. Although some strains carry multiple unique nonsynonymous, nonsense, or frameshift mutations, the combined power of limiting the scope of the search based on genetic markers and of using a comparative analysis among the eighteen genomes provides strong support for the association between mutation and phenotype. For ten of the mutants, the mutant phenotype is recapitulated in classical or gene deletion mutants in Neurospora or other filamentous fungi. From thirteen to 137 nonsense mutations are present in each strain and indel sizes are shown to be highly skewed in gene coding sequence. Significant additional genetic variation was found in the eighteen mutant strains, and this variability defines multiple alleles of many genes. These alleles may be useful in further genetic and molecular analysis of known and yet-to-be-discovered functions and they invite new interpretations of molecular and genetic interactions in classical mutant strains.

  8. Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics.

    Science.gov (United States)

    Munchel, Sarah; Hoang, Yen; Zhao, Yue; Cottrell, Joseph; Klotzle, Brandy; Godwin, Andrew K; Koestler, Devin; Beyerlein, Peter; Fan, Jian-Bing; Bibikova, Marina; Chien, Jeremy

    2015-09-22

    Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes. PMID:26305677

  9. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

    Science.gov (United States)

    Puente, Xose S.; Pinyol, Magda; Quesada, Víctor; Conde, Laura; Ordóñez, Gonzalo R.; Villamor, Neus; Escaramis, Georgia; Jares, Pedro; Beà, Sílvia; González-Díaz, Marcos; Bassaganyas, Laia; Baumann, Tycho; Juan, Manel; López-Guerra, Mónica; Colomer, Dolors; Tubío, José M. C.; López, Cristina; Navarro, Alba; Tornador, Cristian; Aymerich, Marta; Rozman, María; Hernández, Jesús M.; Puente, Diana A.; Freije, José M. P.; Velasco, Gloria; Gutiérrez-Fernández, Ana; Costa, Dolors; Carrió, Anna; Guijarro, Sara; Enjuanes, Anna; Hernández, Lluís; Yagüe, Jordi; Nicolás, Pilar; Romeo-Casabona, Carlos M.; Himmelbauer, Heinz; Castillo, Ester; Dohm, Juliane C.; de Sanjosé, Silvia; Piris, Miguel A.; de Alava, Enrique; Miguel, Jesús San; Royo, Romina; Gelpí, Josep L.; Torrents, David; Orozco, Modesto; Pisano, David G.; Valencia, Alfonso; Guigó, Roderic; Bayés, Mónica; Heath, Simon; Gut, Marta; Klatt, Peter; Marshall, John; Raine, Keiran; Stebbings, Lucy A.; Futreal, P. Andrew; Stratton, Michael R.; Campbell, Peter J.; Gut, Ivo; López-Guillermo, Armando; Estivill, Xavier; Montserrat, Emili; López-Otín, Carlos; Campo, Elías

    2012-01-01

    Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution1,2. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes3,4. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer. PMID:21642962

  10. Whole-Genome Mapping as a Novel High-Resolution Typing Tool for Legionella pneumophila

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    Euser, Sjoerd M.; Landman, Fabian; Bruin, Jacob P.; IJzerman, Ed P.; den Boer, Jeroen W.; Schouls, Leo M.

    2015-01-01

    Legionella is the causative agent for Legionnaires' disease (LD) and is responsible for several large outbreaks in the world. More than 90% of LD cases are caused by Legionella pneumophila, and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of L. pneumophila mainly depends on sequence-based typing (SBT). However, studies have shown that in some outbreak situations, SBT does not have sufficient discriminatory power to distinguish between related and nonrelated L. pneumophila isolates. In this study, we used a novel high-resolution typing technique, called whole-genome mapping (WGM), to differentiate between epidemiologically related and nonrelated L. pneumophila isolates. Assessment of the method by various validation experiments showed highly reproducible results, and WGM was able to confirm two well-documented Dutch L. pneumophila outbreaks. Comparison of whole-genome maps of the two outbreaks together with WGMs of epidemiologically nonrelated L. pneumophila isolates showed major differences between the maps, and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of L. pneumophila in real time since the turnaround time from culture to comparison of the L. pneumophila maps is less than 24 h. PMID:26202110

  11. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    Science.gov (United States)

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  12. Characterization of Genomic Variants Associated with Scout and Recruit Behavioral Castes in Honey Bees Using Whole-Genome Sequencing.

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    Bruce R Southey

    Full Text Available Among forager honey bees, scouts seek new resources and return to the colony, enlisting recruits to collect these resources. Differentially expressed genes between these behaviors and genetic variability in scouting phenotypes have been reported. Whole-genome sequencing of 44 Apis mellifera scouts and recruits was undertaken to detect variants and further understand the genetic architecture underlying the behavioral differences between scouts and recruits. The median coverage depth in recruits and scouts was 10.01 and 10.7 X, respectively. Representation of bacterial species among the unmapped reads reflected a more diverse microbiome in scouts than recruits. Overall, 1,412,705 polymorphic positions were analyzed for associations with scouting behavior, and 212 significant (p-value 1000 bp apart from each other. A number of these variants were mapped to ncRNA LOC100578102, solute carrier family 12 member 6-like gene, and LOC100576965 (meprin and TRAF-C homology domain containing gene. Functional categories represented among the genes corresponding to significant variants included: neuronal function, exoskeleton, immune response, salivary gland development, and enzymatic food processing. These categories offer a glimpse into the molecular support to the behaviors of scouts and recruits. The level of association between genomic variants and scouting behavior observed in this study may be linked to the honey bee's genomic plasticity and fluidity of transition between castes.

  13. Characterization of Genomic Variants Associated with Scout and Recruit Behavioral Castes in Honey Bees Using Whole-Genome Sequencing.

    Science.gov (United States)

    Southey, Bruce R; Zhu, Ping; Carr-Markell, Morgan K; Liang, Zhengzheng S; Zayed, Amro; Li, Ruiqiang; Robinson, Gene E; Rodriguez-Zas, Sandra L

    2016-01-01

    Among forager honey bees, scouts seek new resources and return to the colony, enlisting recruits to collect these resources. Differentially expressed genes between these behaviors and genetic variability in scouting phenotypes have been reported. Whole-genome sequencing of 44 Apis mellifera scouts and recruits was undertaken to detect variants and further understand the genetic architecture underlying the behavioral differences between scouts and recruits. The median coverage depth in recruits and scouts was 10.01 and 10.7 X, respectively. Representation of bacterial species among the unmapped reads reflected a more diverse microbiome in scouts than recruits. Overall, 1,412,705 polymorphic positions were analyzed for associations with scouting behavior, and 212 significant (p-value 1000 bp apart from each other. A number of these variants were mapped to ncRNA LOC100578102, solute carrier family 12 member 6-like gene, and LOC100576965 (meprin and TRAF-C homology domain containing gene). Functional categories represented among the genes corresponding to significant variants included: neuronal function, exoskeleton, immune response, salivary gland development, and enzymatic food processing. These categories offer a glimpse into the molecular support to the behaviors of scouts and recruits. The level of association between genomic variants and scouting behavior observed in this study may be linked to the honey bee's genomic plasticity and fluidity of transition between castes. PMID:26784945

  14. High-resolution Whole-Genome Analysis of Skull Base Chordomas Implicates FHIT Loss in Chordoma Pathogenesis12

    Science.gov (United States)

    Diaz, Roberto Jose; Guduk, Mustafa; Romagnuolo, Rocco; Smith, Christian A; Northcott, Paul; Shih, David; Berisha, Fitim; Flanagan, Adrienne; Munoz, David G; Cusimano, Michael D; Pamir, M Necmettin; Rutka, James T

    2012-01-01

    Chordoma is a rare tumor arising in the sacrum, clivus, or vertebrae. It is often not completely resectable and shows a high incidence of recurrence and progression with shortened patient survival and impaired quality of life. Chemotherapeutic options are limited to investigational therapies at present. Therefore, adjuvant therapy for control of tumor recurrence and progression is of great interest, especially in skull base lesions where complete tumor resection is often not possible because of the proximity of cranial nerves. To understand the extent of genetic instability and associated chromosomal and gene losses or gains in skull base chordoma, we undertook whole-genome single-nucleotide polymorphism microarray analysis of flash frozen surgical chordoma specimens, 21 from the clivus and 1 from C1 to C2 vertebrae. We confirm the presence of a deletion at 9p involving CDKN2A, CDKN2B, and MTAP but at a much lower rate (22%) than previously reported for sacral chordoma. At a similar frequency (21%), we found aneuploidy of chromosome 3. Tissue microarray immunohistochemistry demonstrated absent or reduced fragile histidine triad (FHIT) protein expression in 98% of sacral chordomas and 67%of skull base chordomas. Our data suggest that chromosome 3 aneuploidy and epigenetic regulation of FHIT contribute to loss of the FHIT tumor suppressor in chordoma. The finding that FHIT is lost in a majority of chordomas provides new insight into chordoma pathogenesis and points to a potential new therapeutic target for this challenging neoplasm. PMID:23019410

  15. High-resolution Whole-Genome Analysis of Skull Base Chordomas Implicates FHIT Loss in Chordoma Pathogenesis

    Directory of Open Access Journals (Sweden)

    Roberto Jose Diaz

    2012-09-01

    Full Text Available Chordoma is a rare tumor arising in the sacrum, clivus, or vertebrae. It is often not completely resectable and shows a high incidence of recurrence and progression with shortened patient survival and impaired quality of life. Chemotherapeutic options are limited to investigational therapies at present. Therefore, adjuvant therapy for control of tumor recurrence and progression is of great interest, especially in skull base lesions where complete tumor resection is often not possible because of the proximity of cranial nerves. To understand the extent of genetic instability and associated chromosomal and gene losses or gains in skull base chordoma, we undertook whole-genome single-nucleotide polymorphism microarray analysis of flash frozen surgical chordoma specimens, 21 from the clivus and 1 from C1 to C2 vertebrae. We confirm the presence of a deletion at 9p involving CDKN2A, CDKN2B, and MTAP but at a much lower rate (22% than previously reported for sacral chordoma. At a similar frequency (21%, we found aneuploidy of chromosome 3. Tissue microarray immunohistochemistry demonstrated absent or reduced fragile histidine triad (FHIT protein expression in 98% of sacral chordomas and 67%of skull base chordomas. Our data suggest that chromosome 3 aneuploidy and epigenetic regulation of FHIT contribute to loss of the FHIT tumor suppressor in chordoma. The finding that FHIT is lost in a majority of chordomas provides new insight into chordoma pathogenesis and points to a potential new therapeutic target for this challenging neoplasm.

  16. High-resolution whole-genome analysis of skull base chordomas implicates FHIT loss in chordoma pathogenesis.

    Science.gov (United States)

    Diaz, Roberto Jose; Guduk, Mustafa; Romagnuolo, Rocco; Smith, Christian A; Northcott, Paul; Shih, David; Berisha, Fitim; Flanagan, Adrienne; Munoz, David G; Cusimano, Michael D; Pamir, M Necmettin; Rutka, James T

    2012-09-01

    Chordoma is a rare tumor arising in the sacrum, clivus, or vertebrae. It is often not completely resectable and shows a high incidence of recurrence and progression with shortened patient survival and impaired quality of life. Chemotherapeutic options are limited to investigational therapies at present. Therefore, adjuvant therapy for control of tumor recurrence and progression is of great interest, especially in skull base lesions where complete tumor resection is often not possible because of the proximity of cranial nerves. To understand the extent of genetic instability and associated chromosomal and gene losses or gains in skull base chordoma, we undertook whole-genome single-nucleotide polymorphism microarray analysis of flash frozen surgical chordoma specimens, 21 from the clivus and 1 from C1 to C2 vertebrae. We confirm the presence of a deletion at 9p involving CDKN2A, CDKN2B, and MTAP but at a much lower rate (22%) than previously reported for sacral chordoma. At a similar frequency (21%), we found aneuploidy of chromosome 3. Tissue microarray immunohistochemistry demonstrated absent or reduced fragile histidine triad (FHIT) protein expression in 98% of sacral chordomas and 67%of skull base chordomas. Our data suggest that chromosome 3 aneuploidy and epigenetic regulation of FHIT contribute to loss of the FHIT tumor suppressor in chordoma. The finding that FHIT is lost in a majority of chordomas provides new insight into chordoma pathogenesis and points to a potential new therapeutic target for this challenging neoplasm. PMID:23019410

  17. Single site suppressors of a fission yeast temperature-sensitive mutant in cdc48 identified by whole genome sequencing.

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    Irina N Marinova

    Full Text Available The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors.

  18. Gene expression profiling reveals novel protective effects of Aminaphtone on ECV304 endothelial cells.

    Science.gov (United States)

    Salazar, Giulia; Bellocchi, Chiara; Todoerti, Katia; Saporiti, Federica; Piacentini, Luca; Scorza, Raffaella; Colombo, Gualtiero I

    2016-07-01

    Aminaphtone, a drug used in the treatment of chronic venous insufficiency (CVI), showed a remarkable role in the modulation of several vasoactive factors, like endothelin-1 and adhesion molecules. We analysed in vitro the effects of Aminaphtone on whole-genome gene expression and production of different inflammatory proteins. ECV-304 endothelial cells were stimulated with IL-1β 100U/ml in the presence or absence of Aminaphtone 6μg/ml. Gene expression profiles were compared at 1, 3, and 6h after stimulation by microarray. Supernatants of ECV-304 cultures were analysed at 3, 6, 12, and 24h by multiplex ELISA for production of several cytokine and chemokines. Microarrays showed a significant down-regulation at all times of a wide range of inflammatory genes. Aminaphtone appeared also able to modulate the regulation of immune response process (down-regulating cytokine biosynthesis, transcripts involved in lymphocyte differentiation and cell proliferation, and cytokine-cytokine receptor interaction) and to regulate genes engaged in homeostasis, secretion, body fluid levels, response to hypoxia, cell division, and cell-to-cell communication and signalling. Results were confirmed and extended analysing the secretome, which showed significant reduction of the release of 14 cytokines and chemokines. These effects are predicted to be mediated by interaction with different transcription factors. Aminaphtone was able to modulate the expression of inflammatory molecules relevant to the pathogenesis of several conditions in which the endothelial dysfunction is the main player and early event, like scleroderma, lung fibrosis, or atherosclerosis. PMID:27083548

  19. Global genetic response in a cancer cell: self-organized coherent expression dynamics.

    Science.gov (United States)

    Tsuchiya, Masa; Hashimoto, Midori; Takenaka, Yoshiko; Motoike, Ikuko N; Yoshikawa, Kenichi

    2014-01-01

    Understanding the basic mechanism of the spatio-temporal self-control of genome-wide gene expression engaged with the complex epigenetic molecular assembly is one of major challenges in current biological science. In this study, the genome-wide dynamical profile of gene expression was analyzed for MCF-7 breast cancer cells induced by two distinct ErbB receptor ligands: epidermal growth factor (EGF) and heregulin (HRG), which drive cell proliferation and differentiation, respectively. We focused our attention to elucidate how global genetic responses emerge and to decipher what is an underlying principle for dynamic self-control of genome-wide gene expression. The whole mRNA expression was classified into about a hundred groups according to the root mean square fluctuation (rmsf). These expression groups showed characteristic time-dependent correlations, indicating the existence of collective behaviors on the ensemble of genes with respect to mRNA expression and also to temporal changes in expression. All-or-none responses were observed for HRG and EGF (biphasic statistics) at around 10-20 min. The emergence of time-dependent collective behaviors of expression occurred through bifurcation of a coherent expression state (CES). In the ensemble of mRNA expression, the self-organized CESs reveals distinct characteristic expression domains for biphasic statistics, which exhibits notably the presence of criticality in the expression profile as a route for genomic transition. In time-dependent changes in the expression domains, the dynamics of CES reveals that the temporal development of the characteristic domains is characterized as autonomous bistable switch, which exhibits dynamic criticality (the temporal development of criticality) in the genome-wide coherent expression dynamics. It is expected that elucidation of the biophysical origin for such critical behavior sheds light on the underlying mechanism of the control of whole genome. PMID:24831017

  20. dbWGFP: a database and web server of human whole-genome single nucleotide variants and their functional predictions.

    Science.gov (United States)

    Wu, Jiaxin; Wu, Mengmeng; Li, Lianshuo; Liu, Zhuo; Zeng, Wanwen; Jiang, Rui

    2016-01-01

    The recent advancement of the next generation sequencing technology has enabled the fast and low-cost detection of all genetic variants spreading across the entire human genome, making the application of whole-genome sequencing a tendency in the study of disease-causing genetic variants. Nevertheless, there still lacks a repository that collects predictions of functionally damaging effects of human genetic variants, though it has been well recognized that such predictions play a central role in the analysis of whole-genome sequencing data. To fill this gap, we developed a database named dbWGFP (a database and web server of human whole-genome single nucleotide variants and their functional predictions) that contains functional predictions and annotations of nearly 8.58 billion possible human whole-genome single nucleotide variants. Specifically, this database integrates 48 functional predictions calculated by 17 popular computational methods and 44 valuable annotations obtained from various data sources. Standalone software, user-friendly query services and free downloads of this database are available at http://bioinfo.au.tsinghua.edu.cn/dbwgfp. dbWGFP provides a valuable resource for the analysis of whole-genome sequencing, exome sequencing and SNP array data, thereby complementing existing data sources and computational resources in deciphering genetic bases of human inherited diseases. PMID:26989155

  1. Whole-Genome Transcriptional Analysis of Chemolithoautotrophic Thiosulfate Oxidation by Thiobacillus denitrificans Under Aerobic vs. Denitrifying Conditions

    Energy Technology Data Exchange (ETDEWEB)

    Beller, H R; Letain, T E; Chakicherla, A; Kane, S R; Legler, T C; Coleman, M A

    2006-04-22

    Thiobacillus denitrificans is one of the few known obligate chemolithoautotrophic bacteria capable of energetically coupling thiosulfate oxidation to denitrification as well as aerobic respiration. As very little is known about the differential expression of genes associated with ke chemolithoautotrophic functions (such as sulfur-compound oxidation and CO2 fixation) under aerobic versus denitrifying conditions, we conducted whole-genome, cDNA microarray studies to explore this topic systematically. The microarrays identified 277 genes (approximately ten percent of the genome) as differentially expressed using Robust Multi-array Average statistical analysis and a 2-fold cutoff. Genes upregulated (ca. 6- to 150-fold) under aerobic conditions included a cluster of genes associated with iron acquisition (e.g., siderophore-related genes), a cluster of cytochrome cbb3 oxidase genes, cbbL and cbbS (encoding the large and small subunits of form I ribulose 1,5-bisphosphate carboxylase/oxygenase, or RubisCO), and multiple molecular chaperone genes. Genes upregulated (ca. 4- to 95-fold) under denitrifying conditions included nar, nir, and nor genes (associated respectively with nitrate reductase, nitrite reductase, and nitric oxide reductase, which catalyze successive steps of denitrification), cbbM (encoding form II RubisCO), and genes involved with sulfur-compound oxidation (including two physically separated but highly similar copies of sulfide:quinone oxidoreductase and of dsrC, associated with dissimilatory sulfite reductase). Among genes associated with denitrification, relative expression levels (i.e., degree of upregulation with nitrate) tended to decrease in the order nar > nir > nor > nos. Reverse transcription, quantitative PCR analysis was used to validate these trends.

  2. Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates

    KAUST Repository

    Black, PA

    2015-10-24

    Background Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. Methods Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. Results Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30 % to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. Conclusions Our study demonstrated true levels of genetic diversity

  3. Use of Whole Genome Sequencing and Patient Interviews To Link a Case of Sporadic Listeriosis to Consumption of Prepackaged Lettuce.

    Science.gov (United States)

    Jackson, K A; Stroika, S; Katz, L S; Beal, J; Brandt, E; Nadon, C; Reimer, A; Major, B; Conrad, A; Tarr, C; Jackson, B R; Mody, R K

    2016-05-01

    We report on a case of listeriosis in a patient who probably consumed a prepackaged romaine lettuce-containing product recalled for Listeria monocytogenes contamination. Although definitive epidemiological information demonstrating exposure to the specific recalled product was lacking, the patient reported consumption of a prepackaged romaine lettuce-containing product of either the recalled brand or a different brand. A multinational investigation found that patient and food isolates from the recalled product were indistinguishable by pulsed-field gel electrophoresis and were highly related by whole genome sequencing, differing by four alleles by whole genome multilocus sequence typing and by five high-quality single nucleotide polymorphisms, suggesting a common source. To our knowledge, this is the first time prepackaged lettuce has been identified as a likely source for listeriosis. This investigation highlights the power of whole genome sequencing, as well as the continued need for timely and thorough epidemiological exposure data to identify sources of foodborne infections. PMID:27296429

  4. A Bacterial Analysis Platform: An Integrated System for Analysing Bacterial Whole Genome Sequencing Data for Clinical Diagnostics and Surveillance

    DEFF Research Database (Denmark)

    Thomsen, Martin Christen Frølund; Ahrenfeldt, Johanne; Bellod Cisneros, Jose Luis;

    2016-01-01

    and antimicrobial resistance genes. A short printable report for each sample will be provided and an Excel spreadsheet containing all the metadata and a summary of the results for all submitted samples can be downloaded. The pipeline was benchmarked using datasets previously used to test the...... web-based tools we developed a single pipeline for batch uploading of whole genome sequencing data from multiple bacterial isolates. The pipeline will automatically identify the bacterial species and, if applicable, assemble the genome, identify the multilocus sequence type, plasmids, virulence genes...... platform was developed and made publicly available, providing easy-to-use automated analysis of bacterial whole genome sequencing data. The platform may be of immediate relevance as a guide for investigators using whole genome sequencing for clinical diagnostics and surveillance. The platform is freely...

  5. Identification of genomic regions associated with female fertility in Danish Jersey using whole genome sequence data

    DEFF Research Database (Denmark)

    Höglund, Johanna; Guldbrandtsen, Bernt; Lund, Mogens Sandø;

    2015-01-01

    Background: Female fertility is an important trait in cattle breeding programs. In the Nordic countries selection is based on a fertility index (FTI). The fertility index is a weighted combination of four female fertility traits estimated breeding values for number of inseminations per conception...... sires from Denmark with official breeding values for female fertility traits. The association analyses were carried out in two steps: first the cattle genome was scanned for quantitative trait loci using a sire model for FTI using imputed whole genome sequence variants; second the significant...... cows on BTA20, BTA23 and BTA25, IFL for heifers on BTA7 and QTL9-2 on BTA9, NRR for heifers on BTA7 and BTA23, and NRR for cows on BTA23. Conclusion: The genome wide association study presented here revealed 6 genomic regions associated with FTI. Screening these 6 QTL regions for the underlying female...

  6. Hepatitis C virus whole genome sequencing: Current methods/issues and future challenges.

    Science.gov (United States)

    Trémeaux, Pauline; Caporossi, Alban; Thélu, Marie-Ange; Blum, Michael; Leroy, Vincent; Morand, Patrice; Larrat, Sylvie

    2016-10-01

    Therapy for hepatitis C is currently undergoing a revolution. The arrival of new antiviral agents targeting viral proteins reinforces the need for a better knowledge of the viral strains infecting each patient. Hepatitis C virus (HCV) whole genome sequencing provides essential information for precise typing, study of the viral natural history or identification of resistance-associated variants. First performed with Sanger sequencing, the arrival of next-generation sequencing (NGS) has simplified the technical process and provided more detailed data on the nature and evolution of viral quasi-species. We will review the different techniques used for HCV complete genome sequencing and their applications, both before and after the apparition of NGS. The progress brought by new and future technologies will also be discussed, as well as the remaining difficulties, largely due to the genomic variability. PMID:27068766

  7. Whole genome sequencing of emerging multidrug resistant Candida auris isolates in India demonstrates low genetic variation.

    Science.gov (United States)

    Sharma, C; Kumar, N; Pandey, R; Meis, J F; Chowdhary, A

    2016-09-01

    Candida auris is an emerging multidrug resistant yeast that causes nosocomial fungaemia and deep-seated infections. Notably, the emergence of this yeast is alarming as it exhibits resistance to azoles, amphotericin B and caspofungin, which may lead to clinical failure in patients. The multigene phylogeny and amplified fragment length polymorphism typing methods report the C. auris population as clonal. Here, using whole genome sequencing analysis, we decipher for the first time that C. auris strains from four Indian hospitals were highly related, suggesting clonal transmission. Further, all C. auris isolates originated from cases of fungaemia and were resistant to fluconazole (MIC >64 mg/L). PMID:27617098

  8. Practical Value of Food Pathogen Traceability through Building a Whole-Genome Sequencing Network and Database.

    Science.gov (United States)

    Allard, Marc W; Strain, Errol; Melka, David; Bunning, Kelly; Musser, Steven M; Brown, Eric W; Timme, Ruth

    2016-08-01

    The FDA has created a United States-based open-source whole-genome sequencing network of state, federal, international, and commercial partners. The GenomeTrakr network represents a first-of-its-kind distributed genomic food shield for characterizing and tracing foodborne outbreak pathogens back to their sources. The GenomeTrakr network is leading investigations of outbreaks of foodborne illnesses and compliance actions with more accurate and rapid recalls of contaminated foods as well as more effective monitoring of preventive controls for food manufacturing environments. An expanded network would serve to provide an international rapid surveillance system for pathogen traceback, which is critical to support an effective public health response to bacterial outbreaks. PMID:27008877

  9. Multiplex SNP analysis on whole genome amplified DNA from archived dried bloodspots, a validation study

    DEFF Research Database (Denmark)

    Tvedegaard, Kristine C.; Parner, Erik; Hooper, Craig W.;

    Multiplex SNP analysis on whole genome amplified DNA from archived dried bloodspots, a validation study Kristine C. Tvedegaard,1 Erik Parner,1 Craig W. Hooper,2 Jørn Atterman,1 Niels Gregersen3, Poul Thorsen,1 1Institute of Public Health, NANEA at Department of Epidemiology, University of Aarhus...... further development of allele specific primer extension (ASPE) for multiplex SNP analysis based on the Luminex 100 IS platform. It uses isobases (isoC and isoG) and the software MultiCode-PLx platform for data analysis and data handling. We validate the EraGen multicode system in two 6-plex assays used on.......3-100%, repeatability ranged from 99.2-99.7% and robustness ranged from 94.1-99.3%. CONCLUSION: The Multi-Code System is a highly sensitive and specific method for multiplex SNP analysis on WGA DNA from archived dried bloodspots....

  10. Rapid high resolution genotyping of Francisella tularensis by whole genome sequence comparison of annotated genes ("MLST+".

    Directory of Open Access Journals (Sweden)

    Markus H Antwerpen

    Full Text Available The zoonotic disease tularemia is caused by the bacterium Francisella tularensis. This pathogen is considered as a category A select agent with potential to be misused in bioterrorism. Molecular typing based on DNA-sequence like canSNP-typing or MLVA has become the accepted standard for this organism. Due to the organism's highly clonal nature, the current typing methods have reached their limit of discrimination for classifying closely related subpopulations within the subspecies F. tularensis ssp. holarctica. We introduce a new gene-by-gene approach, MLST+, based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains and apply this approach to investigate an epidemic of lethal tularemia among non-human primates in two animal facilities in Germany. Due to the high resolution of MLST+ we are able to demonstrate that three independent clones of this highly infectious pathogen were responsible for these spatially and temporally restricted outbreaks.

  11. Whole-genome sequencing of 234 bulls facilitates mapping of monogenic and complex traits in cattle

    DEFF Research Database (Denmark)

    Daetwyler, Hans D; Capitan, Aurélien; Pausch, Hubert;

    2014-01-01

    The 1000 bull genomes project supports the goal of accelerating the rates of genetic gain in domestic cattle while at the same time considering animal health and welfare by providing the annotated sequence variants and genotypes of key ancestor bulls. In the first phase of the 1000 bull genomes...... project, we sequenced the whole genomes of 234 cattle to an average of 8.3-fold coverage. This sequencing includes data for 129 individuals from the global Holstein-Friesian population, 43 individuals from the Fleckvieh breed and 15 individuals from the Jersey breed. We identified a total of 28.3 million...... variants, with an average of 1.44 heterozygous sites per kilobase for each individual. We demonstrate the use of this database in identifying a recessive mutation underlying embryonic death and a dominant mutation underlying lethal chrondrodysplasia. We also performed genome-wide association studies for...

  12. A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing

    Science.gov (United States)

    Alioto, Tyler S.; Buchhalter, Ivo; Derdak, Sophia; Hutter, Barbara; Eldridge, Matthew D.; Hovig, Eivind; Heisler, Lawrence E.; Beck, Timothy A.; Simpson, Jared T.; Tonon, Laurie; Sertier, Anne-Sophie; Patch, Ann-Marie; Jäger, Natalie; Ginsbach, Philip; Drews, Ruben; Paramasivam, Nagarajan; Kabbe, Rolf; Chotewutmontri, Sasithorn; Diessl, Nicolle; Previti, Christopher; Schmidt, Sabine; Brors, Benedikt; Feuerbach, Lars; Heinold, Michael; Gröbner, Susanne; Korshunov, Andrey; Tarpey, Patrick S.; Butler, Adam P.; Hinton, Jonathan; Jones, David; Menzies, Andrew; Raine, Keiran; Shepherd, Rebecca; Stebbings, Lucy; Teague, Jon W.; Ribeca, Paolo; Giner, Francesc Castro; Beltran, Sergi; Raineri, Emanuele; Dabad, Marc; Heath, Simon C.; Gut, Marta; Denroche, Robert E.; Harding, Nicholas J.; Yamaguchi, Takafumi N.; Fujimoto, Akihiro; Nakagawa, Hidewaki; Quesada, Víctor; Valdés-Mas, Rafael; Nakken, Sigve; Vodák, Daniel; Bower, Lawrence; Lynch, Andrew G.; Anderson, Charlotte L.; Waddell, Nicola; Pearson, John V.; Grimmond, Sean M.; Peto, Myron; Spellman, Paul; He, Minghui; Kandoth, Cyriac; Lee, Semin; Zhang, John; Létourneau, Louis; Ma, Singer; Seth, Sahil; Torrents, David; Xi, Liu; Wheeler, David A.; López-Otín, Carlos; Campo, Elías; Campbell, Peter J.; Boutros, Paul C.; Puente, Xose S.; Gerhard, Daniela S.; Pfister, Stefan M.; McPherson, John D.; Hudson, Thomas J.; Schlesner, Matthias; Lichter, Peter; Eils, Roland; Jones, David T. W.; Gut, Ivo G.

    2015-01-01

    As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy. PMID:26647970

  13. The effect of whole genome amplification on samples originating from more than one donor

    DEFF Research Database (Denmark)

    Thacker, C.R.; Balogh, M.K.; Børsting, Claus;

    2006-01-01

    In this study, the GenomiPhi(TM) DNA Amplification Kit (Amersham Biosciences) was used to investigate the potential of whole genome amplification (WGA) when considering samples originating from more than one donor. DNA was extracted from blood samples, quantified and normalised before being mixed...... ratios were found to match the expected peak ratios regardless of the starting concentration of DNA. With samples mixed in the ratio of 1:7 and 1:15, and when the concentration of starting material was at the manufacturer's lower limit, too few minor component peaks were found to allow for statistical...... analysis. With an initial template exceeding 1 ng/[mu]L there was an increase in problems associated with profile interpretation but the results obtained indicated that mixture proportions could be quantifiably maintained...

  14. A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome.

    Science.gov (United States)

    Chapman, Jarrod A; Mascher, Martin; Buluç, Aydın; Barry, Kerrie; Georganas, Evangelos; Session, Adam; Strnadova, Veronika; Jenkins, Jerry; Sehgal, Sunish; Oliker, Leonid; Schmutz, Jeremy; Yelick, Katherine A; Scholz, Uwe; Waugh, Robbie; Poland, Jesse A; Muehlbauer, Gary J; Stein, Nils; Rokhsar, Daniel S

    2015-01-01

    Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population. PMID:25637298

  15. Identification of cancer-driver genes in focal genomic alterations from whole genome sequencing data.

    Science.gov (United States)

    Jang, Ho; Hur, Youngmi; Lee, Hyunju

    2016-01-01

    DNA copy number alterations (CNAs) are the main genomic events that occur during the initiation and development of cancer. Distinguishing driver aberrant regions from passenger regions, which might contain candidate target genes for cancer therapies, is an important issue. Several methods for identifying cancer-driver genes from multiple cancer patients have been developed for single nucleotide polymorphism (SNP) arrays. However, for NGS data, methods for the SNP array cannot be directly applied because of different characteristics of NGS such as higher resolutions of data without predefined probes and incorrectly mapped reads to reference genomes. In this study, we developed a wavelet-based method for identification of focal genomic alterations for sequencing data (WIFA-Seq). We applied WIFA-Seq to whole genome sequencing data from glioblastoma multiforme, ovarian serous cystadenocarcinoma and lung adenocarcinoma, and identified focal genomic alterations, which contain candidate cancer-related genes as well as previously known cancer-driver genes. PMID:27156852

  16. Whole genome sequencing provides an unambiguous link between Salmonella Dublin outbreak strain and a historical isolate.

    Science.gov (United States)

    Mohammed, M; Delappe, N; O'Connor, J; McKeown, P; Garvey, P; Cormican, M

    2016-02-01

    Salmonella enterica subsp. enterica serovar Dublin is an uncommon cause of human salmonellosis; however, a relatively high proportion of cases are associated with invasive disease. The serotype is associated with cattle. A geographically diffuse outbreak of S. Dublin involving nine patients occurred in Ireland in 2013. The source of infection was not identified. Typing of outbreak associated isolates by pulsed-field gel electrophoresis (PFGE) was of limited value because PFGE has limited discriminatory power for S. Dublin. Whole genome sequencing (WGS) showed conclusively that the isolates were closely related to each other, to an apparently unrelated isolate from 2011 and distinct from other isolates that were not readily distinguishable by PFGE. PMID:26165314

  17. Mapping genomic features to functional traits through microbial whole genome sequences.

    Science.gov (United States)

    Zhang, Wei; Zeng, Erliang; Liu, Dan; Jones, Stuart E; Emrich, Scott

    2014-01-01

    Recently, the utility of trait-based approaches for microbial communities has been identified. Increasing availability of whole genome sequences provide the opportunity to explore the genetic foundations of a variety of functional traits. We proposed a machine learning framework to quantitatively link the genomic features with functional traits. Genes from bacteria genomes belonging to different functional traits were grouped to Cluster of Orthologs (COGs), and were used as features. Then, TF-IDF technique from the text mining domain was applied to transform the data to accommodate the abundance and importance of each COG. After TF-IDF processing, COGs were ranked using feature selection methods to identify their relevance to the functional trait of interest. Extensive experimental results demonstrated that functional trait related genes can be detected using our method. Further, the method has the potential to provide novel biological insights. PMID:24989863

  18. Genomic Epidemiology: Whole-Genome-Sequencing-Powered Surveillance and Outbreak Investigation of Foodborne Bacterial Pathogens.

    Science.gov (United States)

    Deng, Xiangyu; den Bakker, Henk C; Hendriksen, Rene S

    2016-01-01

    As we are approaching the twentieth anniversary of PulseNet, a network of public health and regulatory laboratories that has changed the landscape of foodborne illness surveillance through molecular subtyping, public health microbiology is undergoing another transformation brought about by so-called next-generation sequencing (NGS) technologies that have made whole-genome sequencing (WGS) of foodborne bacterial pathogens a realistic and superior alternative to traditional subtyping methods. Routine, real-time, and widespread application of WGS in food safety and public health is on the horizon. Technological, operational, and policy challenges are still present and being addressed by an international and multidisciplinary community of researchers, public health practitioners, and other stakeholders. PMID:26772415

  19. How accurately is ncRNA aligned within whole-genome multiple alignments?

    Directory of Open Access Journals (Sweden)

    Ruzzo Walter L

    2007-10-01

    Full Text Available Abstract Background Multiple alignment of homologous DNA sequences is of great interest to biologists since it provides a window into evolutionary processes. At present, the accuracy of whole-genome multiple alignments, particularly in noncoding regions, has not been thoroughly evaluated. Results We evaluate the alignment accuracy of certain noncoding regions using noncoding RNA alignments from Rfam as a reference. We inspect the MULTIZ 17-vertebrate alignment from the UCSC Genome Browser for all the human sequences in the Rfam seed alignments. In particular, we find 638 instances of chimeric and partial alignments to human noncoding RNA elements, of which at least 225 can be improved by straightforward means. As a byproduct of our procedure, we predict many novel instances of known ncRNA families that are suggested by the alignment. Conclusion MULTIZ does a fairly accurate job of aligning these genomes in these difficult regions. However, our experiments indicate that better alignments exist in some regions.

  20. Diversity through duplication: whole-genome sequencing reveals novel gene retrocopies in the human population.

    Science.gov (United States)

    Richardson, Sandra R; Salvador-Palomeque, Carmen; Faulkner, Geoffrey J

    2014-05-01

    Gene retrocopies are generated by reverse transcription and genomic integration of mRNA. As such, retrocopies present an important exception to the central dogma of molecular biology, and have substantially impacted the functional landscape of the metazoan genome. While an estimated 8,000-17,000 retrocopies exist in the human genome reference sequence, the extent of variation between individuals in terms of retrocopy content has remained largely unexplored. Three recent studies by Abyzov et al., Ewing et al. and Schrider et al. have exploited 1,000 Genomes Project Consortium data, as well as other sources of whole-genome sequencing data, to uncover novel gene retrocopies. Here, we compare the methods and results of these three studies, highlight the impact of retrocopies in human diversity and genome evolution, and speculate on the potential for somatic gene retrocopies to impact cancer etiology and genetic diversity among individual neurons in the mammalian brain. PMID:24615986

  1. Clinical decision support for whole genome sequence information leveraging a service-oriented architecture: a prototype.

    Science.gov (United States)

    Welch, Brandon M; Rodriguez-Loya, Salvador; Eilbeck, Karen; Kawamoto, Kensaku

    2014-01-01

    Whole genome sequence (WGS) information could soon be routinely available to clinicians to support the personalized care of their patients. At such time, clinical decision support (CDS) integrated into the clinical workflow will likely be necessary to support genome-guided clinical care. Nevertheless, developing CDS capabilities for WGS information presents many unique challenges that need to be overcome for such approaches to be effective. In this manuscript, we describe the development of a prototype CDS system that is capable of providing genome-guided CDS at the point of care and within the clinical workflow. To demonstrate the functionality of this prototype, we implemented a clinical scenario of a hypothetical patient at high risk for Lynch Syndrome based on his genomic information. We demonstrate that this system can effectively use service-oriented architecture principles and standards-based components to deliver point of care CDS for WGS information in real-time. PMID:25954430

  2. Identification of cancer-driver genes in focal genomic alterations from whole genome sequencing data

    Science.gov (United States)

    Jang, Ho; Hur, Youngmi; Lee, Hyunju

    2016-01-01

    DNA copy number alterations (CNAs) are the main genomic events that occur during the initiation and development of cancer. Distinguishing driver aberrant regions from passenger regions, which might contain candidate target genes for cancer therapies, is an important issue. Several methods for identifying cancer-driver genes from multiple cancer patients have been developed for single nucleotide polymorphism (SNP) arrays. However, for NGS data, methods for the SNP array cannot be directly applied because of different characteristics of NGS such as higher resolutions of data without predefined probes and incorrectly mapped reads to reference genomes. In this study, we developed a wavelet-based method for identification of focal genomic alterations for sequencing data (WIFA-Seq). We applied WIFA-Seq to whole genome sequencing data from glioblastoma multiforme, ovarian serous cystadenocarcinoma and lung adenocarcinoma, and identified focal genomic alterations, which contain candidate cancer-related genes as well as previously known cancer-driver genes. PMID:27156852

  3. A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing.

    Science.gov (United States)

    Alioto, Tyler S; Buchhalter, Ivo; Derdak, Sophia; Hutter, Barbara; Eldridge, Matthew D; Hovig, Eivind; Heisler, Lawrence E; Beck, Timothy A; Simpson, Jared T; Tonon, Laurie; Sertier, Anne-Sophie; Patch, Ann-Marie; Jäger, Natalie; Ginsbach, Philip; Drews, Ruben; Paramasivam, Nagarajan; Kabbe, Rolf; Chotewutmontri, Sasithorn; Diessl, Nicolle; Previti, Christopher; Schmidt, Sabine; Brors, Benedikt; Feuerbach, Lars; Heinold, Michael; Gröbner, Susanne; Korshunov, Andrey; Tarpey, Patrick S; Butler, Adam P; Hinton, Jonathan; Jones, David; Menzies, Andrew; Raine, Keiran; Shepherd, Rebecca; Stebbings, Lucy; Teague, Jon W; Ribeca, Paolo; Giner, Francesc Castro; Beltran, Sergi; Raineri, Emanuele; Dabad, Marc; Heath, Simon C; Gut, Marta; Denroche, Robert E; Harding, Nicholas J; Yamaguchi, Takafumi N; Fujimoto, Akihiro; Nakagawa, Hidewaki; Quesada, Víctor; Valdés-Mas, Rafael; Nakken, Sigve; Vodák, Daniel; Bower, Lawrence; Lynch, Andrew G; Anderson, Charlotte L; Waddell, Nicola; Pearson, John V; Grimmond, Sean M; Peto, Myron; Spellman, Paul; He, Minghui; Kandoth, Cyriac; Lee, Semin; Zhang, John; Létourneau, Louis; Ma, Singer; Seth, Sahil; Torrents, David; Xi, Liu; Wheeler, David A; López-Otín, Carlos; Campo, Elías; Campbell, Peter J; Boutros, Paul C; Puente, Xose S; Gerhard, Daniela S; Pfister, Stefan M; McPherson, John D; Hudson, Thomas J; Schlesner, Matthias; Lichter, Peter; Eils, Roland; Jones, David T W; Gut, Ivo G

    2015-01-01

    As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼ 100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy. PMID:26647970

  4. Rapid determination of anti-tuberculosis drug resistance from whole-genome sequences

    KAUST Repository

    Coll, Francesc

    2015-05-27

    Mycobacterium tuberculosis drug resistance (DR) challenges effective tuberculosis disease control. Current molecular tests examine limited numbers of mutations, and although whole genome sequencing approaches could fully characterise DR, data complexity has restricted their clinical application. A library (1,325 mutations) predictive of DR for 15 anti-tuberculosis drugs was compiled and validated for 11 of them using genomic-phenotypic data from 792 strains. A rapid online ‘TB-Profiler’ tool was developed to report DR and strain-type profiles directly from raw sequences. Using our DR mutation library, in silico diagnostic accuracy was superior to some commercial diagnostics and alternative databases. The library will facilitate sequence-based drug-susceptibility testing.

  5. Overview of HBV whole genome data in public repositories and the Chinese HBV reference sequences

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The number of Hepatitis B virus (HBV) whole genomic sequences in public nucleotide databases (GenBank, EMBL, and DDBJ) had reached 866 by January 1, 2007. Coming from 46 countries and regions, these sequences were categorized as eight genotypes (A-H). With the statistical and phylogenetic analysis on all available complete genomic data of HBV, we here present an overview of HBV sequences in public databases. From all registered 229 HBV genomes in Chinese regions as well as 59 sequencing data from our research group, we report the establishment of reference sequences of HBV strains prevailing in China. These analyses provide clues for the effects of HBV genotypes in host clinical progressions, geographic distribution of the infection, and the viral evolutionary history. Moreover, the viral sequence reference would be helpful in the identification of various HBV mutations. Based on the analysis of various public databases,we suggest that the Chinese HBV database with the clinical information should be constructed.

  6. A strategic stakeholder approach for addressing further analysis requests in whole genome sequencing research.

    Science.gov (United States)

    Thornock, Bradley Steven O

    2016-01-01

    Whole genome sequencing (WGS) can be a cost-effective and efficient means of diagnosis for some children, but it also raises a number of ethical concerns. One such concern is how researchers derive and communicate results from WGS, including future requests for further analysis of stored sequences. The purpose of this paper is to think about what is at stake, and for whom, in any solution that is developed to deal with such requests. To accomplish this task, this paper will utilize stakeholder theory, a common method used in business ethics. Several scenarios that connect stakeholder concerns and WGS will also posited and analyzed. This paper concludes by developing criteria composed of a series of questions that researchers can answer in order to more effectively address requests for further analysis of stored sequences. PMID:27091475

  7. Bioinformatics Workflow for Clinical Whole Genome Sequencing at Partners HealthCare Personalized Medicine

    Directory of Open Access Journals (Sweden)

    Ellen A. Tsai

    2016-02-01

    Full Text Available Effective implementation of precision medicine will be enhanced by a thorough understanding of each patient’s genetic composition to better treat his or her presenting symptoms or mitigate the onset of disease. This ideally includes the sequence information of a complete genome for each individual. At Partners HealthCare Personalized Medicine, we have developed a clinical process for whole genome sequencing (WGS with application in both healthy individuals and those with disease. In this manuscript, we will describe our bioinformatics strategy to efficiently process and deliver genomic data to geneticists for clinical interpretation. We describe the handling of data from FASTQ to the final variant list for clinical review for the final report. We will also discuss our methodology for validating this workflow and the cost implications of running WGS.

  8. Phylogenetics and differentiation of Salmonella Newport lineages by whole genome sequencing.

    Directory of Open Access Journals (Sweden)

    Guojie Cao

    Full Text Available Salmonella Newport has ranked in the top three Salmonella serotypes associated with foodborne outbreaks from 1995 to 2011 in the United States. In the current study, we selected 26 S. Newport strains isolated from diverse sources and geographic locations and then conducted 454 shotgun pyrosequencing procedures to obtain 16-24 × coverage of high quality draft genomes for each strain. Comparative genomic analysis of 28 S. Newport strains (including 2 reference genomes and 15 outgroup genomes identified more than 140,000 informative SNPs. A resulting phylogenetic tree consisted of four sublineages and indicated that S. Newport had a clear geographic structure. Strains from Asia were divergent from those from the Americas. Our findings demonstrated that analysis using whole genome sequencing data resulted in a more accurate picture of phylogeny compared to that using single genes or small sets of genes. We selected loci around the mutS gene of S. Newport to differentiate distinct lineages, including those between invH and mutS genes at the 3' end of Salmonella Pathogenicity Island 1 (SPI-1, ste fimbrial operon, and Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR associated-proteins (cas. These genes in the outgroup genomes held high similarity with either S. Newport Lineage II or III at the same loci. S. Newport Lineages II and III have different evolutionary histories in this region and our data demonstrated genetic flow and homologous recombination events around mutS. The findings suggested that S. Newport Lineages II and III diverged early in the serotype evolution and have evolved largely independently. Moreover, we identified genes that could delineate sublineages within the phylogenetic tree and that could be used as potential biomarkers for trace-back investigations during outbreaks. Thus, whole genome sequencing data enabled us to better understand the genetic background of pathogenicity and evolutionary history of S

  9. A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis.

    Directory of Open Access Journals (Sweden)

    Peter G Kroth

    Full Text Available BACKGROUND: Diatoms are unicellular algae responsible for approximately 20% of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids. METHODOLOGY/PRINCIPAL FINDINGS: The whole genome sequence of the diatom Phaeodactylum tricornutum has recently been completed. We identified and annotated genes for enzymes involved in carbohydrate pathways based on extensive EST support and comparison to the whole genome sequence of a second diatom, Thalassiosira pseudonana. Protein localization to mitochondria was predicted based on identified similarities to mitochondrial localization motifs in other eukaryotes, whereas protein localization to plastids was based on the presence of signal peptide motifs in combination with plastid localization motifs previously shown to be required in diatoms. We identified genes potentially involved in a C4-like photosynthesis in P. tricornutum and, on the basis of sequence-based putative localization of relevant proteins, discuss possible differences in carbon concentrating mechanisms and CO(2 fixation between the two diatoms. We also identified genes encoding enzymes involved in photorespiration with one interesting exception: glycerate kinase was not found in either P. tricornutum or T. pseudonana. Various Calvin cycle enzymes were found in up to five different isoforms, distributed between plastids, mitochondria and the cytosol. Diatoms store energy either as lipids or as chrysolaminaran (a beta-1,3-glucan outside of the plastids. We identified various beta-glucanases and large membrane-bound glucan synthases. Interestingly most of the glucanases appear to contain C-terminal anchor domains that may attach the enzymes to membranes. CONCLUSIONS/SIGNIFICANCE: Here we present a detailed synthesis of carbohydrate metabolism in diatoms based on the genome sequences of Thalassiosira pseudonana and Phaeodactylum tricornutum

  10. Navigating Microbiological Food Safety in the Era of Whole-Genome Sequencing.

    Science.gov (United States)

    Ronholm, J; Nasheri, Neda; Petronella, Nicholas; Pagotto, Franco

    2016-10-01

    The epidemiological investigation of a foodborne outbreak, including identification of related cases, source attribution, and development of intervention strategies, relies heavily on the ability to subtype the etiological agent at a high enough resolution to differentiate related from nonrelated cases. Historically, several different molecular subtyping methods have been used for this purpose; however, emerging techniques, such as single nucleotide polymorphism (SNP)-based techniques, that use whole-genome sequencing (WGS) offer a resolution that was previously not possible. With WGS, unlike traditional subtyping methods that lack complete information, data can be used to elucidate phylogenetic relationships and disease-causing lineages can be tracked and monitored over time. The subtyping resolution and evolutionary context provided by WGS data allow investigators to connect related illnesses that would be missed by traditional techniques. The added advantage of data generated by WGS is that these data can also be used for secondary analyses, such as virulence gene detection, antibiotic resistance gene profiling, synteny comparisons, mobile genetic element identification, and geographic attribution. In addition, several software packages are now available to generate in silico results for traditional molecular subtyping methods from the whole-genome sequence, allowing for efficient comparison with historical databases. Metagenomic approaches using next-generation sequencing have also been successful in the detection of nonculturable foodborne pathogens. This review addresses state-of-the-art techniques in microbial WGS and analysis and then discusses how this technology can be used to help support food safety investigations. Retrospective outbreak investigations using WGS are presented to provide organism-specific examples of the benefits, and challenges, associated with WGS in comparison to traditional molecular subtyping techniques. PMID:27559074

  11. Light whole genome sequence for SNP discovery across domestic cat breeds

    Directory of Open Access Journals (Sweden)

    Driscoll Carlos

    2010-06-01

    Full Text Available Abstract Background The domestic cat has offered enormous genomic potential in the veterinary description of over 250 hereditary disease models as well as the occurrence of several deadly feline viruses (feline leukemia virus -- FeLV, feline coronavirus -- FECV, feline immunodeficiency virus - FIV that are homologues to human scourges (cancer, SARS, and AIDS respectively. However, to realize this bio-medical potential, a high density single nucleotide polymorphism (SNP map is required in order to accomplish disease and phenotype association discovery. Description To remedy this, we generated 3,178,297 paired fosmid-end Sanger sequence reads from seven cats, and combined these data with the publicly available 2X cat whole genome sequence. All sequence reads were assembled together to form a 3X whole genome assembly allowing the discovery of over three million SNPs. To reduce potential false positive SNPs due to the low coverage assembly, a low upper-limit was placed on sequence coverage and a high lower-limit on the quality of the discrepant bases at a potential variant site. In all domestic cats of different breeds: female Abyssinian, female American shorthair, male Cornish Rex, female European Burmese, female Persian, female Siamese, a male Ragdoll and a female African wildcat were sequenced lightly. We report a total of 964 k common SNPs suitable for a domestic cat SNP genotyping array and an additional 900 k SNPs detected between African wildcat and domestic cats breeds. An empirical sampling of 94 discovered SNPs were tested in the sequenced cats resulting in a SNP validation rate of 99%. Conclusions These data provide a large collection of mapped feline SNPs across the cat genome that will allow for the development of SNP genotyping platforms for mapping feline diseases.

  12. Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks.

    Science.gov (United States)

    Rusconi, Brigida; Sanjar, Fatemeh; Koenig, Sara S K; Mammel, Mark K; Tarr, Phillip I; Eppinger, Mark

    2016-01-01

    Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and

  13. Whole genome investigation of a divergent clade of the pathogen Streptococcus suis

    Directory of Open Access Journals (Sweden)

    Abiyad eBaig

    2015-11-01

    Full Text Available Streptococcus suis is a major porcine and zoonotic pathogen responsible for significant economic losses in the pig industry and an increasing number of human cases. Multiple isolates of S. suis show marked genomic diversity. Here we report the analysis of whole genome sequences of nine pig isolates that caused disease typical of S. suis and had phenotypic characteristics of S. suis, but their genomes were divergent from those of many other S. suis isolates. Comparison of protein sequences predicted from divergent genomes with those from normal S. suis reduced the size of core genome from 793 to only 397 genes. Divergence was clear if phylogenetic analysis was performed on reduced core genes and MLST alleles. Phylogenies based on certain other genes (16S rRNA, sodA, recN and cpn60 did not show divergence for all isolates, suggesting recombination between some divergent isolates with normal S. suis for these genes. Indeed, there is evidence of recent recombination between the divergent and normal S. suis genomes for 249 of 397 core genes. In addition, phylogenetic analysis based on the 16S rRNA gene and 132 genes that were conserved between the divergent isolates and representatives of the broader Streptococcus genus showed that divergent isolates were more closely related to S. suis. Six out of nine divergent isolates possessed a S. suis-like capsule region with variation in capsular gene sequences but the remaining three did not have a discrete capsule locus. The majority (40/70, of virulence-associated genes in normal S. suis were present in the divergent genomes. Overall, the divergent isolates extend the current diversity of S. suis species but the phenotypic similarities and the large amount of gene exchange with normal S. suis gives insufficient evidence to assign these isolates to a new species or subspecies. Further sampling and whole genome analysis of more isolates is warranted to understand the diversity of the species.

  14. Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks

    Science.gov (United States)

    Rusconi, Brigida; Sanjar, Fatemeh; Koenig, Sara S. K.; Mammel, Mark K.; Tarr, Phillip I.; Eppinger, Mark

    2016-01-01

    Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and

  15. Evaluation of whole genome sequencing for outbreak detection of Salmonella enterica.

    Directory of Open Access Journals (Sweden)

    Pimlapas Leekitcharoenphon

    Full Text Available Salmonella enterica is a common cause of minor and large food borne outbreaks. To achieve successful and nearly 'real-time' monitoring and identification of outbreaks, reliable sub-typing is essential. Whole genome sequencing (WGS shows great promises for using as a routine epidemiological typing tool. Here we evaluate WGS for typing of S. Typhimurium including different approaches for analyzing and comparing the data. A collection of 34 S. Typhimurium isolates was sequenced. This consisted of 18 isolates from six outbreaks and 16 epidemiologically unrelated background strains. In addition, 8 S. Enteritidis and 5 S. Derby were also sequenced and used for comparison. A number of different bioinformatics approaches were applied on the data; including pan-genome tree, k-mer tree, nucleotide difference tree and SNP tree. The outcome of each approach was evaluated in relation to the association of the isolates to specific outbreaks. The pan-genome tree clustered 65% of the S. Typhimurium isolates according to the pre-defined epidemiology, the k-mer tree 88%, the nucleotide difference tree 100% and the SNP tree 100% of the strains within S. Typhimurium. The resulting outcome of the four phylogenetic analyses were also compared to PFGE revealing that WGS typing achieved the greater performance than the traditional method. In conclusion, for S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS. These approaches were also superior to the more classical typing method, PFGE. Our study also indicates that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.

  16. Insights on cryoprotectant toxicity from gene expression profiling of endothelial cells exposed to ethylene glycol.

    Science.gov (United States)

    Cordeiro, Rui Martins; Stirling, Soren; Fahy, Gregory M; de Magalhães, João Pedro

    2015-12-01

    Cryopreservation consists of preserving living cells or tissues generally at -80 °C or below and has many current applications in cell and tissue banking, and future potential for organ banking. Cryoprotective agents such as ethylene glycol (EG) are required for successful cryopreservation of most living systems, but have toxic side effects whose mechanisms remain largely unknown. In this work, we investigated the mechanisms of toxicity of ethylene glycol in human umbilical vein endothelial cells (HUVECs) as a model of the vascular endothelium in perfused organs. Exposing cells to 60% v/v EG for 2 h at 4 °C resulted in only a slight decrease in subsequent cell growth, suggesting only modest toxicity of EG for this cell type. Gene expression analysis with whole genome microarrays revealed signatures indicative of a generalized stress response at 24 h after EG exposure and a trend toward partial recovery at 72 h. The observed changes involved signalling pathways, glycoproteins, and genes involved in extracellular and transmembrane functions, the latter suggesting potential effects of ethylene glycol on membranes. These results continue to develop a new paradigm for understanding cryoprotectant toxicity and reveal molecular signatures helpful for future experiments in more completely elucidating the toxic effects of ethylene glycol in vascular endothelial cells and other cell types. PMID:26471925

  17. Whole genome sequencing of sugarbeet and identification of differentially expressed genes regulating beet curly top resistance

    Science.gov (United States)

    The genome of KDH13 doubled haploid line has been sequenced using Illumina HiSeq2000 NGS platform. This line (PI663862) was released by USDA-ARS as a genetic stock resistant to beet curly top. Sequencing of a standard paired end and a 2kb-insert mate-pair genomic libraries, constructed from a leaf ...

  18. Transcriptome sequencing and whole genome expression profiling of chrysanthemum under dehydration stress

    OpenAIRE

    Xu, Yanjie; Gao, Shan; Yang, Yingjie; Huang, Mingyun; Cheng, Lina; Wei, Qian; Fei, Zhangjun; Gao, Junping; Hong, Bo

    2013-01-01

    Background Chrysanthemum is one of the most important ornamental crops in the world and drought stress seriously limits its production and distribution. In order to generate a functional genomics resource and obtain a deeper understanding of the molecular mechanisms regarding chrysanthemum responses to dehydration stress, we performed large-scale transcriptome sequencing of chrysanthemum plants under dehydration stress using the Illumina sequencing technology. Results Two cDNA libraries const...

  19. Whole-genome sequencing identifies emergence of a quinolone resistance mutation in a case of Stenotrophomonas maltophilia bacteremia.

    Science.gov (United States)

    Pak, Theodore R; Altman, Deena R; Attie, Oliver; Sebra, Robert; Hamula, Camille L; Lewis, Martha; Deikus, Gintaras; Newman, Leah C; Fang, Gang; Hand, Jonathan; Patel, Gopi; Wallach, Fran; Schadt, Eric E; Huprikar, Shirish; van Bakel, Harm; Kasarskis, Andrew; Bashir, Ali

    2015-11-01

    Whole-genome sequences for Stenotrophomonas maltophilia serial isolates from a bacteremic patient before and after development of levofloxacin resistance were assembled de novo and differed by one single-nucleotide variant in smeT, a repressor for multidrug efflux operon smeDEF. Along with sequenced isolates from five contemporaneous cases, they displayed considerable diversity compared against all published complete genomes. Whole-genome sequencing and complete assembly can conclusively identify resistance mechanisms emerging in S. maltophilia strains during clinical therapy. PMID:26324280

  20. Regulatory divergence of homeologous Atlantic salmon elovl5 genes following the salmonid-specific whole-genome duplication.

    Science.gov (United States)

    Carmona-Antoñanzas, Greta; Zheng, Xiaozhong; Tocher, Douglas R; Leaver, Michael J

    2016-10-10

    Fatty acyl elongase 5 (elovl5) is a critical enzyme in the vertebrate biosynthetic pathway which produces the physiologically essential long-chain polyunsaturated fatty acids (LC-PUFA), docosahexenoic acid (DHA), and eicosapentenoic acid (EPA) from 18 carbon fatty acids precursors. In contrast to most other vertebrates, Atlantic salmon possess two copies of elovl5 (elovl5a and elovl5b) as a result of a whole-genome duplication (WGD) which occurred at the base of the salmonid lineage. WGDs have had a major influence on vertebrate evolution, providing extra genetic material, enabling neofunctionalization to accelerate adaptation and speciation. However, little is known about the mechanisms by which such duplicated homeologous genes diverge. Here we show that homeologous Atlantic salmon elovl5a and elovl5b genes have been asymmetrically colonised by transposon-like elements. Identical locations and identities of insertions are also present in the rainbow trout duplicate elovl5 genes, but not in the nearest extant representative preduplicated teleost, the northern pike. Both elovl5 salmon duplicates possessed conserved regulatory elements that promoted Srebp1- and Srebp2-dependent transcription, and differences in the magnitude of Srebp response between promoters could be attributed to a tandem duplication of SRE and NF-Y cofactor binding sites in elovl5b. Furthermore, an insertion in the promoter region of elovl5a confers responsiveness to Lxr/Rxr transcriptional activation. Our results indicate that most, but not all, transposon mobilisation into elovl5 genes occurred after the split from the common ancestor of pike and salmon, but before more recent salmonid speciations, and that divergence of elovl5 regulatory regions have enabled neofuntionalization by promoting differential expression of these homeologous genes. PMID:27374149

  1. Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly

    DEFF Research Database (Denmark)

    Li, Yingrui; Zheng, Hancheng; Luo, Ruibang; Wu, Honglong; Zhu, Hongmei; Li, Ruiqiang; Cao, Hongzhi; Wu, Boxin; Huang, Shujia; Shao, Haojing; Ma, Hanzhou; Zhang, Fan; Feng, Shuijian; Zhang, Wei; Du, Hongli; Tian, Geng; Li, Jingxiang; Zhang, Xiuqing; Li, Songgang; Bolund, Lars; Kristiansen, Karsten; de Smith, Adam J; Blakemore, Alexandra I F; Coin, Lachlan J M; Yang, Huanming; Wang, Jian; Wang, Jun

    2011-01-01

    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...

  2. Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing

    DEFF Research Database (Denmark)

    Bjorn-Mortensen, K; Zallet, J; Lillebaek, T;

    2015-01-01

    Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such as Mycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with...

  3. Whole genome sequencing of Candidatus Liberibacter asiaticus strain A4 from Guangdong, China, and strain HHCA from California

    Science.gov (United States)

    “Candidatus Liberibacter asiaticus” is associated with citrus Huanglongbing (HLB) in both China and the United States. While HLB has been known for over a century in Guangdong, China, the disease was first discovered in California in 2012. To better study the “old” and “new” HLBs, whole genomes of “...

  4. Understanding the Quorum-Sensing Bacterium Pantoea stewartii Strain M009 with Whole-Genome Sequencing Analysis.

    Science.gov (United States)

    Tan, Wen-Si; Chang, Chien-Yi; Yin, Wai-Fong; Chan, Kok-Gan

    2015-01-01

    Pantoea stewartii is known to be the causative agent of Stewart's wilt, which usually affects sweet corn (Zea mays) with the corn flea beetle as the transmission vector. In this work, we present the whole-genome sequence of Pantoea stewartii strain M009, isolated from a Malaysian tropical rainforest waterfall. PMID:25635007

  5. Understanding the Quorum-Sensing Bacterium Pantoea stewartii Strain M009 with Whole-Genome Sequencing Analysis

    OpenAIRE

    Tan, Wen-Si; Chang, Chien-Yi; Yin, Wai-Fong; Chan, Kok-Gan

    2015-01-01

    Pantoea stewartii is known to be the causative agent of Stewart’s wilt, which usually affects sweet corn (Zea mays) with the corn flea beetle as the transmission vector. In this work, we present the whole-genome sequence of Pantoea stewartii strain M009, isolated from a Malaysian tropical rainforest waterfall.

  6. Detection and Whole-Genome Sequencing of Carbapenemase-Producing Aeromonas hydrophila Isolates from Routine Perirectal Surveillance Culture.

    Science.gov (United States)

    Hughes, Heather Y; Conlan, Sean P; Lau, Anna F; Dekker, John P; Michelin, Angela V; Youn, Jung-Ho; Henderson, David K; Frank, Karen M; Segre, Julia A; Palmore, Tara N

    2016-04-01

    Perirectal surveillance cultures and a stool culture grewAeromonasspecies from three patients over a 6-week period and were without epidemiological links. Detection of theblaKPC-2gene in one isolate prompted inclusion of non-Enterobacteriaceaein our surveillance culture workup. Whole-genome sequencing confirmed that the isolates were unrelated and provided data forAeromonasreference genomes. PMID:26888898

  7. Identifying Rare Variation in Cases of Schizophrenia in the Isolated Population of the Faroe Islands using Whole-genome Sequencing

    DEFF Research Database (Denmark)

    Als, Thomas Damm; Lescai, Francesco; Dahl, Hans;

    studies aiming to map risk variants involved in complex traits. We aim at utilizing samples of cases and controls of the isolated population of the Faroe Islands to conduct whole-genome-sequence analysis in order to identify rare genetic variants associated with schizophrenia. We will search for rare...

  8. Whole-genome sequence of Clostridium lituseburense L74, isolated from the larval gut of the rhinoceros beetle, Trypoxylus dichotomus

    OpenAIRE

    Yookyung Lee; Sooyeon Lim; Moon-Soo Rhee; Dong-Ho Chang; Byoung-Chan Kim

    2016-01-01

    Clostridium lituseburense L74 was isolated from the larval gut of the rhinoceros beetle, Trypoxylus dichotomus collected in Yeong-dong, Chuncheongbuk-do, South Korea and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession NZ_LITJ00000000.

  9. Whole-genome sequence of Clostridium lituseburense L74, isolated from the larval gut of the rhinoceros beetle, Trypoxylus dichotomus.

    Science.gov (United States)

    Lee, Yookyung; Lim, Sooyeon; Rhee, Moon-Soo; Chang, Dong-Ho; Kim, Byoung-Chan

    2016-03-01

    Clostridium lituseburense L74 was isolated from the larval gut of the rhinoceros beetle, Trypoxylus dichotomus collected in Yeong-dong, Chuncheongbuk-do, South Korea and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession NZ_LITJ00000000. PMID:26981432

  10. Whole-Genome Shotgun Sequence of Bacillus amyloliquefaciens Strain UASWS BA1, a Bacterium Antagonistic to Plant Pathogenic Fungi

    OpenAIRE

    Lefort, F; Calmin, G.; Pelleteret, P.; Farinelli, L.; Osteras, M; Crovadore, J.

    2014-01-01

    We report here the whole-genome shotgun sequence of Bacillus amyloliquefaciens strain UASWS BA1, isolated from inner wood tissues of a decaying Platanus × acerifolia tree. This strain proved to be antagonistic to several plant pathogenic fungi and oomycetes and can be developed as a biological control agent in agriculture.

  11. Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis

    DEFF Research Database (Denmark)

    Winkel, Bo G; Hollegaard, Mads Vilhelm; Olesen, Morten S; Svendsen, Jesper H; Haunsø, Stig; Hougaard, David M; Tfelt-Hansen, Jacob

    2011-01-01

    The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare m...

  12. Investigating Salmonella Eko from Various Sources in Nigeria by Whole Genome Sequencing to Identify the Source of Human Infections.

    Science.gov (United States)

    Leekitcharoenphon, Pimlapas; Raufu, Ibrahim; Nielsen, Mette T; Rosenqvist Lund, Birthe S; Ameh, James A; Ambali, Abdul G; Sørensen, Gitte; Le Hello, Simon; Aarestrup, Frank M; Hendriksen, Rene S

    2016-01-01

    Twenty-six Salmonella enterica serovar Eko isolated from various sources in Nigeria were investigated by whole genome sequencing to identify the source of human infections. Diversity among the isolates was observed and camel and cattle were identified as the primary reservoirs and the most likely source of the human infections. PMID:27228329

  13. Investigating Salmonella Eko from Various Sources in Nigeria by Whole Genome Sequencing to Identify the Source of Human Infections

    DEFF Research Database (Denmark)

    Leekitcharoenphon, Pimlapas; Raufu, Ibrahim; Thorup Nielsen, Mette;

    2016-01-01

    Twenty-six Salmonella enterica serovar Eko isolated from various sources in Nigeria were investigated by whole genome sequencing to identify the source of human infections. Diversity among the isolates was observed and camel and cattle were identified as the primary reservoirs and the most likely...

  14. Investigating Salmonella Eko from Various Sources in Nigeria by Whole Genome Sequencing to Identify the Source of Human Infections.

    Directory of Open Access Journals (Sweden)

    Pimlapas Leekitcharoenphon

    Full Text Available Twenty-six Salmonella enterica serovar Eko isolated from various sources in Nigeria were investigated by whole genome sequencing to identify the source of human infections. Diversity among the isolates was observed and camel and cattle were identified as the primary reservoirs and the most likely source of the human infections.

  15. Comparing Whole-Genome Sequencing with Sanger Sequencing for spa Typing of Methicillin-Resistant Staphylococcus aureus

    DEFF Research Database (Denmark)

    Bartels, Mette Damkjaer; Petersen, Andreas; Worning, Peder;

    2014-01-01

    spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and an...

  16. Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture

    Science.gov (United States)

    Zheng, Hou-Feng; Forgetta, Vincenzo; Hsu, Yi-Hsiang; Estrada, Karol; Rosello-Diez, Alberto; Leo, Paul J; Dahia, Chitra L; Park-Min, Kyung Hyun; Tobias, Jonathan H; Kooperberg, Charles; Kleinman, Aaron; Styrkarsdottir, Unnur; Liu, Ching-Ti; Uggla, Charlotta; Evans, Daniel S; Nielson, Carrie M; Walter, Klaudia; Pettersson-Kymmer, Ulrika; McCarthy, Shane; Eriksson, Joel; Kwan, Tony; Jhamai, Mila; Trajanoska, Katerina; Memari, Yasin; Min, Josine; Huang, Jie; Danecek, Petr; Wilmot, Beth; Li, Rui; Chou, Wen-Chi; Mokry, Lauren E; Moayyeri, Alireza; Claussnitzer, Melina; Cheng, Chia-Ho; Cheung, Warren; Medina-Gómez, Carolina; Ge, Bing; Chen, Shu-Huang; Choi, Kwangbom; Oei, Ling; Fraser, James; Kraaij, Robert; Hibbs, Matthew A; Gregson, Celia L; Paquette, Denis; Hofman, Albert; Wibom, Carl; Tranah, Gregory J; Marshall, Mhairi; Gardiner, Brooke B; Cremin, Katie; Auer, Paul; Hsu, Li; Ring, Sue; Tung, Joyce Y; Thorleifsson, Gudmar; Enneman, Anke W; van Schoor, Natasja M; de Groot, Lisette C.P.G.M.; van der Velde, Nathalie; Melin, Beatrice; Kemp, John P; Christiansen, Claus; Sayers, Adrian; Zhou, Yanhua; Calderari, Sophie; van Rooij, Jeroen; Carlson, Chris; Peters, Ulrike; Berlivet, Soizik; Dostie, Josée; Uitterlinden, Andre G; Williams, Stephen R.; Farber, Charles; Grinberg, Daniel; LaCroix, Andrea Z; Haessler, Jeff; Chasman, Daniel I; Giulianini, Franco; Rose, Lynda M; Ridker, Paul M; Eisman, John A; Nguyen, Tuan V; Center, Jacqueline R; Nogues, Xavier; Garcia-Giralt, Natalia; Launer, Lenore L; Gudnason, Vilmunder; Mellström, Dan; Vandenput, Liesbeth; Karlsson, Magnus K; Ljunggren, Östen; Svensson, Olle; Hallmans, Göran; Rousseau, François; Giroux, Sylvie; Bussière, Johanne; Arp, Pascal P; Koromani, Fjorda; Prince, Richard L; Lewis, Joshua R; Langdahl, Bente L; Hermann, A Pernille; Jensen, Jens-Erik B; Kaptoge, Stephen; Khaw, Kay-Tee; Reeve, Jonathan; Formosa, Melissa M; Xuereb-Anastasi, Angela; Åkesson, Kristina; McGuigan, Fiona E; Garg, Gaurav; Olmos, Jose M; Zarrabeitia, Maria T; Riancho, Jose A; Ralston, Stuart H; Alonso, Nerea; Jiang, Xi; Goltzman, David; Pastinen, Tomi; Grundberg, Elin; Gauguier, Dominique; Orwoll, Eric S; Karasik, David; Davey-Smith, George; Smith, Albert V; Siggeirsdottir, Kristin; Harris, Tamara B; Zillikens, M Carola; van Meurs, Joyce BJ; Thorsteinsdottir, Unnur; Maurano, Matthew T; Timpson, Nicholas J; Soranzo, Nicole; Durbin, Richard; Wilson, Scott G; Ntzani, Evangelia E; Brown, Matthew A; Stefansson, Kari; Hinds, David A; Spector, Tim; Cupples, L Adrienne; Ohlsson, Claes; Greenwood, Celia MT; Jackson, Rebecca D; Rowe, David W; Loomis, Cynthia A; Evans, David M; Ackert-Bicknell, Cheryl L; Joyner, Alexandra L; Duncan, Emma L; Kiel, Douglas P; Rivadeneira, Fernando; Richards, J Brent

    2016-01-01

    SUMMARY The extent to which low-frequency (minor allele frequency [MAF] between 1–5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is largely unknown. Bone mineral density (BMD) is highly heritable, is a major predictor of osteoporotic fractures and has been previously associated with common genetic variants1–8, and rare, population-specific, coding variants9. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n=2,882 from UK10K), whole-exome sequencing (n= 3,549), deep imputation of genotyped samples using a combined UK10K/1000Genomes reference panel (n=26,534), and de-novo replication genotyping (n= 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size 4-fold larger than the mean of previously reported common variants for lumbar spine BMD8 (rs11692564[T], MAF = 1.7%, replication effect size = +0.20 standard deviations [SD], Pmeta = 2×10−14), which was also associated with a decreased risk of fracture (OR = 0.85; P = 2×10−11; ncases = 98,742 and ncontrols = 409,511). Using an En1Cre/flox mouse model, we observed that conditional loss of En1 results in low bone mass, likely as a consequence of high bone turn-over. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817[T], MAF = 1.1%, replication effect size = +0.39 SD, Pmeta = 1×10−11). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of

  17. Quantitative trait loci markers derived from whole genome sequence data increases the reliability of genomic prediction.

    Science.gov (United States)

    Brøndum, R F; Su, G; Janss, L; Sahana, G; Guldbrandtsen, B; Boichard, D; Lund, M S

    2015-06-01

    This study investigated the effect on the reliability of genomic prediction when a small number of significant variants from single marker analysis based on whole genome sequence data were added to the regular 54k single nucleotide polymorphism (SNP) array data. The extra markers were selected with the aim of augmenting the custom low-density Illumina BovineLD SNP chip (San Diego, CA) used in the Nordic countries. The single-marker analysis was done breed-wise on all 16 index traits included in the breeding goals for Nordic Holstein, Danish Jersey, and Nordic Red cattle plus the total merit index itself. Depending on the trait's economic weight, 15, 10, or 5 quantitative trait loci (QTL) were selected per trait per breed and 3 to 5 markers were selected to tag each QTL. After removing duplicate markers (same marker selected for more than one trait or breed) and filtering for high pairwise linkage disequilibrium and assaying performance on the array, a total of 1,623 QTL markers were selected for inclusion on the custom chip. Genomic prediction analyses were performed for Nordic and French Holstein and Nordic Red animals using either a genomic BLUP or a Bayesian variable selection model. When using the genomic BLUP model including the QTL markers in the analysis, reliability was increased by up to 4 percentage points for production traits in Nordic Holstein animals, up to 3 percentage points for Nordic Reds, and up to 5 percentage points for French Holstein. Smaller gains of up to 1 percentage point was observed for mastitis, but only a 0.5 percentage point increase was seen for fertility. When using a Bayesian model accuracies were generally higher with only 54k data compared with the genomic BLUP approach, but increases in reliability were relatively smaller when QTL markers were included. Results from this study indicate that the reliability of genomic prediction can be increased by including markers significant in genome-wide association studies on whole genome

  18. Whole-Genome Transcriptional Analysis of Escherichia coli during Heat Inactivation Processes Related to Industrial Cooking

    OpenAIRE

    Guernec, A.; Robichaud-Rincon, P.; Saucier, L.

    2013-01-01

    Escherichia coli K-12 was grown to the stationary phase, for maximum physiological resistance, in brain heart infusion (BHI) broth at 37°C. Cells were then heated at 58°C or 60°C to reach a process lethality value (Fo7010) of 2 or 3 or to a core temperature of 71°C (control industrial cooking temperature). Growth recovery and cell membrane integrity were evaluated immediately after heating, and a global transcription analysis was performed using gene expression microarrays. Only cells heated ...

  19. Genetic characterization of 2006-2008 isolates of Chikungunya virus from Kerala, South India, by whole genome sequence analysis.

    Science.gov (United States)

    Sreekumar, E; Issac, Aneesh; Nair, Sajith; Hariharan, Ramkumar; Janki, M B; Arathy, D S; Regu, R; Mathew, Thomas; Anoop, M; Niyas, K P; Pillai, M R

    2010-02-01

    Chikungunya virus (CHIKV), a positive-stranded alphavirus, causes epidemic febrile infections characterized by severe and prolonged arthralgia. In the present study, six CHIKV isolates (2006 RGCB03, RGCB05; 2007 RGCB80, RGCB120; 2008 RGCB355, RGCB356) from three consecutive Chikungunya outbreaks in Kerala, South India, were analyzed for genetic variations by sequencing the 11798 bp whole genome of the virus. A total of 37 novel mutations were identified and they were predominant in the 2007 and 2008 isolates among the six isolates studied. The previously identified E1 A226V critical mutation, which enhances mosquito adaptability, was present in the 2007 and 2008 samples. An important observation was the presence of two coding region substitutions, leading to nsP2 L539S and E2 K252Q change. These were identified in three isolates (2007 RGCB80 and RGCB120; 2008 RGCB355) by full-genome analysis, and also in 13 of the 31 additional samples (42%), obtained from various parts of the state, by sequencing the corresponding genomic regions. These mutations showed 100% co-occurrence in all these samples. In phylogenetic analysis, formation of a new genetic clade by these isolates within the East, Central and South African (ECSA) genotypes was observed. Homology modeling followed by mapping revealed that at least 20 of the identified mutations fall into functionally significant domains of the viral proteins and are predicted to affect protein structure. Eighteen of the identified mutations in structural proteins, including the E2 K252Q change, are predicted to disrupt T-cell epitope immunogenicity. Our study reveals that CHIK virus with novel genetic changes were present in the severe Chikungunya outbreaks in 2007 and 2008 in South India. PMID:19851853

  20. A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor negative and positive breast cancers

    Science.gov (United States)

    Weigelt, Britta; Wilkerson, Paul M; Manie, Elodie; Grigoriadis, Anita; A’Hern, Roger; van der Groep, Petra; Kozarewa, Iwanka; Popova, Tatiana; Mariani, Odette; Turaljic, Samra; Furney, Simon J; Marais, Richard; Rodruigues, Daniel-Nava; Flora, Adriana C; Wai, Patty; Pawar, Vidya; McDade, Simon; Carroll, Jason; Stoppa-Lyonnet, Dominique; Green, Andrew R; Ellis, Ian O; Swanton, Charles; van Diest, Paul; Delattre, Olivier; Lord, Christopher J; Foulkes, William D; Vincent-Salomon, Anne; Ashworth, Alan; Stern, Marc Henri; Reis-Filho, Jorge S

    2016-01-01

    BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In >80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative, however up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterise the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations and somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (i.e. DAPK3, TMEM135 and GATA4). PMID:22362584

  1. Whole Genome Sequencing of Mycobacterium africanum Strains from Mali Provides Insights into the Mechanisms of Geographic Restriction

    Science.gov (United States)

    Maiga, Mamoudou; Abeel, Thomas; Shea, Terrance; Desjardins, Christopher A.; Diarra, Bassirou; Baya, Bocar; Sanogo, Moumine; Diallo, Souleymane; Earl, Ashlee M.; Bishai, William R.

    2016-01-01

    Background Mycobacterium africanum, made up of lineages 5 and 6 within the Mycobacterium tuberculosis complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system. Methodology/Principal Findings We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes. Conclusions This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity. PMID:26751217

  2. Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201 subsurface samples

    Directory of Open Access Journals (Sweden)

    Amanda eMartino

    2012-01-01

    Full Text Available A degenerate PCR-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. The method, which we have called Random Amplification Metagenomic PCR (RAMP, involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3’ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10X. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin, and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa show that community analysis can be sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low biomass samples.

  3. Impacts of Whole-Genome Triplication on MIRNA Evolution in Brassica rapa.

    Science.gov (United States)

    Sun, Chao; Wu, Jian; Liang, Jianli; Schnable, James C; Yang, Wencai; Cheng, Feng; Wang, Xiaowu

    2015-11-01

    MicroRNAs (miRNAs) are a class of short non-coding, endogenous RNAs that play essential roles in eukaryotes. Although the influence of whole-genome triplication (WGT) on protein-coding genes has been well documented in Brassica rapa, little is known about its impacts on MIRNAs. In this study, through generating a comprehensive annotation of 680 MIRNAs for B. rapa, we analyzed the evolutionary characteristics of these MIRNAs from different aspects in B. rapa. First, while MIRNAs and genes show similar patterns of biased distribution among subgenomes of B. rapa, we found that MIRNAs are much more overretained than genes following fractionation after WGT. Second, multiple-copy MIRNAs show significant sequence conservation than that of single-copy MIRNAs, which is opposite to that of genes. This indicates that increased purifying selection is acting upon these highly retained multiple-copy MIRNAs and their functional importance over singleton MIRNAs. Furthermore, we found the extensive divergence between pairs of miRNAs and their target genes following the WGT in B. rapa. In summary, our study provides a valuable resource for exploring MIRNA in B. rapa and highlights the impacts of WGT on the evolution of MIRNA. PMID:26527651

  4. Whole-genome sequencing identifies a recurrent functional synonymous mutation in melanoma

    Science.gov (United States)

    Gartner, Jared J.; Parker, Stephen C. J.; Prickett, Todd D.; Dutton-Regester, Ken; Stitzel, Michael L.; Lin, Jimmy C.; Davis, Sean; Simhadri, Vijaya L.; Jha, Sujata; Katagiri, Nobuko; Gotea, Valer; Teer, Jamie K.; Morken, Mario A.; Bhanot, Umesh K.; Chen, Guo; Elnitski, Laura L.; Davies, Michael A.; Gershenwald, Jeffrey E.; Carter, Hannah; Karchin, Rachel; Robinson, William; Robinson, Steven; Rosenberg, Steven A.; Collins, Francis S.; Parmigiani, Giovanni; Komar, Anton A.; Kimchi-Sarfaty, Chava; Hayward, Nicholas K.; Margulies, Elliott H.; Samuels, Yardena

    2013-01-01

    Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683–691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This mutation led to increased BCL2L12 mRNA and protein levels because of differential targeting of WT and mutant BCL2L12 by hsa-miR-671–5p. Protein made from mutant BCL2L12 transcript bound p53, inhibited UV-induced apoptosis more efficiently than WT BCL2L12, and reduced endogenous p53 target gene transcription. This report shows selection of a recurrent somatic synonymous mutation in cancer. Our data indicate that silent alterations have a role to play in human cancer, emphasizing the importance of their investigation in future cancer genome studies. PMID:23901115

  5. Neuropeptide evolution: Chelicerate neurohormone and neuropeptide genes may reflect one or more whole genome duplications.

    Science.gov (United States)

    Veenstra, Jan A

    2016-04-01

    Four genomes and two transcriptomes from six Chelicerate species were analyzed for the presence of neuropeptide and neurohormone precursors and their GPCRs. The genome from the spider Stegodyphus mimosarum yielded 87 neuropeptide precursors and 120 neuropeptide GPCRs. Many neuropeptide transcripts were also found in the transcriptomes of three other spiders, Latrodectus hesperus, Parasteatoda tepidariorum and Acanthoscurria geniculata. For the scorpion Mesobuthus martensii the numbers are 79 and 93 respectively. The very small genome of the house dust mite, Dermatophagoides farinae, on the other hand contains a much smaller number of such genes. A few new putative Arthropod neuropeptide genes were discovered. Thus, both spiders and the scorpion have an achatin gene and in spiders there are two different genes encoding myosuppressin-like peptides while spiders also have two genes encoding novel LGamides. Another finding is the presence of trissin in spiders and scorpions, while neuropeptide genes that seem to be orthologs of Lottia LFRYamide and Platynereis CCRFamide were also found. Such genes were also found in various insect species, but seem to be lacking from the Holometabola. The Chelicerate neuropeptide and neuropeptide GPCR genes often have paralogs. As the large majority of these are probably not due to local gene duplications, is plausible that they reflect the effects of one or more ancient whole genome duplications. PMID:26928473

  6. Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures.

    Science.gov (United States)

    Votintseva, Antonina A; Pankhurst, Louise J; Anson, Luke W; Morgan, Marcus R; Gascoyne-Binzi, Deborah; Walker, Timothy M; Quan, T Phuong; Wyllie, David H; Del Ojo Elias, Carlos; Wilcox, Mark; Walker, A Sarah; Peto, Tim E A; Crook, Derrick W

    2015-04-01

    We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with 90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools. PMID:25631807

  7. Genomic view of bipolar disorder revealed by whole genome sequencing in a genetic isolate.

    Directory of Open Access Journals (Sweden)

    Benjamin Georgi

    2014-03-01

    Full Text Available Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder, causative genetic risk factors remain elusive. We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree. Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects, comprising 18 parent-child trios. This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings. Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks, each of which included dozens of predicted deleterious variants. Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts. However, despite the in-depth genomic characterization of this unique, large and multigenerational pedigree from a genetic isolate, there was no convergence of evidence implicating a particular set of risk loci or common pathways. The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders.

  8. Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

    Science.gov (United States)

    Nik-Zainal, Serena; Davies, Helen; Staaf, Johan; Ramakrishna, Manasa; Glodzik, Dominik; Zou, Xueqing; Martincorena, Inigo; Alexandrov, Ludmil B; Martin, Sancha; Wedge, David C; Van Loo, Peter; Ju, Young Seok; Smid, Marcel; Brinkman, Arie B; Morganella, Sandro; Aure, Miriam R; Lingjærde, Ole Christian; Langerød, Anita; Ringnér, Markus; Ahn, Sung-Min; Boyault, Sandrine; Brock, Jane E; Broeks, Annegien; Butler, Adam; Desmedt, Christine; Dirix, Luc; Dronov, Serge; Fatima, Aquila; Foekens, John A; Gerstung, Moritz; Hooijer, Gerrit K J; Jang, Se Jin; Jones, David R; Kim, Hyung-Yong; King, Tari A; Krishnamurthy, Savitri; Lee, Hee Jin; Lee, Jeong-Yeon; Li, Yilong; McLaren, Stuart; Menzies, Andrew; Mustonen, Ville; O'Meara, Sarah; Pauporté, Iris; Pivot, Xavier; Purdie, Colin A; Raine, Keiran; Ramakrishnan, Kamna; Rodríguez-González, F Germán; Romieu, Gilles; Sieuwerts, Anieta M; Simpson, Peter T; Shepherd, Rebecca; Stebbings, Lucy; Stefansson, Olafur A; Teague, Jon; Tommasi, Stefania; Treilleux, Isabelle; Van den Eynden, Gert G; Vermeulen, Peter; Vincent-Salomon, Anne; Yates, Lucy; Caldas, Carlos; van't Veer, Laura; Tutt, Andrew; Knappskog, Stian; Tan, Benita Kiat Tee; Jonkers, Jos; Borg, Åke; Ueno, Naoto T; Sotiriou, Christos; Viari, Alain; Futreal, P Andrew; Campbell, Peter J; Span, Paul N; Van Laere, Steven; Lakhani, Sunil R; Eyfjord, Jorunn E; Thompson, Alastair M; Birney, Ewan; Stunnenberg, Hendrik G; van de Vijver, Marc J; Martens, John W M; Børresen-Dale, Anne-Lise; Richardson, Andrea L; Kong, Gu; Thomas, Gilles; Stratton, Michael R

    2016-06-01

    We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer. PMID:27135926

  9. Kuwaiti population subgroup of nomadic Bedouin ancestry—Whole genome sequence and analysis

    Directory of Open Access Journals (Sweden)

    Sumi Elsa John

    2015-03-01

    Full Text Available Kuwaiti native population comprises three distinct genetic subgroups of Persian, “city-dwelling” Saudi Arabian tribe, and nomadic “tent-dwelling” Bedouin ancestry. Bedouin subgroup is characterized by presence of 17% African ancestry; it owes it origin to nomadic tribes of the deserts of Arabian Peninsula and North Africa. By sequencing whole genome of a Kuwaiti male from this subgroup at 41X coverage, we report 3,752,878 SNPs, 411,839 indels, and 8451 structural variations. Neighbor-joining tree, based on shared variant positions carrying disease-risk alleles between the Bedouin and other continental genomes, places Bedouin genome at the nexus of African, Asian, and European genomes in concordance with geographical location of Kuwait and Peninsula. In congruence with participant's medical history for morbid obesity and bronchial asthma, risk alleles are seen at deleterious SNPs associated with obesity and asthma. Many of the observed deleterious ‘novel’ variants lie in genes associated with autosomal recessive disorders characteristic of the region.

  10. BALSA: integrated secondary analysis for whole-genome and whole-exome sequencing, accelerated by GPU

    Directory of Open Access Journals (Sweden)

    Ruibang Luo

    2014-06-01

    Full Text Available This paper reports an integrated solution, called BALSA, for the secondary analysis of next generation sequencing data; it exploits the computational power of GPU and an intricate memory management to give a fast and accurate analysis. From raw reads to variants (including SNPs and Indels, BALSA, using just a single computing node with a commodity GPU board, takes 5.5 h to process 50-fold whole genome sequencing (∼750 million 100 bp paired-end reads, or just 25 min for 210-fold whole exome sequencing. BALSA’s speed is rooted at its parallel algorithms to effectively exploit a GPU to speed up processes like alignment, realignment and statistical testing. BALSA incorporates a 16-genotype model to support the calling of SNPs and Indels and achieves competitive variant calling accuracy and sensitivity when compared to the ensemble of six popular variant callers. BALSA also supports efficient identification of somatic SNVs and CNVs; experiments showed that BALSA recovers all the previously validated somatic SNVs and CNVs, and it is more sensitive for somatic Indel detection. BALSA outputs variants in VCF format. A pileup-like SNAPSHOT format, while maintaining the same fidelity as BAM in variant calling, enables efficient storage and indexing, and facilitates the App development of downstream analyses. BALSA is available at: http://sourceforge.net/p/balsa.

  11. Digital Droplet Multiple Displacement Amplification (ddMDA for Whole Genome Sequencing of Limited DNA Samples.

    Directory of Open Access Journals (Sweden)

    Minsoung Rhee

    Full Text Available Multiple displacement amplification (MDA is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet, ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

  12. Whole genome and transcriptome sequencing of matched primary and peritoneal metastatic gastric carcinoma.

    Science.gov (United States)

    Zhang, J; Huang, J Y; Chen, Y N; Yuan, F; Zhang, H; Yan, F H; Wang, M J; Wang, G; Su, M; Lu, G; Huang, Y; Dai, H; Ji, J; Zhang, J; Zhang, J N; Jiang, Y N; Chen, S J; Zhu, Z G; Yu, Y Y

    2015-01-01

    Gastric cancer is one of the most aggressive cancers and is the second leading cause of cancer death worldwide. Approximately 40% of global gastric cancer cases occur in China, with peritoneal metastasis being the prevalent form of recurrence and metastasis in advanced disease. Currently, there are limited clinical approaches for predicting and treatment of peritoneal metastasis, resulting in a 6-month average survival time. By comprehensive genome analysis will uncover the pathogenesis of peritoneal metastasis. Here we describe a comprehensive whole-genome and transcriptome sequencing analysis of one advanced gastric cancer case, including non-cancerous mucosa, primary cancer and matched peritoneal metastatic cancer. The peripheral blood is used as normal control. We identified 27 mutated genes, of which 19 genes are reported in COSMIC database (ZNF208, CRNN, ATXN3, DCTN1, RP1L1, PRB4, PRB1, MUC4, HS6ST3, MUC17, JAM2, ITGAD, IREB2, IQUB, CORO1B, CCDC121, AKAP2, ACAN and ACADL), and eight genes have not previously been described in gastric cancer (CCDC178, ARMC4, TUBB6, PLIN4, PKLR, PDZD2, DMBT1and DAB1).Additionally,GPX4 and MPND in 19q13.3-13.4 region, is characterized as a novel fusion-gene. This study disclosed novel biological markers and tumorigenic pathways that would predict gastric cancer occurring peritoneal metastasis. PMID:26330360

  13. Whole genome sequencing provides insights into the genetic determinants of invasiveness in Salmonella Dublin.

    Science.gov (United States)

    Mohammed, M; Cormican, M

    2016-08-01

    Salmonella enterica subsp. enterica serovar Dublin (S. Dublin) is one of the non-typhoidal Salmonella (NTS); however, a relatively high proportion of human infections are associated with invasive disease. We applied whole genome sequencing to representative invasive and non-invasive clinical isolates of S. Dublin to determine the genomic variations among them and to investigate the underlying genetic determinants associated with invasiveness in S. Dublin. Although no particular genomic variation was found to differentiate in invasive and non-invasive isolates four virulence factors were detected within the genome of all isolates including two different type VI secretion systems (T6SS) encoded on two Salmonella pathogenicity islands (SPI), including SPI-6 (T6SSSPI-6) and SPI-19 (T6SSSPI-19), an intact lambdoid prophage (Gifsy-2-like prophage) that contributes significantly to the virulence and pathogenesis of Salmonella serotypes in addition to a virulence plasmid. These four virulence factors may all contribute to the potential of S. Dublin to cause invasive disease in humans. PMID:26996313

  14. Prospective Whole-Genome Sequencing Enhances National Surveillance of Listeria monocytogenes.

    Science.gov (United States)

    Kwong, Jason C; Mercoulia, Karolina; Tomita, Takehiro; Easton, Marion; Li, Hua Y; Bulach, Dieter M; Stinear, Timothy P; Seemann, Torsten; Howden, Benjamin P

    2016-02-01

    Whole-genome sequencing (WGS) has emerged as a powerful tool for comparing bacterial isolates in outbreak detection and investigation. Here we demonstrate that WGS performed prospectively for national epidemiologic surveillance of Listeria monocytogenes has the capacity to be superior to our current approaches using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), binary typing, and serotyping. Initially 423 L. monocytogenes isolates underwent WGS, and comparisons uncovered a diverse genetic population structure derived from three distinct lineages. MLST, binary typing, and serotyping results inferred in silico from the WGS data were highly concordant (>99%) with laboratory typing performed in parallel. However, WGS was able to identify distinct nested clusters within groups of isolates that were otherwise indistinguishable using our current typing methods. Routine WGS was then used for prospective epidemiologic surveillance on a further 97 L. monocytogenes isolates over a 12-month period, which provided a greater level of discrimination than that of conventional typing for inferring linkage to point source outbreaks. A risk-based alert system based on WGS similarity was used to inform epidemiologists required to act on the data. Our experience shows that WGS can be adopted for prospective L. monocytogenes surveillance and investigated for other pathogens relevant to public health. PMID:26607978

  15. Whole genome nucleosome sequencing identifies novel types of forensic markers in degraded DNA samples.

    Science.gov (United States)

    Dong, Chun-Nan; Yang, Ya-Dong; Li, Shu-Jin; Yang, Ya-Ran; Zhang, Xiao-Jing; Fang, Xiang-Dong; Yan, Jiang-Wei; Cong, Bin

    2016-01-01

    In the case of mass disasters, missing persons and forensic caseworks, highly degraded biological samples are often encountered. It can be a challenge to analyze and interpret the DNA profiles from these samples. Here we provide a new strategy to solve the problem by taking advantage of the intrinsic structural properties of DNA. We have assessed the in vivo positions of more than 35 million putative nucleosome cores in human leukocytes using high-throughput whole genome sequencing, and identified 2,462 single nucleotide variations (SNVs), 128 insertion-deletion polymorphisms (indels). After comparing the sequence reads with 44 STR loci commonly used in forensics, five STRs (TH01, TPOX, D18S51, DYS391, and D10S1248)were matched. We compared these "nucleosome protected STRs" (NPSTRs) with five other non-NPSTRs using mini-STR primer design, real-time PCR, and capillary gel electrophoresis on artificially degraded DNA. Moreover, genotyping performance of the five NPSTRs and five non-NPSTRs was also tested with real casework samples. All results show that loci located in nucleosomes are more likely to be successfully genotyped in degraded samples. In conclusion, after further strict validation, these markers could be incorporated into future forensic and paleontology identification kits, resulting in higher discriminatory power for certain degraded sample types. PMID:27189082

  16. A Recent Whole-Genome Duplication Divides Populations of a Globally Distributed Microsporidian.

    Science.gov (United States)

    Williams, Tom A; Nakjang, Sirintra; Campbell, Scott E; Freeman, Mark A; Eydal, Matthías; Moore, Karen; Hirt, Robert P; Embley, T Martin; Williams, Bryony A P

    2016-08-01

    The Microsporidia are a major group of intracellular fungi and important parasites of animals including insects, fish, and immunocompromised humans. Microsporidian genomes have undergone extreme reductive evolution but there are major differences in genome size and structure within the group: some are prokaryote-like in size and organisation (marine microsporidian infecting goosefish worldwide. Our analysis revealed that population structure across the Atlantic Ocean is associated with a conserved difference in ploidy, with American and Canadian isolates sharing an ancestral whole genome duplication that was followed by widespread pseudogenisation and sorting-out of paralogue pairs. While past analyses have suggested de novo gene formation of microsporidian-specific genes, we found evidence for the origin of new genes from noncoding sequence since the divergence of these populations. Some of these genes experience selective constraint, suggesting the evolution of new functions and local host adaptation. Combining our data with published microsporidian genomes, we show that nucleotide composition across the phylum is shaped by a mutational bias favoring A and T nucleotides, which is opposed by an evolutionary force favoring an increase in genomic GC content. This study reveals ongoing dramatic reorganization of genome structure and the evolution of new gene functions in modern microsporidians despite extensive genomic streamlining in their common ancestor. PMID:27189558

  17. Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity.

    Science.gov (United States)

    Dulak, Austin M; Stojanov, Petar; Peng, Shouyong; Lawrence, Michael S; Fox, Cameron; Stewart, Chip; Bandla, Santhoshi; Imamura, Yu; Schumacher, Steven E; Shefler, Erica; McKenna, Aaron; Carter, Scott L; Cibulskis, Kristian; Sivachenko, Andrey; Saksena, Gordon; Voet, Douglas; Ramos, Alex H; Auclair, Daniel; Thompson, Kristin; Sougnez, Carrie; Onofrio, Robert C; Guiducci, Candace; Beroukhim, Rameen; Zhou, Zhongren; Lin, Lin; Lin, Jules; Reddy, Rishindra; Chang, Andrew; Landrenau, Rodney; Pennathur, Arjun; Ogino, Shuji; Luketich, James D; Golub, Todd R; Gabriel, Stacey B; Lander, Eric S; Beer, David G; Godfrey, Tony E; Getz, Gad; Bass, Adam J

    2013-05-01

    The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a 5-year survival rate of ~15%, the identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole-exome sequencing of 149 EAC tumor-normal pairs, 15 of which have also been subjected to whole-genome sequencing. We identify a mutational signature defined by a high prevalence of A>C transversions at AA dinucleotides. Statistical analysis of exome data identified 26 significantly mutated genes. Of these genes, five (TP53, CDKN2A, SMAD4, ARID1A and PIK3CA) have previously been implicated in EAC. The new significantly mutated genes include chromatin-modifying factors and candidate contributors SPG20, TLR4, ELMO1 and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 identifies increased cellular invasion. Therefore, we suggest the potential activation of the RAC1 pathway as a contributor to EAC tumorigenesis. PMID:23525077

  18. Whole genome nucleosome sequencing identifies novel types of forensic markers in degraded DNA samples

    Science.gov (United States)

    Dong, Chun-nan; Yang, Ya-dong; Li, Shu-jin; Yang, Ya-ran; Zhang, Xiao-jing; Fang, Xiang-dong; Yan, Jiang-wei; Cong, Bin

    2016-01-01

    In the case of mass disasters, missing persons and forensic caseworks, highly degraded biological samples are often encountered. It can be a challenge to analyze and interpret the DNA profiles from these samples. Here we provide a new strategy to solve the problem by taking advantage of the intrinsic structural properties of DNA. We have assessed the in vivo positions of more than 35 million putative nucleosome cores in human leukocytes using high-throughput whole genome sequencing, and identified 2,462 single nucleotide variations (SNVs), 128 insertion-deletion polymorphisms (indels). After comparing the sequence reads with 44 STR loci commonly used in forensics, five STRs (TH01, TPOX, D18S51, DYS391, and D10S1248)were matched. We compared these “nucleosome protected STRs” (NPSTRs) with five other non-NPSTRs using mini-STR primer design, real-time PCR, and capillary gel electrophoresis on artificially degraded DNA. Moreover, genotyping performance of the five NPSTRs and five non-NPSTRs was also tested with real casework samples. All results show that loci located in nucleosomes are more likely to be successfully genotyped in degraded samples. In conclusion, after further strict validation, these markers could be incorporated into future forensic and paleontology identification kits, resulting in higher discriminatory power for certain degraded sample types. PMID:27189082

  19. Automated whole-genome multiple alignment of rat, mouse, and human

    Energy Technology Data Exchange (ETDEWEB)

    Brudno, Michael; Poliakov, Alexander; Salamov, Asaf; Cooper, Gregory M.; Sidow, Arend; Rubin, Edward M.; Solovyev, Victor; Batzoglou, Serafim; Dubchak, Inna

    2004-07-04

    We have built a whole genome multiple alignment of the three currently available mammalian genomes using a fully automated pipeline which combines the local/global approach of the Berkeley Genome Pipeline and the LAGAN program. The strategy is based on progressive alignment, and consists of two main steps: (1) alignment of the mouse and rat genomes; and (2) alignment of human to either the mouse-rat alignments from step 1, or the remaining unaligned mouse and rat sequences. The resulting alignments demonstrate high sensitivity, with 87% of all human gene-coding areas aligned in both mouse and rat. The specificity is also high: <7% of the rat contigs are aligned to multiple places in human and 97% of all alignments with human sequence > 100kb agree with a three-way synteny map built independently using predicted exons in the three genomes. At the nucleotide level <1% of the rat nucleotides are mapped to multiple places in the human sequence in the alignment; and 96.5% of human nucleotides within all alignments agree with the synteny map. The alignments are publicly available online, with visualization through the novel Multi-VISTA browser that we also present.

  20. Whole-genome sequencing of a laboratory-evolved yeast strain

    Directory of Open Access Journals (Sweden)

    Dunham Maitreya J

    2010-02-01

    Full Text Available Abstract Background Experimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. However, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. New DNA sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined. Results We sequenced >93.5% of the genome of a laboratory-evolved strain of the yeast Saccharomyces cerevisiae and its ancestor at >28× depth. Both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. Applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5× gene amplification. These boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement. Conclusions This study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. In addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity.

  1. A human genome-wide library of local phylogeny predictions for whole-genome inference problems

    Directory of Open Access Journals (Sweden)

    Schwartz Russell

    2008-08-01

    Full Text Available Abstract Background Many common inference problems in computational genetics depend on inferring aspects of the evolutionary history of a data set given a set of observed modern sequences. Detailed predictions of the full phylogenies are therefore of value in improving our ability to make further inferences about population history and sources of genetic variation. Making phylogenetic predictions on the scale needed for whole-genome analysis is, however, extremely computationally demanding. Results In order to facilitate phylogeny-based predictions on a genomic scale, we develop a library of maximum parsimony phylogenies within local regions spanning all autosomal human chromosomes based on Haplotype Map variation data. We demonstrate the utility of this library for population genetic inferences by examining a tree statistic we call 'imperfection,' which measures the reuse of variant sites within a phylogeny. This statistic is significantly predictive of recombination rate, shows additional regional and population-specific conservation, and allows us to identify outlier genes likely to have experienced unusual amounts of variation in recent human history. Conclusion Recent theoretical advances in algorithms for phylogenetic tree reconstruction have made it possible to perform large-scale inferences of local maximum parsimony phylogenies from single nucleotide polymorphism (SNP data. As results from the imperfection statistic demonstrate, phylogeny predictions encode substantial information useful for detecting genomic features and population history. This data set should serve as a platform for many kinds of inferences one may wish to make about human population history and genetic variation.

  2. Whole-genome sequencing of multidrug-resistant Mycobacterium tuberculosis isolates from Myanmar.

    Science.gov (United States)

    Aung, Htin Lin; Tun, Thanda; Moradigaravand, Danesh; Köser, Claudio U; Nyunt, Wint Wint; Aung, Si Thu; Lwin, Thandar; Thinn, Kyi Kyi; Crump, John A; Parkhill, Julian; Peacock, Sharon J; Cook, Gregory M; Hill, Philip C

    2016-09-01

    Drug-resistant tuberculosis (TB) is a major health threat in Myanmar. An initial study was conducted to explore the potential utility of whole-genome sequencing (WGS) for the diagnosis and management of drug-resistant TB in Myanmar. Fourteen multidrug-resistant Mycobacterium tuberculosis isolates were sequenced. Known resistance genes for a total of nine antibiotics commonly used in the treatment of drug-susceptible and multidrug-resistant TB (MDR-TB) in Myanmar were interrogated through WGS. All 14 isolates were MDR-TB, consistent with the results of phenotypic drug susceptibility testing (DST), and the Beijing lineage predominated. Based on the results of WGS, 9 of the 14 isolates were potentially resistant to at least one of the drugs used in the standard MDR-TB regimen but for which phenotypic DST is not conducted in Myanmar. This study highlights a need for the introduction of second-line DST as part of routine TB diagnosis in Myanmar as well as new classes of TB drugs to construct effective regimens. PMID:27530852

  3. Use of Whole Genome Sequencing for Diagnosis and Discovery in the Cancer Genetics Clinic

    Directory of Open Access Journals (Sweden)

    Samantha B. Foley

    2015-01-01

    Full Text Available Despite the potential of whole-genome sequencing (WGS to improve patient diagnosis and care, the empirical value of WGS in the cancer genetics clinic is unknown. We performed WGS on members of two cohorts of cancer genetics patients: those with BRCA1/2 mutations (n = 176 and those without (n = 82. Initial analysis of potentially pathogenic variants (PPVs, defined as nonsynonymous variants with allele frequency < 1% in ESP6500 in 163 clinically-relevant genes suggested that WGS will provide useful clinical results. This is despite the fact that a majority of PPVs were novel missense variants likely to be classified as variants of unknown significance (VUS. Furthermore, previously reported pathogenic missense variants did not always associate with their predicted diseases in our patients. This suggests that the clinical use of WGS will require large-scale efforts to consolidate WGS and patient data to improve accuracy of interpretation of rare variants. While loss-of-function (LoF variants represented only a small fraction of PPVs, WGS identified additional cancer risk LoF PPVs in patients with known BRCA1/2 mutations and led to cancer risk diagnoses in 21% of non-BRCA cancer genetics patients after expanding our analysis to 3209 ClinVar genes. These data illustrate how WGS can be used to improve our ability to discover patients' cancer genetic risks.

  4. Whole-Genome Screening of Newborns? The Constitutional Boundaries of State Newborn Screening Programs

    Science.gov (United States)

    King, Jaime S.; Smith, Monica E.

    2016-01-01

    State newborn screening (NBS) programs routinely screen nearly all of the 4 million newborns in the United States each year for ~30 primary conditions and a number of secondary conditions. NBS could be on the cusp of an unprecedented expansion as a result of advances in whole-genome sequencing (WGS). As WGS becomes cheaper and easier and as our knowledge and understanding of human genetics expand, the question of whether WGS has a role to play in state NBS programs becomes increasingly relevant and complex. As geneticists and state public health officials begin to contemplate the technical and procedural details of whether WGS could benefit existing NBS programs, this is an opportune time to revisit the legal framework of state NBS programs. In this article, we examine the constitutional underpinnings of state-mandated NBS and explore the range of current state statutes and regulations that govern the programs. We consider the legal refinements that will be needed to keep state NBS programs within constitutional bounds, focusing on 2 areas of concern: consent procedures and the criteria used to select new conditions for NBS panels. We conclude by providing options for states to consider when contemplating the use of WGS for NBS. PMID:26729704

  5. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins.

    Science.gov (United States)

    Croucher, Nicholas J; Page, Andrew J; Connor, Thomas R; Delaney, Aidan J; Keane, Jacqueline A; Bentley, Stephen D; Parkhill, Julian; Harris, Simon R

    2015-02-18

    The emergence of new sequencing technologies has facilitated the use of bacterial whole genome alignments for evolutionary studies and outbreak analyses. These datasets, of increasing size, often include examples of multiple different mechanisms of horizontal sequence transfer resulting in substantial alterations to prokaryotic chromosomes. The impact of these processes demands rapid and flexible approaches able to account for recombination when reconstructing isolates' recent diversification. Gubbins is an iterative algorithm that uses spatial scanning statistics to identify loci containing elevated densities of base substitutions suggestive of horizontal sequence transfer while concurrently constructing a maximum likelihood phylogeny based on the putative point mutations outside these regions of high sequence diversity. Simulations demonstrate the algorithm generates highly accurate reconstructions under realistically parameterized models of bacterial evolution, and achieves convergence in only a few hours on alignments of hundreds of bacterial genome sequences. Gubbins is appropriate for reconstructing the recent evolutionary history of a variety of haploid genotype alignments, as it makes no assumptions about the underlying mechanism of recombination. The software is freely available for download at github.com/sanger-pathogens/Gubbins, implemented in Python and C and supported on Linux and Mac OS X. PMID:25414349

  6. A proposed clinical decision support architecture capable of supporting whole genome sequence information.

    Science.gov (United States)

    Welch, Brandon M; Loya, Salvador Rodriguez; Eilbeck, Karen; Kawamoto, Kensaku

    2014-04-01

    Whole genome sequence (WGS) information may soon be widely available to help clinicians personalize the care and treatment of patients. However, considerable barriers exist, which may hinder the effective utilization of WGS information in a routine clinical care setting. Clinical decision support (CDS) offers a potential solution to overcome such barriers and to facilitate the effective use of WGS information in the clinic. However, genomic information is complex and will require significant considerations when developing CDS capabilities. As such, this manuscript lays out a conceptual framework for a CDS architecture designed to deliver WGS-guided CDS within the clinical workflow. To handle the complexity and breadth of WGS information, the proposed CDS framework leverages service-oriented capabilities and orchestrates the interaction of several independently-managed components. These independently-managed components include the genome variant knowledge base, the genome database, the CDS knowledge base, a CDS controller and the electronic health record (EHR). A key design feature is that genome data can be stored separately from the EHR. This paper describes in detail: (1) each component of the architecture; (2) the interaction of the components; and (3) how the architecture attempts to overcome the challenges associated with WGS information. We believe that service-oriented CDS capabilities will be essential to using WGS information for personalized medicine. PMID:25411644

  7. Allele-specific copy-number discovery from whole-genome and whole-exome sequencing.

    Science.gov (United States)

    Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J; Szatkiewicz, Jin P

    2015-08-18

    Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. PMID:25883151

  8. Two Rounds of Whole Genome Duplication in the AncestralVertebrate

    Energy Technology Data Exchange (ETDEWEB)

    Dehal, Paramvir; Boore, Jeffrey L.

    2005-04-12

    The hypothesis that the relatively large and complex vertebrate genome was created by two ancient, whole genome duplications has been hotly debated, but remains unresolved. We reconstructed the evolutionary relationships of all gene families from the complete gene sets of a tunicate, fish, mouse, and human, then determined when each gene duplicated relative to the evolutionary tree of the organisms. We confirmed the results of earlier studies that there remains little signal of these events in numbers of duplicated genes, gene tree topology, or the number of genes per multigene family. However, when we plotted the genomic map positions of only the subset of paralogous genes that were duplicated prior to the fish-tetrapod split, their global physical organization provides unmistakable evidence of two distinct genome duplication events early in vertebrate evolution indicated by clear patterns of 4-way paralogous regions covering a large part of the human genome. Our results highlight the potential for these large-scale genomic events to have driven the evolutionary success of the vertebrate lineage.

  9. A Recent Whole-Genome Duplication Divides Populations of a Globally Distributed Microsporidian

    Science.gov (United States)

    Williams, Tom A.; Nakjang, Sirintra; Campbell, Scott E.; Freeman, Mark A.; Eydal, Matthías; Moore, Karen; Hirt, Robert P.; Embley, T. Martin; Williams, Bryony A. P.

    2016-01-01

    The Microsporidia are a major group of intracellular fungi and important parasites of animals including insects, fish, and immunocompromised humans. Microsporidian genomes have undergone extreme reductive evolution but there are major differences in genome size and structure within the group: some are prokaryote-like in size and organisation (difference in ploidy, with American and Canadian isolates sharing an ancestral whole genome duplication that was followed by widespread pseudogenisation and sorting-out of paralogue pairs. While past analyses have suggested de novo gene formation of microsporidian-specific genes, we found evidence for the origin of new genes from noncoding sequence since the divergence of these populations. Some of these genes experience selective constraint, suggesting the evolution of new functions and local host adaptation. Combining our data with published microsporidian genomes, we show that nucleotide composition across the phylum is shaped by a mutational bias favoring A and T nucleotides, which is opposed by an evolutionary force favoring an increase in genomic GC content. This study reveals ongoing dramatic reorganization of genome structure and the evolution of new gene functions in modern microsporidians despite extensive genomic streamlining in their common ancestor. PMID:27189558

  10. Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples

    Science.gov (United States)

    Rhee, Minsoung; Light, Yooli K.; Meagher, Robert J.; Singh, Anup K.

    2016-01-01

    Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology. PMID:27144304

  11. Isolation and whole genome sequencing of a Ruminococcus-like bacterium, associated with irritable bowel syndrome.

    Science.gov (United States)

    Hynönen, Ulla; Rasinkangas, Pia; Satokari, Reetta; Paulin, Lars; de Vos, Willem M; Pietilä, Taija E; Kant, Ravi; Palva, Airi

    2016-06-01

    In our previous studies on the intestinal microbiota in irritable bowel syndrome (IBS), we identified a bacterial phylotype with higher abundance in patients suffering from diarrhea than in healthy controls. In the present work, we have isolated in pure culture strain RT94, belonging to this phylotype, determined its whole genome sequence and performed an extensive genomic analysis and phenotypical testing. This revealed strain RT94 to be a strict anaerobe apparently belonging to a novel species with only 94% similarity in the 16S rRNA gene sequence to the closest relatives Ruminococcus torques and Ruminococcus lactaris. The G + C content of strain RT94 is 45.2 mol% and the major long-chain cellular fatty acids are C16:0, C18:0 and C14:0. The isolate is metabolically versatile but not a mucus or cellulose utilizer. It produces acetate, ethanol, succinate, lactate and formate, but very little butyrate, as end products of glucose metabolism. The mechanisms underlying the association of strain RT94 with diarrhea-type IBS are discussed. PMID:26946362

  12. Unique features of a Japanese 'Candidatus Liberibacter asiaticus' strain revealed by whole genome sequencing.

    Directory of Open Access Journals (Sweden)

    Hiroshi Katoh

    Full Text Available Citrus greening (huanglongbing is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with phloem-limited bacteria of three species of α-Proteobacteria, namely, 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus', and 'Ca. L. africanus'. Recent findings suggested that some Japanese strains lack the bacteriophage-type DNA polymerase region (DNA pol, in contrast to the Floridian psy62 strain. The whole genome sequence of the pol-negative 'Ca. L. asiaticus' Japanese isolate Ishi-1 was determined by metagenomic analysis of DNA extracted from 'Ca. L. asiaticus'-infected psyllids and leaf midribs. The 1.19-Mb genome has an average 36.32% GC content. Annotation revealed 13 operons encoding rRNA and 44 tRNA genes, but no typical bacterial pathogenesis-related genes were located within the genome, similar to the Floridian psy62 and Chinese gxpsy. In contrast to other 'Ca. L. asiaticus' strains, the genome of the Japanese Ishi-1 strain lacks a prophage-related region.

  13. Bonus Organisms in High-Throughput Eukaryotic Whole-Genome Shorgun Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Pangilinan, Jasmyn; Shapiro, Harris; Tu, Hank; Platt, Darren

    2006-02-06

    The DOE Joint Genome Institute has sequenced over 50 eukaryotic genomes, ranging in size from 15 MB to 1.6 GB, over a wide range of organism types. In the course of doing so, it has become clear that a substantial fraction of these data sets contains bonus organisms, usually prokaryotes, in addition to the desired genome. While some of these additional organisms are extraneous contamination, they are sometimes symbionts, and so can be of biological interest. Therefore, it is desirable to assemble the bonus organisms along with the main genome. This transforms the problem into one of metagenomic assembly, which is considerably more challenging than traditional whole-genome shotgun (WGS) assembly. The different organisms will usually be present at different sequence depths, which is difficult to handle in most WGS assemblers. In addition, with multiple distinct genomes present, chimerism can produce cross-organism combinations. Finally, there is no guarantee that only a single bonus organism will be present. For example, one JGI project contained at least two different prokaryotic contaminants, plus a 145 KB plasmid of unknown origin. We have developed techniques to routinely identify and handle such bonus organisms in a high-throughput sequencing environment. Approaches include screening and partitioning the unassembled data, and iterative subassemblies. These methods are applicable not only to bonus organisms, but also to desired components such as organelles. These procedures have the additional benefit of identifying, and allowing for the removal of, cloning artifacts such as E.coli and spurious vector inclusions.

  14. De novo assembly of the carrot mitochondrial genome using next generation sequencing of whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome

    Directory of Open Access Journals (Sweden)

    Iorizzo Massimo

    2012-05-01

    Full Text Available Abstract Background Sequence analysis of organelle genomes has revealed important aspects of plant cell evolution. The scope of this study was to develop an approach for de novo assembly of the carrot mitochondrial genome using next generation sequence data from total genomic DNA. Results Sequencing data from a carrot 454 whole genome library were used to develop a de novo assembly of the mitochondrial genome. Development of a new bioinformatic tool allowed visualizing contig connections and elucidation of the de novo assembly. Southern hybridization demonstrated recombination across two large repeats. Genome annotation allowed identification of 44 protein coding genes, three rRNA and 17 tRNA. Identification of the plastid genome sequence allowed organelle genome comparison. Mitochondrial intergenic sequence analysis allowed detection of a fragment of DNA specific to the carrot plastid genome. PCR amplification and sequence analysis across different Apiaceae species revealed consistent conservation of this fragment in the mitochondrial genomes and an insertion in Daucus plastid genomes, giving evidence of a mitochondrial to plastid transfer of DNA. Sequence similarity with a retrotransposon element suggests a possibility that a transposon-like event transferred this sequence into the plastid genome. Conclusions This study confirmed that whole genome sequencing is a practical approach for de novo assembly of higher plant mitochondrial genomes. In addition, a new aspect of intercompartmental genome interaction was reported providing the first evidence for DNA transfer into an angiosperm plastid genome. The approach used here could be used more broadly to sequence and assemble mitochondrial genomes of diverse species. This information will allow us to better understand intercompartmental interactions and cell evolution.

  15. Whole-genome sequencing to establish relapse or re-infection with Mycobacterium tuberculosis: a retrospective observational study

    Science.gov (United States)

    Bryant, Josephine M; Harris, Simon R; Parkhill, Julian; Dawson, Rodney; Diacon, Andreas H; van Helden, Paul; Pym, Alex; Mahayiddin, Aziah A; Chuchottaworn, Charoen; Sanne, Ian M; Louw, Cheryl; Boeree, Martin J; Hoelscher, Michael; McHugh, Timothy D; Bateson, Anna L C; Hunt, Robert D; Mwaigwisya, Solomon; Wright, Laura; Gillespie, Stephen H; Bentley, Stephen D

    2013-01-01

    Summary Background Recurrence of tuberculosis after treatment makes management difficult and is a key factor for determining treatment efficacy. Two processes can cause recurrence: relapse of the primary infection or re-infection with an exogenous strain. Although re-infection can and does occur, its importance to tuberculosis epidemiology and its biological basis is still debated. We used whole-genome sequencing—which is more accurate than conventional typing used to date—to assess the frequency of recurrence and to gain insight into the biological basis of re-infection. Methods We assessed patients from the REMoxTB trial—a randomised controlled trial of tuberculosis treatment that enrolled previously untreated participants with Mycobacterium tuberculosis infection from Malaysia, South Africa, and Thailand. We did whole-genome sequencing and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing of pairs of isolates taken by sputum sampling: one from before treatment and another from either the end of failed treatment at 17 weeks or later or from a recurrent infection. We compared the number and location of SNPs between isolates collected at baseline and recurrence. Findings We assessed 47 pairs of isolates. Whole-genome sequencing identified 33 cases with little genetic distance (0–6 SNPs) between strains, deemed relapses, and three cases for which the genetic distance ranged from 1306 to 1419 SNPs, deemed re-infections. Six cases of relapse and six cases of mixed infection were classified differently by whole-genome sequencing and MIRU-VNTR. We detected five single positive isolates (positive culture followed by at least two negative cultures) without clinical evidence of disease. Interpretation Whole-genome sequencing enables the differentiation of relapse and re-infection cases with greater resolution than do genotyping methods used at present, such as MIRU-VNTR, and provides insights into the biology of

  16. Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

    Science.gov (United States)

    Song, Jie; Hu, Yajie; Hu, Yunguang; Wang, Jingjing; Zhang, Xiaolong; Wang, Lichun; Guo, Lei; Wang, Yancui; Ning, Ruotong; Liao, Yun; Zhang, Ying; Zheng, Huiwen; Shi, Haijing; He, Zhanlong; Li, Qihan; Liu, Longding

    2016-03-01

    Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus. PMID:26775814

  17. Whole-genome approach implicates CD44 in cellular resistance to carboplatin

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    Shukla Sunita J

    2009-01-01

    Full Text Available Abstract Carboplatin is a chemotherapeutic agent used in the management of many cancers, yet treatment is limited by resistance and toxicities. To achieve a better understanding of the genetic contribution to carboplatin resistance or toxicities, lymphoblastoid cell lines from 34 large Centre d'Etude du Polymorphisme Humain pedigrees were utilised to evaluate interindividual variation in carboplatin cytotoxicity. Significant heritability, ranging from 0.17-0.36 (p = 1 × 10-7 to 9 × 10-4, was found for cell growth inhibition following 72-hour treatment at each carboplatin concentration (10, 20, 40 and 80 μM and IC50 (concentration for 50 per cent cell growth inhibition. Linkage analysis revealed 11 regions with logarithm of odds (LOD scores greater than 1.5. The highest LOD score on chromosome 11 (LOD = 3.36, p = 4.2 × 10-5 encompasses 65 genes within the 1 LOD confidence interval for the carboplatin IC50. We further analysed the IC50 phenotype with a linkage-directed association analysis using 71 unrelated HapMap and Perlegen cell lines and identified 18 single nucleotide polymorphisms within eight genes that were significantly associated with the carboplatin IC50 (p -5; false discovery rate 50 values of the eight associated genes, which identified the most significant correlation between CD44 expression and IC50 (r2 = 0.20; p = 6 × 10-4. The quantitative real-time polymerase chain reaction further confirmed a statistically significant difference in CD44 expression levels between carboplatin-resistant and -sensitive cell lines (p = 5.9 × 10-3. Knockdown of CD44 expression through small interfering RNA resulted in increased cellular sensitivity to carboplatin (p CD44 as being important in conferring cellular resistance to carboplatin.

  18. Whole genome evaluation of horizontal transfers in the pathogenic fungus Aspergillus fumigatus

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    Deschavanne Patrick

    2010-03-01

    Full Text Available Abstract Background Numerous cases of horizontal transfers (HTs have been described for eukaryote genomes, but in contrast to prokaryote genomes, no whole genome evaluation of HTs has been carried out. This is mainly due to a lack of parametric methods specially designed to take the intrinsic heterogeneity of eukaryote genomes into account. We applied a simple and tested method based on local variations of genomic signatures to analyze the genome of the pathogenic fungus Aspergillus fumigatus. Results We detected 189 atypical regions containing 214 genes, accounting for about 1 Mb of DNA sequences. However, the fraction of atypical DNA detected was smaller than the average amount detected in the same conditions in prokaryote genomes (3.1% vs 5.6%. It appeared that about one third of these regions contained no annotated genes, a proportion far greater than in prokaryote genomes. When analyzing the origin of these HTs by comparing their signatures to a home made database of species signatures, 3 groups of donor species emerged: bacteria (40%, fungi (25%, and viruses (22%. It is to be noticed that though inter-domain exchanges are confirmed, we only put in evidence very few exchanges between eukaryotic kingdoms. Conclusions In conclusion, we demonstrated that HTs are not negligible in eukaryote genomes, bearing in mind that in our stringent conditions this amount is a floor value, though of a lesser extent than in prokaryote genomes. The biological mechanisms underlying those transfers remain to be elucidated as well as the biological functions of the transferred genes.

  19. Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing

    Science.gov (United States)

    Hua, Xing; Zeller, Georg; Sunagawa, Shinichi; Voigt, Anita Y.; Hercog, Rajna; Goedert, James J.; Shi, Jianxin; Bork, Peer; Sinha, Rashmi

    2016-01-01

    Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect

  20. Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata.

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    Marco Fracassetti

    Full Text Available Sequencing pooled DNA of multiple individuals from a population instead of sequencing individuals separately has become popular due to its cost-effectiveness and simple wet-lab protocol, although some criticism of this approach remains. Here we validated a protocol for pooled whole-genome re-sequencing (Pool-seq of Arabidopsis lyrata libraries prepared with low amounts of DNA (1.6 ng per individual. The validation was based on comparing single nucleotide polymorphism (SNP frequencies obtained by pooling with those obtained by individual-based Genotyping By Sequencing (GBS. Furthermore, we investigated the effect of sample number, sequencing depth per individual and variant caller on population SNP frequency estimates. For Pool-seq data, we compared frequency estimates from two SNP callers, VarScan and Snape; the former employs a frequentist SNP calling approach while the latter uses a Bayesian approach. Results revealed concordance correlation coefficients well above 0.8, confirming that Pool-seq is a valid method for acquiring population-level SNP frequency data. Higher accuracy was achieved by pooling more samples (25 compared to 14 and working with higher sequencing depth (4.1× per individual compared to 1.4× per individual, which increased the concordance correlation coefficient to 0.955. The Bayesian-based SNP caller produced somewhat higher concordance correlation coefficients, particularly at low sequencing depth. We recommend pooling at least 25 individuals combined with sequencing at a depth of 100× to produce satisfactory frequency estimates for common SNPs (minor allele frequency above 0.05.

  1. Whole Genome Analysis of Leptospira licerasiae Provides Insight into Leptospiral Evolution and Pathogenicity

    Science.gov (United States)

    Selengut, Jeremy D.; Harkins, Derek M.; Patra, Kailash P.; Moreno, Angelo; Lehmann, Jason S.; Purushe, Janaki; Sanka, Ravi; Torres, Michael; Webster, Nicholas J.; Vinetz, Joseph M.; Matthias, Michael A.

    2012-01-01

    The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835) provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae) that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010T and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT). Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for its infectiousness

  2. Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing.

    Science.gov (United States)

    Vogtmann, Emily; Hua, Xing; Zeller, Georg; Sunagawa, Shinichi; Voigt, Anita Y; Hercog, Rajna; Goedert, James J; Shi, Jianxin; Bork, Peer; Sinha, Rashmi

    2016-01-01

    Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect

  3. Whole-genome SNP association analysis of reproduction traits in the Finnish Landrace pig breed

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    Uimari Pekka

    2011-12-01

    Full Text Available Abstract Background Good genetic progress for pig reproduction traits has been achieved using a quantitative genetics-based multi-trait BLUP evaluation system. At present, whole-genome single nucleotide polymorphisms (SNP panels provide a new tool for pig selection. The purpose of this study was to identify SNP associated with reproduction traits in the Finnish Landrace pig breed using the Illumina PorcineSNP60 BeadChip. Methods Association of each SNP with different traits was tested with a weighted linear model, using SNP genotype as a covariate and animal as a random variable. Deregressed estimated breeding values of the progeny tested boars were used as the dependent variable and weights were based on their reliabilities. Statistical significance of the associations was based on Bonferroni-corrected P-values. Results Deregressed estimated breeding values were available for 328 genotyped boars. Of the 62 163 SNP in the chip, 57 868 SNP had a call rate > 0.9 and 7 632 SNP were monomorphic. Statistically significant results (P-value P-value P-value = 1.69E-08 more than unfavourable double homozygote animals. A region on chromosome 9 (66 Mb was statistically significant for piglet mortality between birth and weaning in later parity (0.44 piglets between homozygotes, P-value = 6.94E-08. Conclusions Three separate regions on chromosome 9 gave significant results for litter size and pig mortality. The frequencies of favourable alleles of the significant SNP are moderate in the Finnish Landrace population and these SNP are thus valuable candidates for possible marker-assisted selection.

  4. Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform

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    Shepherd Lara D

    2010-09-01

    Full Text Available Abstract Background Complete chloroplast genome sequences provide a valuable source of molecular markers for studies in molecular ecology and evolution of plants. To obtain complete genome sequences, recent studies have made use of the polymerase chain reaction to amplify overlapping fragments from conserved gene loci. However, this approach is time consuming and can be more difficult to implement where gene organisation differs among plants. An alternative approach is to first isolate chloroplasts and then use the capacity of high-throughput sequencing to obtain complete genome sequences. We report our findings from studies of the latter approach, which used a simple chloroplast isolation procedure, multiply-primed rolling circle amplification of chloroplast DNA, Illumina Genome Analyzer II sequencing, and de novo assembly of paired-end sequence reads. Results A modified rapid chloroplast isolation protocol was used to obtain plant DNA that was enriched for chloroplast DNA, but nevertheless contained nuclear and mitochondrial DNA. Multiply-primed rolling circle amplification of this mixed template produced sufficient quantities of chloroplast DNA, even when the amount of starting material was small, and improved the template quality for Illumina Genome Analyzer II (hereafter Illumina GAII sequencing. We demonstrate, using independent samples of karaka (Corynocarpus laevigatus, that there is high fidelity in the sequence obtained from this template. Although less than 20% of our sequenced reads could be mapped to chloroplast genome, it was relatively easy to assemble complete chloroplast genome sequences from the mixture of nuclear, mitochondrial and chloroplast reads. Conclusions We report successful whole genome sequencing of chloroplast DNA from karaka, obtained efficiently and with high fidelity.

  5. Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp.

    Science.gov (United States)

    Zhao, S; Tyson, G H; Chen, Y; Li, C; Mukherjee, S; Young, S; Lam, C; Folster, J P; Whichard, J M; McDermott, P F

    2016-01-01

    The objectives of this study were to identify antimicrobial resistance genotypes for Campylobacter and to evaluate the correlation between resistance phenotypes and genotypes using in vitro antimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114 Campylobacter species isolates (82 C. coli and 32 C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, including tet(O), blaOXA-61, catA, lnu(C), aph(2″)-Ib, aph(2″)-Ic, aph(2')-If, aph(2″)-Ig, aph(2″)-Ih, aac(6')-Ie-aph(2″)-Ia, aac(6')-Ie-aph(2″)-If, aac(6')-Im, aadE, sat4, ant(6'), aad9, aph(3')-Ic, and aph(3')-IIIa, and mutations in two housekeeping genes (gyrA and 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs. PMID:26519386

  6. Genome-wide association study for longevity with whole-genome sequencing in 3 cattle breeds.

    Science.gov (United States)

    Zhang, Qianqian; Guldbrandtsen, Bernt; Thomasen, Jørn Rind; Lund, Mogens Sandø; Sahana, Goutam

    2016-09-01

    Longevity is an important economic trait in dairy production. Improvements in longevity could increase the average number of lactations per cow, thereby affecting the profitability of the dairy cattle industry. Improved longevity for cows reduces the replacement cost of stock and enables animals to achieve the highest production period. Moreover, longevity is an indirect indicator of animal welfare. Using whole-genome sequencing variants in 3 dairy cattle breeds, we carried out an association study and identified 7 genomic regions in Holstein and 5 regions in Red Dairy Cattle that were associated with longevity. Meta-analyses of 3 breeds revealed 2 significant genomic regions, located on chromosomes 6 (META-CHR6-88MB) and 18 (META-CHR18-58MB). META-CHR6-88MB overlaps with 2 known genes: neuropeptide G-protein coupled receptor (NPFFR2; 89,052,210-89,059,348 bp) and vitamin D-binding protein precursor (GC; 88,695,940-88,739,180 bp). The NPFFR2 gene was previously identified as a candidate gene for mastitis resistance. META-CHR18-58MB overlaps with zinc finger protein 717 (ZNF717; 58,130,465-58,141,877 bp) and zinc finger protein 613 (ZNF613; 58,115,782-58,117,110 bp), which have been associated with calving difficulties. Information on longevity-associated genomic regions could be used to find causal genes/variants influencing longevity and exploited to improve the reliability of genomic prediction. PMID:27289149

  7. Whole Genome Sequencing Increases Molecular Diagnostic Yield Compared with Current Diagnostic Testing for Inherited Retinal Disease

    Science.gov (United States)

    Ellingford, Jamie M.; Barton, Stephanie; Bhaskar, Sanjeev; Williams, Simon G.; Sergouniotis, Panagiotis I.; O'Sullivan, James; Lamb, Janine A.; Perveen, Rahat; Hall, Georgina; Newman, William G.; Bishop, Paul N.; Roberts, Stephen A.; Leach, Rick; Tearle, Rick; Bayliss, Stuart; Ramsden, Simon C.; Nemeth, Andrea H.; Black, Graeme C.M.

    2016-01-01

    Purpose To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD). Design Case series. Participants A total of 562 patients diagnosed with IRD. Methods We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data. Main Outcome Measures Diagnostic yield of genomic testing. Results Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15–45) uplift in diagnostic yield. Conclusions We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS. PMID:26872967

  8. Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing.

    Directory of Open Access Journals (Sweden)

    Emily Vogtmann

    Full Text Available Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient

  9. Whole genome analysis of Leptospira licerasiae provides insight into leptospiral evolution and pathogenicity.

    Directory of Open Access Journals (Sweden)

    Jessica N Ricaldi

    Full Text Available The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835 provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010(T and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT. Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for

  10. Quantification of trace-level DNA by real-time whole genome amplification.

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    Min-Jung Kang

    Full Text Available Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES, an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

  11. Microbiota present in cystic fibrosis lungs as revealed by whole genome sequencing.

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    Philippe M Hauser

    Full Text Available Determination of the precise composition and variation of microbiota in cystic fibrosis lungs is crucial since chronic inflammation due to microorganisms leads to lung damage and ultimately, death. However, this constitutes a major technical challenge. Culturing of microorganisms does not provide a complete representation of a microbiota, even when using culturomics (high-throughput culture. So far, only PCR-based metagenomics have been investigated. However, these methods are biased towards certain microbial groups, and suffer from uncertain quantification of the different microbial domains. We have explored whole genome sequencing (WGS using the Illumina high-throughput technology applied directly to DNA extracted from sputa obtained from two cystic fibrosis patients. To detect all microorganism groups, we used four procedures for DNA extraction, each with a different lysis protocol. We avoided biases due to whole DNA amplification thanks to the high efficiency of current Illumina technology. Phylogenomic classification of the reads by three different methods produced similar results. Our results suggest that WGS provides, in a single analysis, a better qualitative and quantitative assessment of microbiota compositions than cultures and PCRs. WGS identified a high quantity of Haemophilus spp. (patient 1 or Staphylococcus spp. plus Streptococcus spp. (patient 2 together with low amounts of anaerobic (Veillonella, Prevotella, Fusobacterium and aerobic bacteria (Gemella, Moraxella, Granulicatella. WGS suggested that fungal members represented very low proportions of the microbiota, which were detected by cultures and PCRs because of their selectivity. The future increase of reads' sizes and decrease in cost should ensure the usefulness of WGS for the characterisation of microbiota.

  12. Inference of gorilla demographic and selective history from whole-genome sequence data.

    Science.gov (United States)

    McManus, Kimberly F; Kelley, Joanna L; Song, Shiya; Veeramah, Krishna R; Woerner, August E; Stevison, Laurie S; Ryder, Oliver A; Ape Genome Project, Great; Kidd, Jeffrey M; Wall, Jeffrey D; Bustamante, Carlos D; Hammer, Michael F

    2015-03-01

    Although population-level genomic sequence data have been gathered extensively for humans, similar data from our closest living relatives are just beginning to emerge. Examination of genomic variation within great apes offers many opportunities to increase our understanding of the forces that have differentially shaped the evolutionary history of hominid taxa. Here, we expand upon the work of the Great Ape Genome Project by analyzing medium to high coverage whole-genome sequences from 14 western lowland gorillas (Gorilla gorilla gorilla), 2 eastern lowland gorillas (G. beringei graueri), and a single Cross River individual (G. gorilla diehli). We infer that the ancestors of western and eastern lowland gorillas diverged from a common ancestor approximately 261 ka, and that the ancestors of the Cross River population diverged from the western lowland gorilla lineage approximately 68 ka. Using a diffusion approximation approach to model the genome-wide site frequency spectrum, we infer a history of western lowland gorillas that includes an ancestral population expansion of 1.4-fold around 970 ka and a recent 5.6-fold contraction in population size 23 ka. The latter may correspond to a major reduction in African equatorial forests around the Last Glacial Maximum. We also analyze patterns of variation among western lowland gorillas to identify several genomic regions with strong signatures of recent selective sweeps. We find that processes related to taste, pancreatic and saliva secretion, sodium ion transmembrane transport, and cardiac muscle function are overrepresented in genomic regions predicted to have experienced recent positive selection. PMID:25534031

  13. Examining phylogenetic relationships of Erwinia and Pantoea species using whole genome sequence data.

    Science.gov (United States)

    Zhang, Yucheng; Qiu, Sai

    2015-11-01

    The genera Erwinia and Pantoea contain species that are devastating plant pathogens, non-pathogen epiphytes, and opportunistic human pathogens. However, some controversies persist in the taxonomic classification of these two closely related genera. The phylogenomic analysis of these two genera was investigated via a comprehensive analysis of 25 Erwinia genomes and 23 Pantoea genomes. Single-copy orthologs could be extracted from the Erwinia/Pantoea core-genome to reconstruct the Erwinia/Pantoea phylogeny. This tree has strong bootstrap support for almost all branches. We also estimated the in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) values between each genome; strains from the same species showed ANI values ≥96% and isDDH values >70%. These data confirm that whole genome sequence data provides a powerful tool to resolve the complex taxonomic questions of Erwinia/Pantoea, e.g. Pantoea agglomerans 299R was not clustered into a single group with other P. agglomerans strains, and the ANI values and isDDH values between them were agglomerans 299R should not be classified into the P. agglomerans species. In addition, another strain (Pantoea sp. At_9b) was identified that may represent a novel Pantoea species. We also evaluated the performance of six commonly used housekeeping genes (atpD, carA, gyrB, infB, recA, and rpoB) in phylogenetic inference. A single gene was not enough to obtain a reliable species tree, and it was necessary to use the multilocus sequence analysis of the six marker genes to recover the Erwinia/Pantoea phylogeny. PMID:26296376

  14. Economic evidence on identifying clinically actionable findings with whole-genome sequencing: a scoping review.

    Science.gov (United States)

    Douglas, Michael P; Ladabaum, Uri; Pletcher, Mark J; Marshall, Deborah A; Phillips, Kathryn A

    2016-02-01

    The American College of Medical Genetics and Genomics (ACMG) recommends that mutations in 56 genes for 24 conditions are clinically actionable and should be reported as secondary findings after whole-genome sequencing (WGS). Our aim was to identify published economic evaluations of detecting mutations in these genes among the general population or among targeted/high-risk populations and conditions and identify gaps in knowledge. A targeted PubMed search from 1994 through November 2014 was performed, and we included original, English-language articles reporting cost-effectiveness or a cost-to-utility ratio or net benefits/benefit-cost focused on screening (not treatment) for conditions and genes listed by the ACMG. Articles were screened, classified as targeting a high-risk or general population, and abstracted by two reviewers. General population studies were evaluated for actual cost-effectiveness measures (e.g., incremental cost-effectiveness ratios (ICER)), whereas studies of targeted populations were evaluated for whether at least one scenario proposed was cost-effective (e.g., ICER of ≤$100,000 per life-year or quality-adjusted life-year gained). A total of 607 studies were identified, and 32 relevant studies were included. Identified studies addressed fewer than one-third (7 of 24; 29%) of the ACMG conditions. The cost-effectiveness of screening in the general population was examined for only 2 of 24 conditions (8%). The cost-effectiveness of most genetic findings that the ACMG recommends for return has not been evaluated in economic studies or in the context of screening in the general population. The individual studies do not directly address the cost-effectiveness of WGS. PMID:25996638

  15. An assessment of population structure in eight breeds of cattle using a whole genome SNP panel

    Directory of Open Access Journals (Sweden)

    Gao Chuan

    2008-05-01

    Full Text Available Abstract Background Analyses of population structure and breed diversity have provided insight into the origin and evolution of cattle. Previously, these studies have used a low density of microsatellite markers, however, with the large number of single nucleotide polymorphism markers that are now available, it is possible to perform genome wide population genetic analyses in cattle. In this study, we used a high-density panel of SNP markers to examine population structure and diversity among eight cattle breeds sampled from Bos indicus and Bos taurus. Results Two thousand six hundred and forty one single nucleotide polymorphisms (SNPs spanning all of the bovine autosomal genome were genotyped in Angus, Brahman, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black, Limousin and Nelore cattle. Population structure was examined using the linkage model in the program STRUCTURE and Fst estimates were used to construct a neighbor-joining tree to represent the phylogenetic relationship among these breeds. Conclusion The whole-genome SNP panel identified several levels of population substructure in the set of examined cattle breeds. The greatest level of genetic differentiation was detected between the Bos taurus and Bos indicus breeds. When the Bos indicus breeds were excluded from the analysis, genetic differences among beef versus dairy and European versus Asian breeds were detected among the Bos taurus breeds. Exploration of the number of SNP loci required to differentiate between breeds showed that for 100 SNP loci, individuals could only be correctly clustered into breeds 50% of the time, thus a large number of SNP markers are required to replace the 30 microsatellite markers that are currently commonly used in genetic diversity studies.

  16. Flexible positions, managed hopes: the promissory bioeconomy of a whole genome sequencing cancer study.

    Science.gov (United States)

    Haase, Rachel; Michie, Marsha; Skinner, Debra

    2015-04-01

    Genomic research has rapidly expanded its scope and ambition over the past decade, promoted by both public and private sectors as having the potential to revolutionize clinical medicine. This promissory bioeconomy of genomic research and technology is generated by, and in turn generates, the hopes and expectations shared by investors, researchers and clinicians, patients, and the general public alike. Examinations of such bioeconomies have often focused on the public discourse, media representations, and capital investments that fuel these "regimes of hope," but also crucial are the more intimate contexts of small-scale medical research, and the private hopes, dreams, and disappointments of those involved. Here we examine one local site of production in a university-based clinical research project that sought to identify novel cancer predisposition genes through whole genome sequencing in individuals at high risk for cancer. In-depth interviews with 24 adults who donated samples to the study revealed an ability to shift flexibly between positioning themselves as research participants on the one hand, and as patients or as family members of patients, on the other. Similarly, interviews with members of the research team highlighted the dual nature of their positions as researchers and as clinicians. For both parties, this dual positioning shaped their investment in the project and valuing of its possible outcomes. In their narratives, all parties shifted between these different relational positions as they managed hopes and expectations for the research project. We suggest that this flexibility facilitated study implementation and participation in the face of potential and probable disappointment on one or more fronts, and acted as a key element in the resilience of this local promissory bioeconomy. We conclude that these multiple dimensions of relationality and positionality are inherent and essential in the creation of any complex economy, "bio" or otherwise. PMID

  17. Whole genome sequences of three Treponema pallidum ssp. pertenue strains: yaws and syphilis treponemes differ in less than 0.2% of the genome sequence.

    Directory of Open Access Journals (Sweden)

    Darina Cejková

    2012-01-01

    Full Text Available BACKGROUND: The yaws treponemes, Treponema pallidum ssp. pertenue (TPE strains, are closely related to syphilis causing strains of Treponema pallidum ssp. pallidum (TPA. Both yaws and syphilis are distinguished on the basis of epidemiological characteristics, clinical symptoms, and several genetic signatures of the corresponding causative agents. METHODOLOGY/PRINCIPAL FINDINGS: To precisely define genetic differences between TPA and TPE, high-quality whole genome sequences of three TPE strains (Samoa D, CDC-2, Gauthier were determined using next-generation sequencing techniques. TPE genome sequences were compared to four genomes of TPA strains (Nichols, DAL-1, SS14, Chicago. The genome structure was identical in all three TPE strains with similar length ranging between 1,139,330 bp and 1,139,744 bp. No major genome rearrangements were found when compared to the four TPA genomes. The whole genome nucleotide divergence (d(A between TPA and TPE subspecies was 4.7 and 4.8 times higher than the observed nucleotide diversity (π among TPA and TPE strains, respectively, corresponding to 99.8% identity between TPA and TPE genomes. A set of 97 (9.9% TPE genes encoded proteins containing two or more amino acid replacements or other major sequence changes. The TPE divergent genes were mostly from the group encoding potential virulence factors and genes encoding proteins with unknown function. CONCLUSIONS/SIGNIFICANCE: Hypothetical genes, with genetic differences, consistently found between TPE and TPA strains are candidates for syphilitic treponemes virulence factors. Seventeen TPE genes were predicted under positive selection, and eleven of them coded either for predicted exported proteins or membrane proteins suggesting their possible association with the cell surface. Sequence changes between TPE and TPA strains and changes specific to individual strains represent suitable targets for subspecies- and strain-specific molecular diagnostics.

  18. Whole Genome Sequences of Three Treponema pallidum ssp. pertenue Strains: Yaws and Syphilis Treponemes Differ in Less than 0.2% of the Genome Sequence

    Science.gov (United States)

    Chen, Lei; Pospíšilová, Petra; Strouhal, Michal; Qin, Xiang; Mikalová, Lenka; Norris, Steven J.; Muzny, Donna M.; Gibbs, Richard A.; Fulton, Lucinda L.; Sodergren, Erica; Weinstock, George M.; Šmajs, David

    2012-01-01

    Background The yaws treponemes, Treponema pallidum ssp. pertenue (TPE) strains, are closely related to syphilis causing strains of Treponema pallidum ssp. pallidum (TPA). Both yaws and syphilis are distinguished on the basis of epidemiological characteristics, clinical symptoms, and several genetic signatures of the corresponding causative agents. Methodology/Principal Findings To precisely define genetic differences between TPA and TPE, high-quality whole genome sequences of three TPE strains (Samoa D, CDC-2, Gauthier) were determined using next-generation sequencing techniques. TPE genome sequences were compared to four genomes of TPA strains (Nichols, DAL-1, SS14, Chicago). The genome structure was identical in all three TPE strains with similar length ranging between 1,139,330 bp and 1,139,744 bp. No major genome rearrangements were found when compared to the four TPA genomes. The whole genome nucleotide divergence (dA) between TPA and TPE subspecies was 4.7 and 4.8 times higher than the observed nucleotide diversity (π) among TPA and TPE strains, respectively, corresponding to 99.8% identity between TPA and TPE genomes. A set of 97 (9.9%) TPE genes encoded proteins containing two or more amino acid replacements or other major sequence changes. The TPE divergent genes were mostly from the group encoding potential virulence factors and genes encoding proteins with unknown function. Conclusions/Significance Hypothetical genes, with genetic differences, consistently found between TPE and TPA strains are candidates for syphilitic treponemes virulence factors. Seventeen TPE genes were predicted under positive selection, and eleven of them coded either for predicted exported proteins or membrane proteins suggesting their possible association with the cell surface. Sequence changes between TPE and TPA strains and changes specific to individual strains represent suitable targets for subspecies- and strain-specific molecular diagnostics. PMID:22292095

  19. Whole genome sequence and genome annotation of Colletotrichum acutatum, causal agent of anthracnose in pepper plants in South Korea

    Directory of Open Access Journals (Sweden)

    Joon-Hee Han

    2016-06-01

    Full Text Available Colletotrichum acutatum is a destructive fungal pathogen which causes anthracnose in a wide range of crops. Here we report the whole genome sequence and annotation of C. acutatum strain KC05, isolated from an infected pepper in Kangwon, South Korea. Genomic DNA from the KC05 strain was used for the whole genome sequencing using a PacBio sequencer and the MiSeq system. The KC05 genome was determined to be 52,190,760 bp in size with a G + C content of 51.73% in 27 scaffolds and to contain 13,559 genes with an average length of 1516 bp. Gene prediction and annotation were performed by incorporating RNA-Seq data. The genome sequence of the KC05 was deposited at DDBJ/ENA/GenBank under the accession number LUXP00000000.

  20. Implementation of Nationwide Real-time Whole-genome Sequencing to Enhance Listeriosis Outbreak Detection and Investigation.

    Science.gov (United States)

    Jackson, Brendan R; Tarr, Cheryl; Strain, Errol; Jackson, Kelly A; Conrad, Amanda; Carleton, Heather; Katz, Lee S; Stroika, Steven; Gould, L Hannah; Mody, Rajal K; Silk, Benjamin J; Beal, Jennifer; Chen, Yi; Timme, Ruth; Doyle, Matthew; Fields, Angela; Wise, Matthew; Tillman, Glenn; Defibaugh-Chavez, Stephanie; Kucerova, Zuzana; Sabol, Ashley; Roache, Katie; Trees, Eija; Simmons, Mustafa; Wasilenko, Jamie; Kubota, Kristy; Pouseele, Hannes; Klimke, William; Besser, John; Brown, Eric; Allard, Marc; Gerner-Smidt, Peter

    2016-08-01

    Listeria monocytogenes (Lm) causes severe foodborne illness (listeriosis). Previous molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), were critical in detecting outbreaks that led to food safety improvements and declining incidence, but PFGE provides limited genetic resolution. A multiagency collaboration began performing real-time, whole-genome sequencing (WGS) on all US Lm isolates from patients, food, and the environment in September 2013, posting sequencing data into a public repository. Compared with the year before the project began, WGS, combined with epidemiologic and product trace-back data, detected more listeriosis clusters and solved more outbreaks (2 outbreaks in pre-WGS year, 5 in WGS year 1, and 9 in year 2). Whole-genome multilocus sequence typing and single nucleotide polymorphism analyses provided equivalent phylogenetic relationships relevant to investigations; results were most useful when interpreted in context of epidemiological data. WGS has transformed listeriosis outbreak surveillance and is being implemented for other foodborne pathogens. PMID:27090985

  1. Whole genome sequence and genome annotation of Colletotrichum acutatum, causal agent of anthracnose in pepper plants in South Korea.

    Science.gov (United States)

    Han, Joon-Hee; Chon, Jae-Kyung; Ahn, Jong-Hwa; Choi, Ik-Young; Lee, Yong-Hwan; Kim, Kyoung Su

    2016-06-01

    Colletotrichum acutatum is a destructive fungal pathogen which causes anthracnose in a wide range of crops. Here we report the whole genome sequence and annotation of C. acutatum strain KC05, isolated from an infected pepper in Kangwon, South Korea. Genomic DNA from the KC05 strain was used for the whole genome sequencing using a PacBio sequencer and the MiSeq system. The KC05 genome was determined to be 52,190,760 bp in size with a G + C content of 51.73% in 27 scaffolds and to contain 13,559 genes with an average length of 1516 bp. Gene prediction and annotation were performed by incorporating RNA-Seq data. The genome sequence of the KC05 was deposited at DDBJ/ENA/GenBank under the accession number LUXP00000000. PMID:27114908

  2. Identification of Ohnolog Genes Originating from Whole Genome Duplication in Early Vertebrates, Based on Synteny Comparison across Multiple Genomes

    OpenAIRE

    Priya Singh, Param; Arora, Jatin; Isambert, Hervé

    2015-01-01

    Whole genome duplications (WGD) have now been firmly established in all major eukary-otic kingdoms. In particular, all vertebrates descend from two rounds of WGDs, that occurred in their jawless ancestor some 500 MY ago. Paralogs retained from WGD, also coined 'ohnologs' after Susumu Ohno, have been shown to be typically associated with development, signaling and gene regulation. Ohnologs, which amount to about 20 to 35% of genes in the human genome, have also been shown to be prone to domina...

  3. “We don’t know her history, her background”: Adoptive parents’ perspectives on whole genome sequencing results

    OpenAIRE

    Crouch, Julia; Yu, Joon-Ho; Shankar, Aditi G.; Tabor, Holly K.

    2014-01-01

    Exome sequencing and whole genome sequencing (ES/WGS) can provide parents with a wide range of genetic information about their children, and adoptive parents may have unique issues to consider regarding possible access to this information. The few papers published on adoption and genetics have focused on targeted genetic testing of children in the pre-adoption context. There are no data on adoptive parent perspectives about pediatric ES/WGS, including their preferences about different kinds o...

  4. Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing

    OpenAIRE

    Outhred, Alexander C.; Holmes, Nadine; Sadsad, Rosemarie; Martinez, Elena; Jelfs, Peter; Hill-Cawthorne, Grant A.; Gilbert, Gwendolyn L.; Marais, Ben J.; Sintchenko, Vitali

    2016-01-01

    Background Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways. Methods We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed ...

  5. Arapan-S: a fast and highly accurate whole-genome assembly software for viruses and small genomes

    OpenAIRE

    Sahli Mohammed; Shibuya Tetsuo

    2012-01-01

    Abstract Background Genome assembly is considered to be a challenging problem in computational biology, and has been studied extensively by many researchers. It is extremely difficult to build a general assembler that is able to reconstruct the original sequence instead of many contigs. However, we believe that creating specific assemblers, for solving specific cases, will be much more fruitful than creating general assemblers. Findings In this paper, we present Arapan-S, a whole-genome assem...

  6. What are people willing to pay for whole-genome sequencing information, and who decides what they receive?

    OpenAIRE

    Marshall, DA; Gonzalez, JM; Johnson, FR; Macdonald, KV; Pugh, A; Douglas, MP; Phillips, KA

    2016-01-01

    Whole-genome sequencing (WGS) can be used as a powerful diagnostic tool as well as for screening, but it may lead to anxiety, unnecessary testing, and overtreatment. Current guidelines suggest reporting clinically actionable secondary findings when diagnostic testing is performed. We examined preferences for receiving WGS results.A US nationally representative survey (n = 410 adults) was used to rank preferences for who decides (an expert panel, your doctor, you) which WGS results are reporte...

  7. Implications of using whole genome sequencing to test unselected populations for high risk breast cancer genes: a modelling study

    OpenAIRE

    Warren-Gash, Charlotte; Kroese, Mark; Burton, Hilary; Pharoah, Paul

    2016-01-01

    Background The decision to test for high risk breast cancer gene mutations is traditionally based on risk scores derived from age, family and personal cancer history. Next generation sequencing technologies such as whole genome sequencing (WGS) make wider population testing more feasible. In the UK’s 100,000 Genomes Project, mutations in 16 genes including BRCA1 and BRCA2 are to be actively sought regardless of clinical presentation. The implications of deploying this approach at scale for pa...

  8. Whole-genome sequencing to establish relapse or re-infection with Mycobacterium tuberculosis : a retrospective observational study

    OpenAIRE

    Bryant, Josephine M; Harris, Simon R; Parkhill, Julian; Dawson, Rodney; Diacon, Andreas; van Helden, Paul; Pym, Alex; Ahmad Mahayiddin, Aziah; Chuchottaworn, C; Sanne, Ian M; Louw, Cheryl; Boeree, Martin J.; Hoelscher, Michael; Timothy D. McHugh; Bateson, Anna L C

    2013-01-01

    Summary Background Recurrence of tuberculosis after treatment makes management difficult and is a key factor for determining treatment efficacy. Two processes can cause recurrence: relapse of the primary infection or re-infection with an exogenous strain. Although re-infection can and does occur, its importance to tuberculosis epidemiology and its biological basis is still debated. We used whole-genome sequencing—which is more accurate than conventional typing used to date—to assess the frequ...

  9. Whole-genome sequence of Sunxiuqinia dokdonensis DH1(T), isolated from deep sub-seafloor sediment in Dokdo Island.

    Science.gov (United States)

    Lim, Sooyeon; Chang, Dong-Ho; Kim, Byoung-Chan

    2016-09-01

    Sunxiuqinia dokdonensis DH1(T) was isolated from deep sub-seafloor sediment at a depth of 900 m below the seafloor off Seo-do (the west part of Dokdo Island) in the East Sea of the Republic of Korea and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession LGIA00000000. PMID:27437183

  10. Whole-genome sequence of Sunxiuqinia dokdonensis DH1T, isolated from deep sub-seafloor sediment in Dokdo Island

    Directory of Open Access Journals (Sweden)

    Sooyeon Lim

    2016-09-01

    Full Text Available Sunxiuqinia dokdonensis DH1T was isolated from deep sub-seafloor sediment at a depth of 900 m below the seafloor off Seo-do (the west part of Dokdo Island in the East Sea of the Republic of Korea and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession LGIA00000000.

  11. PhyResSE: a Web Tool Delineating Mycobacterium tuberculosis Antibiotic Resistance and Lineage from Whole-Genome Sequencing Data

    OpenAIRE

    Feuerriegel, Silke; Schleusener, Viola; Beckert, Patrick; Kohl, Thomas A.; Miotto, Paolo; Cirillo, Daniela M; Cabibbe, Andrea M.; Niemann, Stefan; Fellenberg, Kurt

    2015-01-01

    Antibiotic-resistant tuberculosis poses a global threat, causing the deaths of hundreds of thousands of people annually. While whole-genome sequencing (WGS), with its unprecedented level of detail, promises to play an increasingly important role in diagnosis, data analysis is a daunting challenge. Here, we present a simple-to-use web service (free for academic use at http://phyresse.org). Delineating both lineage and resistance, it provides state-of-the-art methodology to life scientists and ...

  12. Whole genome sequencing of Halomonas sp. SUBG004 isolated from Little Rann of Kutch, a desert of India

    OpenAIRE

    Jigna H. Patel; Thaker, Vrinda S.

    2015-01-01

    A salt tolerant strain, designated as SUBG004, was isolated from the desert of India, Little Rann of Kutch. The organism is a Gram-negative, facultatively anaerobic and rod shaped bacterium. Chemotaxonomic and phylogenetic properties were consistent with its classification in the genus Halomonas. Here we report the whole genome sequence of Halomonas sp. SUBG004 deposited in DDBJ/EMBL/GenBank under accession number JPEU0100000 which provides insights for salt stress adaptation through betaine ...

  13. Whole genome sequencing of Halomonas sp. SUBG004 isolated from Little Rann of Kutch, a desert of India.

    Science.gov (United States)

    Patel, Jigna H; Thaker, Vrinda S

    2015-12-01

    A salt tolerant strain, designated as SUBG004, was isolated from the desert of India, Little Rann of Kutch. The organism is a Gram-negative, facultatively anaerobic and rod shaped bacterium. Chemotaxonomic and phylogenetic properties were consistent with its classification in the genus Halomonas. Here we report the whole genome sequence of Halomonas sp. SUBG004 deposited in DDBJ/EMBL/GenBank under accession number JPEU0100000 which provides insights for salt stress adaptation through betaine synthesis. PMID:26697321

  14. Whole genome sequencing of Halomonas sp. SUBG004 isolated from Little Rann of Kutch, a desert of India

    Directory of Open Access Journals (Sweden)

    Jigna H. Patel

    2015-12-01

    Full Text Available A salt tolerant strain, designated as SUBG004, was isolated from the desert of India, Little Rann of Kutch. The organism is a Gram-negative, facultatively anaerobic and rod shaped bacterium. Chemotaxonomic and phylogenetic properties were consistent with its classification in the genus Halomonas. Here we report the whole genome sequence of Halomonas sp. SUBG004 deposited in DDBJ/EMBL/GenBank under accession number JPEU0100000 which provides insights for salt stress adaptation through betaine synthesis.

  15. Whole-genome sequencing of bladder cancers reveals somatic CDKN1A mutations and clinicopathological associations with mutation burden

    OpenAIRE

    Cazier, J.-B.; Rao, S. R.; Mclean, C. M.; A. L. Walker; Wright, B J; Jaeger, E. E. M.; Kartsonaki, C.; Marsden, L.; Yau, C; Camps, C.; Kaisaki, P.; ,; Allan, Christopher; Attar, Moustafa; Bell, John

    2014-01-01

    Bladder cancers are a leading cause of death from malignancy. Molecular markers might predict disease progression and behaviour more accurately than the available prognostic factors. Here we use whole-genome sequencing to identify somatic mutations and chromosomal changes in 14 bladder cancers of different grades and stages. As well as detecting the known bladder cancer driver mutations, we report the identification of recurrent protein-inactivating mutations in CDKN1A and FAT1. The former ar...

  16. Whole-Genome Resequencing Analysis of Hanwoo and Yanbian Cattle to Identify Genome-Wide SNPs and Signatures of Selection

    OpenAIRE

    Choi, Jung-Woo; Choi, Bong-Hwan; Lee, Seung-Hwan; Lee, Seung-Soo; Kim, Hyeong-Cheol; Yu, Dayeong; Chung, Won-Hyong; Lee, Kyung-Tai; Chai, Han-Ha; Cho, Yong-Min; Lim, Dajeong

    2015-01-01

    Over the last 30 years, Hanwoo has been selectively bred to improve economically important traits. Hanwoo is currently the representative Korean native beef cattle breed, and it is believed that it shared an ancestor with a Chinese breed, Yanbian cattle, until the last century. However, these two breeds have experienced different selection pressures during recent decades. Here, we whole-genome sequenced 10 animals each of Hanwoo and Yanbian cattle (20 total) using the Illumina HiSeq 2000 sequ...

  17. Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

    OpenAIRE

    Hadfield, Stephen J.; Pachebat, Justin A; Swain, Martin T; Robinson, Guy; Cameron, Simon JS; Alexander, Jenna; Hegarty, Matthew J.; Elwin, Kristin; Chalmers, Rachel M.

    2015-01-01

    Background Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation...

  18. Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak

    OpenAIRE

    Saelens, Joseph W.; Lau-Bonilla, Dalia; Moller, Anneliese; Medina, Narda; Guzmán, Brenda; Calderón, Maylena; Herrera, Raúl; Sisk, Dana M.; Ana M Xet-Mull; Stout, Jason E.; Arathoon, Eduardo; Samayoa, Blanca; Tobin, David M.

    2015-01-01

    Summary Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from in...

  19. Identifying Gene Disruptions in Novel Balanced de novo Constitutional Translocations in Childhood Cancer Patients by Whole Genome Sequencing

    OpenAIRE

    Ritter, Deborah I.; Haines, Katherine; Cheung, Hannah; Davis, Caleb F.; Lau, Ching C.; Berg, Jonathan S.; Brown, Chester W.; Thompson, Patrick A.; Gibbs, Richard; Wheeler, David A.; Plon, Sharon E.

    2015-01-01

    Purpose We applied whole genome sequencing to children diagnosed with neoplasms and found to carry apparently balanced constitutional translocations, to discover novel genic disruptions. Methods We applied SV calling programs CREST, Break Dancer, SV-STAT and CGAP-CNV, and developed an annotative filtering strategy to achieve nucleotide resolution at the translocations. Results We identified the breakpoints for t(6;12) (p21.1;q24.31) disrupting HNF1A in a patient diagnosed with hepatic adenoma...

  20. Whole Genome Sequencing of Mycobacterium tuberculosis Reveals Slow Growth and Low Mutation Rates during Latent Infections in Humans

    OpenAIRE

    Roberto Colangeli; Vic L Arcus; Cursons, Ray T.; Ali Ruthe; Noel Karalus; Kathy Coley; Manning, Shannon D.; Soyeon Kim; Emily Marchiano; David Alland

    2014-01-01

    Very little is known about the growth and mutation rates of Mycobacterium tuberculosis during latent infection in humans. However, studies in rhesus macaques have suggested that latent infections have mutation rates that are higher than that observed during active tuberculosis disease. Elevated mutation rates are presumed risk factors for the development of drug resistance. Therefore, the investigation of mutation rates during human latency is of high importance. We performed whole genome mut...

  1. Whole-genome sequence of Clostridium lituseburense L74, isolated from the larval gut of the rhinoceros beetle, Trypoxylus dichotomus

    Directory of Open Access Journals (Sweden)

    Yookyung Lee

    2016-03-01

    Full Text Available Clostridium lituseburense L74 was isolated from the larval gut of the rhinoceros beetle, Trypoxylus dichotomus collected in Yeong-dong, Chuncheongbuk-do, South Korea and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession NZ_LITJ00000000.

  2. Whole-genome sequencing reveals small genomic regions of introgression in an introduced crater lake population of threespine stickleback.

    Science.gov (United States)

    Yoshida, Kohta; Miyagi, Ryutaro; Mori, Seiichi; Takahashi, Aya; Makino, Takashi; Toyoda, Atsushi; Fujiyama, Asao; Kitano, Jun

    2016-04-01

    Invasive species pose a major threat to biological diversity. Although introduced populations often experience population bottlenecks, some invasive species are thought to be originated from hybridization between multiple populations or species, which can contribute to the maintenance of high genetic diversity. Recent advances in genome sequencing enable us to trace the evolutionary history of invasive species even at whole-genome level and may help to identify the history of past hybridization that may be overlooked by traditional marker-based analysis. Here, we conducted whole-genome sequencing of eight threespine stickleback (Gasterosteus aculeatus) individuals, four from a recently introduced crater lake population and four of the putative source population. We found that both populations have several small genomic regions with high genetic diversity, which resulted from introgression from a closely related species (Gasterosteus nipponicus). The sizes of the regions were too small to be detected with traditional marker-based analysis or even some reduced-representation sequencing methods. Further amplicon sequencing revealed linkage disequilibrium around an introgression site, which suggests the possibility of selective sweep at the introgression site. Thus, interspecies introgression might predate introduction and increase genetic variation in the source population. Whole-genome sequencing of even a small number of individuals can therefore provide higher resolution inference of history of introduced populations. PMID:27069575

  3. Inferring regulatory elements from a whole genome. An analysis of Helicobacter pylori sigma(80) family of promoter signals.

    Science.gov (United States)

    Vanet, A; Marsan, L; Labigne, A; Sagot, M F

    2000-03-24

    Helicobacter pylori is adapted to life in a unique niche, the gastric epithelium of primates. Its promoters may therefore be different from those of other bacteria. Here, we determine motifs possibly involved in the recognition of such promoter sequences by the RNA polymerase using a new motif identification method. An important feature of this method is that the motifs are sought with the least possible assumptions about what they may look like. The method starts by considering the whole genome of H. pylori and attempts to infer directly from it a description for a family of promoters. Thus, this approach differs from searching for such promoters with a previously established description. The two algorithms are based on the idea of inferring motifs by flexibly comparing words in the sequences with an external object, instead of between themselves. The first algorithm infers single motifs, the second a combination of two motifs separated from one another by strictly defined, sterically constrained distances. Besides independently finding motifs known to be present in other bacteria, such as the Shine-Dalgarno sequence and the TATA-box, this approach suggests the existence in H. pylori of a new, combined motif, TTAAGC, followed optimally 21 bp downstream by TATAAT. Between these two motifs, there is in some cases another, TTTTAA or, less frequently, a repetition of TTAAGC separated optimally from the TATA-box by 12 bp. The combined motif TTAAGCx(21+/-2)TATAAT is present with no errors immediately upstream from the only two copies of the ribosomal 23 S-5 S RNA genes in H. pylori, and with one error upstream from the only two copies of the ribosomal 16 S RNA genes. The operons of both ribosomal RNA molecules are strongly expressed, representing an encouraging sign of the pertinence of the motifs found by the algorithms. In 25 cases out of a possible 30, the combined motif is found with no more than three substitutions immediately upstream from ribosomal proteins, or

  4. Transcriptional control in embryonic Drosophila midline guidance assessed through a whole genome approach

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    Tomancak Pavel

    2007-07-01

    Full Text Available Abstract Background During the development of the Drosophila central nervous system the process of midline crossing is orchestrated by a number of guidance receptors and ligands. Many key axon guidance molecules have been identified in both invertebrates and vertebrates, but the transcriptional regulation of growth cone guidance remains largely unknown. It is established that translational regulation plays a role in midline crossing, and there are indications that transcriptional regulation is also involved. To investigate this issue, we conducted a genome-wide study of transcription in Drosophila embryos using wild type and a number of well-characterized Drosophila guidance mutants and transgenics. We also analyzed a previously published microarray time course of Drosophila embryonic development with an axon guidance focus. Results Using hopach, a novel clustering method which is well suited to microarray data analysis, we identified groups of genes with similar expression patterns across guidance mutants and transgenics. We then systematically characterized the resulting clusters with respect to their relevance to axon guidance using two complementary controlled vocabularies: the Gene Ontology (GO and anatomical annotations of the Atlas of Pattern of Gene Expression (APoGE in situ hybridization database. The analysis indicates that regulation of gene expression does play a role in the process of axon guidance in Drosophila. We also find a strong link between axon guidance and hemocyte migration, a result that agrees with mounting evidence that axon guidance molecules are co-opted in vertebrate vascularization. Cell cyclin activity in the context of axon guidance is also suggested from our array data. RNA and protein expression patterns of cell cyclins in axon guidance mutants and transgenics support this possible link. Conclusion This study provides important insights into the regulation of axon guidance in vivo.

  5. Identification of Cell Cycle-regulated Genes in Fission YeastD⃞

    OpenAIRE

    Peng, Xu; Karuturi, R Krishna Murthy; Miller, Lance D.; Lin, Kui; Jia, Yonghui; Kondu, Pinar; Wang, Long; Wong, Lim-Soon; Liu, Edison T.; Balasubramanian, Mohan K.; Liu, Jianhua

    2005-01-01

    Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we id...

  6. Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda

    Science.gov (United States)

    Ssengooba, Willy; Meehan, Conor J.; Lukoye, Deus; Kasule, George William; Musisi, Kenneth; Joloba, Moses L.; Cobelens, Frank G.; de Jong, Bouke C.

    2016-01-01

    Understanding the circulating Mycobacterium tuberculosis resistance mutations is vital for better TB control strategies, especially to inform a new MDR-TB treatment programme. We complemented the phenotypic drug susceptibility testing (DST) based drug resistance surveys (DRSs) conducted in Uganda between 2008 and 2011 with Whole Genome Sequencing (WGS) of 90 Mycobacterium tuberculosis isolates phenotypically resistant to rifampicin and/or isoniazid to better understand the extent of drug resistance. A total of 31 (34.4 %) patients had MDR-TB, 5 (5.6 %) mono-rifampicin resistance and 54 (60.0 %) mono-isoniazid resistance by phenotypic DST. Pyrazinamide resistance mutations were identified in 32.3% of the MDR-TB patients. Resistance to injectable agents was detected in 4/90 (4.4%), and none to fluoroquinolones or novel drugs. Compensatory mutations in rpoC were identified in two patients. The sensitivity and specificity of drug resistance mutations compared to phenotypic DST were for rpoB 88.6% and 98.1%, katG 60.0% and 100%, fabG1 16.5% and 100%, katG and/or fabG1 71.8% and 100%, embCAB 63.0% and 82.5%, rrs 11.4% and 100%, rpsL 20.5% and 95.7% and rrs and/or rpsL 31.8% and 95.7%. Phylogenetic analysis showed dispersed MDR-TB isolate, with only one cluster of three Beijing family from South West Uganda. Among tuberculosis patients in Uganda, resistance beyond first-line drugs as well as compensatory mutations remain low, and MDR-TB isolates did not arise from a dominant clone. Our findings show the potential use of sequencing for complementing DRSs or surveillance in this setting, with good specificity compared to phenotypic DST. The reported high confidence mutations can be included in molecular assays, and population-based studies can track transmission of MDR-TB including the Beijing family strains in the South West of the country. PMID:26917365

  7. Whole genome resequencing of black Angus and Holstein cattle for SNP and CNV discovery

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    Stothard Paul

    2011-11-01

    Full Text Available Abstract Background One of the goals of livestock genomics research is to identify the genetic differences responsible for variation in phenotypic traits, particularly those of economic importance. Characterizing the genetic variation in livestock species is an important step towards linking genes or genomic regions with phenotypes. The completion of the bovine genome sequence and recent advances in DNA sequencing technology allow for in-depth characterization of the genetic variations present in cattle. Here we describe the whole-genome resequencing of two Bos taurus bulls from distinct breeds for the purpose of identifying and annotating novel forms of genetic variation in cattle. Results The genomes of a Black Angus bull and a Holstein bull were sequenced to 22-fold and 19-fold coverage, respectively, using the ABI SOLiD system. Comparisons of the sequences with the Btau4.0 reference assembly yielded 7 million single nucleotide polymorphisms (SNPs, 24% of which were identified in both animals. Of the total SNPs found in Holstein, Black Angus, and in both animals, 81%, 81%, and 75% respectively are novel. In-depth annotations of the data identified more than 16 thousand distinct non-synonymous SNPs (85% novel between the two datasets. Alignments between the SNP-altered proteins and orthologues from numerous species indicate that many of the SNPs alter well-conserved amino acids. Several SNPs predicted to create or remove stop codons were also found. A comparison between the sequencing SNPs and genotyping results from the BovineHD high-density genotyping chip indicates a detection rate of 91% for homozygous SNPs and 81% for heterozygous SNPs. The false positive rate is estimated to be about 2% for both the Black Angus and Holstein SNP sets, based on follow-up genotyping of 422 and 427 SNPs, respectively. Comparisons of read depth between the two bulls along the reference assembly identified 790 putative copy-number variations (CNVs. Ten

  8. Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

    KAUST Repository

    Ali, Asho

    2015-02-26

    Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyr B mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded

  9. Whole genome duplications and expansion of the vertebrate GATA transcription factor gene family

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    Bowerman Bruce

    2009-08-01

    Full Text Available Abstract Background GATA transcription factors influence many developmental processes, including the specification of embryonic germ layers. The GATA gene family has significantly expanded in many animal lineages: whereas diverse cnidarians have only one GATA transcription factor, six GATA genes have been identified in many vertebrates, five in many insects, and eleven to thirteen in Caenorhabditis nematodes. All bilaterian animal genomes have at least one member each of two classes, GATA123 and GATA456. Results We have identified one GATA123 gene and one GATA456 gene from the genomic sequence of two invertebrate deuterostomes, a cephalochordate (Branchiostoma floridae and a hemichordate (Saccoglossus kowalevskii. We also have confirmed the presence of six GATA genes in all vertebrate genomes, as well as additional GATA genes in teleost fish. Analyses of conserved sequence motifs and of changes to the exon-intron structure, and molecular phylogenetic analyses of these deuterostome GATA genes support their origin from two ancestral deuterostome genes, one GATA 123 and one GATA456. Comparison of the conserved genomic organization across vertebrates identified eighteen paralogous gene families linked to multiple vertebrate GATA genes (GATA paralogons, providing the strongest evidence yet for expansion of vertebrate GATA gene families via genome duplication events. Conclusion From our analysis, we infer the evolutionary birth order and relationships among vertebrate GATA transcription factors, and define their expansion via multiple rounds of whole genome duplication events. As the genomes of four independent invertebrate deuterostome lineages contain single copy GATA123 and GATA456 genes, we infer that the 0R (pre-genome duplication invertebrate deuterostome ancestor also had two GATA genes, one of each class. Synteny analyses identify duplications of paralogous chromosomal regions (paralogons, from single ancestral vertebrate GATA123 and GATA456

  10. Whole Genome Association Studies of Residual Feed Intake and Related Traits in the Pig.

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    Suneel K Onteru

    Full Text Available Residual feed intake (RFI, a measure of feed efficiency, is the difference between observed feed intake and the expected feed requirement predicted from growth and maintenance. Pigs with low RFI have reduced feed costs without compromising their growth. Identification of genes or genetic markers associated with RFI will be useful for marker-assisted selection at an early age of animals with improved feed efficiency.Whole genome association studies (WGAS for RFI, average daily feed intake (ADFI, average daily gain (ADG, back fat (BF and loin muscle area (LMA were performed on 1,400 pigs from the divergently selected ISU-RFI lines, using the Illumina PorcineSNP60 BeadChip. Various statistical methods were applied to find SNPs and genomic regions associated with the traits, including a Bayesian approach using GenSel software, and frequentist approaches such as allele frequency differences between lines, single SNP and haplotype analyses using PLINK software. Single SNP and haplotype analyses showed no significant associations (except for LMA after genomic control and FDR. Bayesian analyses found at least 2 associations for each trait at a false positive probability of 0.5. At generation 8, the RFI selection lines mainly differed in allele frequencies for SNPs near (<0.05 Mb genes that regulate insulin release and leptin functions. The Bayesian approach identified associations of genomic regions containing insulin release genes (e.g., GLP1R, CDKAL, SGMS1 with RFI and ADFI, of regions with energy homeostasis (e.g., MC4R, PGM1, GPR81 and muscle growth related genes (e.g., TGFB1 with ADG, and of fat metabolism genes (e.g., ACOXL, AEBP1 with BF. Specifically, a very highly significantly associated QTL for LMA on SSC7 with skeletal myogenesis genes (e.g., KLHL31 was identified for subsequent fine mapping.Important genomic regions associated with RFI related traits were identified for future validation studies prior to their incorporation in marker

  11. Genetics professionals' opinions of whole-genome sequencing in the newborn period.

    Science.gov (United States)

    Ulm, Elizabeth; Feero, W Gregory; Dineen, Richard; Charrow, Joel; Wicklund, Catherine

    2015-06-01

    Newborn screening (NBS) programs have been successful in identifying infants with rare, treatable, congenital conditions. While current programs rely largely on biochemical analysis, some predict that in the future, genome sequencing may be used as an adjunct. The purpose of this exploratory pilot study was to begin to characterize genetics professionals' opinions of the use of whole-genome sequencing (WGS) in NBS. We surveyed members of the American College of Medical Genetics and Genomics (ACMG) via an electronic survey distributed through email. The survey included questions about results disclosure, the current NBS paradigm, and the current criteria for adding a condition to the screening panel. The response rate was 7.3 % (n = 113/1549). The majority of respondents (85 %, n = 96/113) felt that WGS should not be currently used in NBS, and that if it were used, it should not be mandatory (86.5 %, n = 96/111). However, 75.7 % (n = 84/111) foresee it as a future use of WGS. Respondents felt that accurate interpretation of results (86.5 %, n = 83/96), a more extensive consent process (72.6 %, n = 69/95), pre- (79.2 %, n = 76/96) and post-test (91.6 %, n = 87/95) counseling, and comparable costs (70.8 %, n = 68/96) and turn-around-times (64.6 %, n = 62/96) to current NBS would be important for using WGS in NBS. Participants were in favor of disclosing most types of results at some point in the lifetime. However, the majority (87.3 %, n = 96/110) also indicated that parents should be able to choose what results are disclosed. Overall, respondents foresee NBS as a future use of WGS, but indicated that WGS should not occur within the framework of traditional NBS. They agreed with the current criteria for including a condition on the recommended uniform screening panel (RUSP). Further discussion about these criteria is needed in order to better understand how they could be utilized if WGS is incorporated into NBS. PMID:25348082

  12. Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda.

    Science.gov (United States)

    Ssengooba, Willy; Meehan, Conor J; Lukoye, Deus; Kasule, George William; Musisi, Kenneth; Joloba, Moses L; Cobelens, Frank G; de Jong, Bouke C

    2016-06-01

    Understanding the circulating Mycobacterium tuberculosis resistance mutations is vital for better TB control strategies, especially to inform a new MDR-TB treatment programme. We complemented the phenotypic drug susceptibility testing (DST) based drug resistance surveys (DRSs) conducted in Uganda between 2008 and 2011 with Whole Genome Sequencing (WGS) of 90 Mycobacterium tuberculosis isolates phenotypically resistant to rifampicin and/or isoniazid to better understand the extent of drug resistance. A total of 31 (34.4 %) patients had MDR-TB, 5 (5.6 %) mono-rifampicin resistance and 54 (60.0 %) mono-isoniazid resistance by phenotypic DST. Pyrazinamide resistance mutations were identified in 32.3% of the MDR-TB patients. Resistance to injectable agents was detected in 4/90 (4.4%), and none to fluoroquinolones or novel drugs. Compensatory mutations in rpoC were identified in two patients. The sensitivity and specificity of drug resistance mutations compared to phenotypic DST were for rpoB 88.6% and 98.1%, katG 60.0% and 100%, fabG1 16.5% and 100%, katG and/or fabG1 71.8% and 100%, embCAB 63.0% and 82.5%, rrs 11.4% and 100%, rpsL 20.5% and 95.7% and rrs and/or rpsL 31.8% and 95.7%. Phylogenetic analysis showed dispersed MDR-TB isolate, with only one cluster of three Beijing family from South West Uganda. Among tuberculosis patients in Uganda, resistance beyond first-line drugs as well as compensatory mutations remain low, and MDR-TB isolates did not arise from a dominant clone. Our findings show the potential use of sequencing for complementing DRSs or surveillance in this setting, with good specificity compared to phenotypic DST. The reported high confidence mutations can be included in molecular assays, and population-based studies can track transmission of MDR-TB including the Beijing family strains in the South West of the country. PMID:26917365

  13. Preparation of a phage DNA fragment library for whole genome shotgun sequencing.

    Science.gov (United States)

    Summer, Elizabeth J

    2009-01-01

    The most efficient method to determine the genomic sequence of a dsDNA phage is to use a whole genome shotgun approach (WGSA). Preparation of a library where each genomic fragment has an equal chance of being represented is critical to the success of the WGSA. For many phages, there are regions of the genome likely to be under-represented in the shotgun library, which results in more gaps in the shotgun assembly than predicted by the Poisson distribution. However, as phage genomes are relatively small, this increased number of gaps does not present an insurmountable impediment to using the WGSA. This chapter will focus on construction of a high-quality random library and sequence analysis of this library in a 96-well format. Techniques are described for the mechanical fragmentation of genomic DNA into 2 kb average size fragments, preparation of the fragmented DNA for shotgun cloning, and advice on the choice of cloning vector for library preparation. Protocols for deepwell block culture, plasmid isolation, and sequencing in 96-well format are given. The rationale for determining the total number of random clones from a library to sequence for a 50 and 150 kb genome is explained. The steps involved in going from hundreds of shotgun sequencing traces to generating contigs will be outlined as well as how to close gaps in the sequence by primer walking on phage DNA and PCR-generated templates. Finally, examples will be given of how biological information about the phage genomic termini can be derived by analysis of the organization of individual clones in the shotgun sequence assembly. Specific examples are given for the circularly permuted termini of pac type phages, the direct terminal repeats found in most T7-like phages, variable host DNA at either end as in the Mu-like phages, and the 5' and 3' overhanging ends of cos type phages. The end result of these steps is the entire DNA sequence of a novel phage, ready for gene prediction. PMID:19082550

  14. Whole genome SNP discovery and analysis of genetic diversity in Turkey (Meleagris gallopavo

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    Aslam Muhammad L

    2012-08-01

    whole genome SNP discovery study in turkey resulted in the detection of 5.49 million putative SNPs compared to the reference genome. All commercial lines appear to share a common origin. Presence of different alleles/haplotypes in the SM population highlights that specific haplotypes have been selected in the modern domesticated turkey.

  15. Whole-genome association study identifies STK39 as a hypertension susceptibility gene

    Science.gov (United States)

    Wang, Ying; O'Connell, Jeffrey R.; McArdle, Patrick F.; Wade, James B.; Dorff, Sarah E.; Shah, Sanjiv J.; Shi, Xiaolian; Pan, Lin; Rampersaud, Evadnie; Shen, Haiqing; Kim, James D.; Subramanya, Arohan R.; Steinle, Nanette I.; Parsa, Afshin; Ober, Carole C.; Welling, Paul A.; Chakravarti, Aravinda; Weder, Alan B.; Cooper, Richard S.; Mitchell, Braxton D.; Shuldiner, Alan R.; Chang, Yen-Pei C.

    2009-01-01

    Hypertension places a major burden on individual and public health, but the genetic basis of this complex disorder is poorly understood. We conducted a genome-wide association study of systolic and diastolic blood pressure (SBP and DBP) in Amish subjects and found strong association signals with common variants in a serine/threonine kinase gene, STK39. We confirmed this association in an independent Amish and 4 non-Amish Caucasian samples including the Diabetes Genetics Initiative, Framingham Heart Study, GenNet, and Hutterites (meta-analysis combining all studies: n = 7,125, P 0.09 and were associated with increases of 3.3/1.3 mm Hg in SBP/DBP, respectively, in the Amish subjects and with smaller but consistent effects across the non-Amish studies. Cell-based functional studies showed that STK39 interacts with WNK kinases and cation-chloride cotransporters, mutations in which cause monogenic forms of BP dysregulation. We demonstrate that in vivo, STK39 is expressed in the distal nephron, where it may interact with these proteins. Although none of the associated SNPs alter protein structure, we identified and experimentally confirmed a highly conserved intronic element with allele-specific in vitro transcription activity as a functional candidate for this association. Thus, variants in STK39 may influence BP by increasing STK39 expression and consequently altering renal Na+ excretion, thus unifying rare and common BP-regulating alleles in the same physiological pathway. PMID:19114657

  16. Interferon-beta induces distinct gene expression response patterns in human monocytes versus T cells.

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    Noa Henig

    Full Text Available BACKGROUND: Monocytes, which are key players in innate immunity, are outnumbered by neutrophils and lymphocytes among peripheral white blood cells. The cytokine interferon-β (IFN-β is widely used as an immunomodulatory drug for multiple sclerosis and its functional pathways in peripheral blood mononuclear cells (PBMCs have been previously described. The aim of the present study was to identify novel, cell-specific IFN-β functions and pathways in tumor necrosis factor (TNF-α-activated monocytes that may have been missed in studies using PBMCs. METHODOLOGY/PRINCIPAL FINDINGS: Whole genome gene expression profiles of human monocytes and T cells were compared following in vitro priming to TNF-α and overnight exposure to IFN-β. Statistical analyses of the gene expression data revealed a cell-type-specific change of 699 transcripts, 667 monocyte-specific transcripts, 21 T cell-specific transcripts and 11 transcripts with either a difference in the response direction or a difference in the magnitude of response. RT-PCR revealed a set of differentially expressed genes (DEGs, exhibiting responses to IFN-β that are modulated by TNF-α in monocytes, such as RIPK2 and CD83, but not in T cells or PBMCs. Known IFN-β promoter response elements, such as ISRE, were enriched in T cell DEGs but not in monocyte DEGs. The overall directionality of the gene expression regulation by IFN-β was different in T cells and monocytes, with up-regulation more prevalent in T cells, and a similar extent of up and down-regulation recorded in monocytes. CONCLUSIONS: By focusing on the response of distinct cell types and by evaluating the combined effects of two cytokines with pro and anti-inflammatory activities, we were able to present two new findings First, new IFN-β response pathways and genes, some of which were monocytes specific; second, a cell-specific modulation of the IFN-β response transcriptome by TNF-α.

  17. Expansion of banana (Musa acuminata) gene families involved in ethylene biosynthesis and signalling after lineage-specific whole-genome duplications.

    Science.gov (United States)

    Jourda, Cyril; Cardi, Céline; Mbéguié-A-Mbéguié, Didier; Bocs, Stéphanie; Garsmeur, Olivier; D'Hont, Angélique; Yahiaoui, Nabila

    2014-05-01

    Whole-genome duplications (WGDs) are widespread in plants, and three lineage-specific WGDs occurred in the banana (Musa acuminata) genome. Here, we analysed the impact of WGDs on the evolution of banana gene families involved in ethylene biosynthesis and signalling, a key pathway for banana fruit ripening. Banana ethylene pathway genes were identified using comparative genomics approaches and their duplication modes and expression profiles were analysed. Seven out of 10 banana ethylene gene families evolved through WGD and four of them (1-aminocyclopropane-1-carboxylate synthase (ACS), ethylene-insensitive 3-like (EIL), ethylene-insensitive 3-binding F-box (EBF) and ethylene response factor (ERF)) were preferentially retained. Banana orthologues of AtEIN3 and AtEIL1, two major genes for ethylene signalling in Arabidopsis, were particularly expanded. This expansion was paralleled by that of EBF genes which are responsible for control of EIL protein levels. Gene expression profiles in banana fruits suggested functional redundancy for several MaEBF and MaEIL genes derived from WGD and subfunctionalization for some of them. We propose that EIL and EBF genes were co-retained after WGD in banana to maintain balanced control of EIL protein levels and thus avoid detrimental effects of constitutive ethylene signalling. In the course of evolution, subfunctionalization was favoured to promote finer control of ethylene signalling. PMID:24716518

  18. Whole Genome DNA Sequence Analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks.

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    Mark R Wilson

    Full Text Available Establishing an association between possible food sources and clinical isolates requires discriminating the suspected pathogen from an environmental background, and distinguishing it from other closely-related foodborne pathogens. We used whole genome sequencing (WGS to Salmonella subspecies enterica serotype Tennessee (S. Tennessee to describe genomic diversity across the serovar as well as among and within outbreak clades of strains associated with contaminated peanut butter. We analyzed 71 isolates of S. Tennessee from disparate food, environmental, and clinical sources and 2 other closely-related Salmonella serovars as outgroups (S. Kentucky and S. Cubana, which were also shot-gun sequenced. A whole genome single nucleotide polymorphism (SNP analysis was performed using a maximum likelihood approach to infer phylogenetic relationships. Several monophyletic lineages of S. Tennessee with limited SNP variability were identified that recapitulated several food contamination events. S. Tennessee clades were separated from outgroup salmonellae by more than sixteen thousand SNPs. Intra-serovar diversity of S. Tennessee was small compared to the chosen outgroups (1,153 SNPs, suggesting recent divergence of some S. Tennessee clades. Analysis of all 1,153 SNPs structuring an S. Tennessee peanut butter outbreak cluster revealed that isolates from several food, plant, and clinical isolates were very closely related, as they had only a few SNP differences between them. SNP-based cluster analyses linked specific food sources to several clinical S. Tennessee strains isolated in separate contamination events. Environmental and clinical isolates had very similar whole genome sequences; no markers were found that could be used to discriminate between these sources. Finally, we identified SNPs within variable S. Tennessee genes that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts

  19. Whole Genome DNA Sequence Analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks.

    Science.gov (United States)

    Wilson, Mark R; Brown, Eric; Keys, Chris; Strain, Errol; Luo, Yan; Muruvanda, Tim; Grim, Christopher; Jean-Gilles Beaubrun, Junia; Jarvis, Karen; Ewing, Laura; Gopinath, Gopal; Hanes, Darcy; Allard, Marc W; Musser, Steven

    2016-01-01

    Establishing an association between possible food sources and clinical isolates requires discriminating the suspected pathogen from an environmental background, and distinguishing it from other closely-related foodborne pathogens. We used whole genome sequencing (WGS) to Salmonella subspecies enterica serotype Tennessee (S. Tennessee) to describe genomic diversity across the serovar as well as among and within outbreak clades of strains associated with contaminated peanut butter. We analyzed 71 isolates of S. Tennessee from disparate food, environmental, and clinical sources and 2 other closely-related Salmonella serovars as outgroups (S. Kentucky and S. Cubana), which were also shot-gun sequenced. A whole genome single nucleotide polymorphism (SNP) analysis was performed using a maximum likelihood approach to infer phylogenetic relationships. Several monophyletic lineages of S. Tennessee with limited SNP variability were identified that recapitulated several food contamination events. S. Tennessee clades were separated from outgroup salmonellae by more than sixteen thousand SNPs. Intra-serovar diversity of S. Tennessee was small compared to the chosen outgroups (1,153 SNPs), suggesting recent divergence of some S. Tennessee clades. Analysis of all 1,153 SNPs structuring an S. Tennessee peanut butter outbreak cluster revealed that isolates from several food, plant, and clinical isolates were very closely related, as they had only a few SNP differences between them. SNP-based cluster analyses linked specific food sources to several clinical S. Tennessee strains isolated in separate contamination events. Environmental and clinical isolates had very similar whole genome sequences; no markers were found that could be used to discriminate between these sources. Finally, we identified SNPs within variable S. Tennessee genes that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future

  20. Cell signaling and transcription factor genes expressed during whole body regeneration in a colonial chordate

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    Rinkevich Baruch

    2008-10-01

    Full Text Available Abstract Background The restoration of adults from fragments of blood vessels in botryllid ascidians (termed whole body regeneration [WBR] represents an inimitable event in the chordates, which is poorly understood on the mechanistic level. Results To elucidate mechanisms underlying this phenomenon, a subtracted EST library for early WBR stages was previously assembled, revealing 76 putative genes belonging to major signaling pathways, including Notch/Delta, JAK/STAT, protein kinases, nuclear receptors, Ras oncogene family members, G-Protein coupled receptor (GPCR and transforming growth factor beta (TGF-β signaling. RT-PCR on selected transcripts documented specific up-regulation in only regenerating fragments, pointing to a broad activation of these signaling pathways at onset of WBR. The followed-up expression pattern of seven representative transcripts from JAK/STAT signaling (Bl-STAT, the Ras oncogene family (Bl-Rap1A, Bl-Rab-33, the protein kinase family (Bl-Mnk, Bl-Cnot, Bl-Slit and Bl-Bax inhibitor, revealed systemic and site specific activations during WBR in a sub-population of circulatory cells. Conclusion WBR in the non-vertebrate chordate Botrylloides leachi is a multifaceted phenomenon, presided by a complex array of cell signaling and transcription factors. Above results, provide a first insight into the whole genome molecular machinery of this unique regeneration process, and reveal the broad participation of cell signaling and transcription factors in the process. While regeneration involves the participation of specific cell populations, WBR signals are systemically expressed at the organism level.

  1. Fatal Cases of Influenza A(H3N2) in Children: Insights from Whole Genome Sequence Analysis

    OpenAIRE

    Monica Galiano; Johnson, Benjamin F.; Richard Myers; Joanna Ellis; Rod Daniels; Maria Zambon

    2012-01-01

    During the Northern Hemisphere winter of 2003-2004 the emergence of a novel influenza antigenic variant, A/Fujian/411/2002-like(H3N2), was associated with an unusually high number of fatalities in children. Seventeen fatal cases in the UK were laboratory confirmed for Fujian/411-like viruses. To look for phylogenetic patterns and genetic markers that might be associated with increased virulence, sequencing and phylogenetic analysis of the whole genomes of 63 viruses isolated from fatal cases ...

  2. Enterobacter asburiae Strain L1: Complete Genome and Whole Genome Optical Mapping Analysis of a Quorum Sensing Bacterium

    OpenAIRE

    Yin Yin Lau; Wai-Fong Yin; Kok-Gan Chan

    2014-01-01

    Enterobacter asburiae L1 is a quorum sensing bacterium isolated from lettuce leaves. In this study, for the first time, the complete genome of E. asburiae L1 was sequenced using the single molecule real time sequencer (PacBio RSII) and the whole genome sequence was verified by using optical genome mapping (OpGen) technology. In our previous study, E. asburiae L1 has been reported to produce AHLs, suggesting the possibility of virulence factor regulation which is quorum sensing dependent. This...

  3. ReAS: Recovery of ancestral sequences for transposable elements from the unassembled reads of a whole genome shotgun.

    Directory of Open Access Journals (Sweden)

    Ruiqiang Li

    2005-09-01

    Full Text Available We describe an algorithm, ReAS, to recover ancestral sequences for transposable elements (TEs from the unassembled reads of a whole genome shotgun. The main assumptions are that these TEs must exist at high copy numbers across the genome and must not be so old that they are no longer recognizable in comparison to their ancestral sequences. Tested on the japonica rice genome, ReAS was able to reconstruct all of the high copy sequences in the Repbase repository of known TEs, and increase the effectiveness of RepeatMasker in identifying TEs from genome sequences.

  4. Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak.

    Science.gov (United States)

    Saelens, Joseph W; Lau-Bonilla, Dalia; Moller, Anneliese; Medina, Narda; Guzmán, Brenda; Calderón, Maylena; Herrera, Raúl; Sisk, Dana M; Xet-Mull, Ana M; Stout, Jason E; Arathoon, Eduardo; Samayoa, Blanca; Tobin, David M

    2015-12-01

    Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City. PMID:26542222

  5. Cell Free DNA of Tumor Origin Induces a 'Metastatic' Expression Profile in HT-29 Cancer Cell Line.

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    István Fűri

    Full Text Available Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions.To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts.DNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin, DNA methyltransferase 3a (DNMT3a and NFκB (for treated HDFα cells.Administration of tumor derived DNA on HT29 cells resulted in significant (p<0.05 mRNA level alteration in 118 genes (logFc≥1, p≤0.05, including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A, metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1, tumor biomarker (CEACAM5, metabolic genes (i.e. INSIG1, LIPG, messenger molecule genes (i.e. DAPP, CREB3L2. Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05, including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β, STING pathway (ADAR, IRF7, CXCL10, CASP1 and the FGF2 gene.DNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from healthy colonic epithelium induced TLR9 and STING signaling

  6. Genome-wide profiling to analyze the effects of FXR activation on mouse renal proximal tubular cells

    OpenAIRE

    Gui, Ting; Gai, Zhibo

    2015-01-01

    To assess the effect of farnesoid X receptor (FXR), a bile acid nuclear receptor, on renal proximal tubular cells, primary cultured mouse kidney proximal tubular cells were treated with GW4064 (a FXR agonist) or DMSO (as controls) overnight. Analysis of gene expression in the proximal tubular cells by whole genome microarrays indicated that FXR activation induced genes involved in fatty acid degradation and oxidation reduction. Among them, genes involved in glutathione metabolism were mostly ...

  7. Construction and evaluation of a whole genome microarray of Chlamydomonas reinhardtii

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    Toepel Jörg

    2011-11-01

    Full Text Available Abstract Background Chlamydomonas reinhardtii is widely accepted as a model organism regarding photosynthesis, circadian rhythm, cell mobility, phototaxis, and biotechnology. The complete annotation of the genome allows transcriptomic studies, however a new microarray platform was needed. Based on the completed annotation of Chlamydomonas reinhardtii a new microarray on an Agilent platform was designed using an extended JGI 3.1 genome data set which included 15000 transcript models. Results In total 44000 probes were determined (3 independent probes per transcript model covering 93% of the transcriptome. Alignment studies with the recently published AUGUSTUS 10.2 annotation confirmed 11000 transcript models resulting in a very good coverage of 70% of the transcriptome (17000. Following the estimation of 10000 predicted genes in Chlamydomonas reinhardtii our new microarray, nevertheless, covers the expected genome by 90-95%. Conclusions To demonstrate the capabilities of the new microarray, we analyzed transcript levels for cultures grown under nitrogen as well as sulfate limitation, and compared the results with recently published microarray and RNA-seq data. We could thereby confirm previous results derived from data on nutrient-starvation induced gene expression of a group of genes related to protein transport and adaptation of the metabolism as well as genes related to efficient light harvesting, light energy distribution and photosynthetic electron transport.

  8. Molecular characterization of c-Abl/c-Src kinase inhibitors targeted against murine tumour progenitor cells that express stem cell markers.

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    Thomas Kruewel

    Full Text Available BACKGROUND: The non-receptor tyrosine kinases c-Abl and c-Src are overexpressed in various solid human tumours. Inhibition of their hyperactivity represents a molecular rationale in the combat of cancerous diseases. Here we examined the effects of a new family of pyrazolo [3,4-d] pyrimidines on a panel of 11 different murine lung tumour progenitor cell lines, that express stem cell markers, as well as on the human lung adenocarcinoma cell line A549, the human hepatoma cell line HepG2 and the human colon cancer cell line CaCo2 to obtain insight into the mode of action of these experimental drugs. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with the dual kinase inhibitors blocked c-Abl and c-Src kinase activity efficiently in the nanomolar range, induced apoptosis, reduced cell viability and caused cell cycle arrest predominantly at G0/G1 phase while western blot analysis confirmed repressed protein expression of c-Abl and c-Src as well as the interacting partners p38 mitogen activated protein kinase, heterogenous ribonucleoprotein K, cyclin dependent kinase 1 and further proteins that are crucial for tumour progression. Importantly, a significant repression of the epidermal growth factor receptor was observed while whole genome gene expression analysis evidenced regulation of many cell cycle regulated genes as well integrin and focal adhesion kinase (FAK signalling to impact cytoskeleton dynamics, migration, invasion and metastasis. CONCLUSIONS/SIGNIFICANCE: Our experiments and recently published in vivo engraftment studies with various tumour cell lines revealed the dual kinase inhibitors to be efficient in their antitumour activity.

  9. Low-coverage, whole-genome sequencing of Artocarpus camansi (Moraceae) for phylogenetic marker development and gene discovery1

    Science.gov (United States)

    Gardner, Elliot M.; Johnson, Matthew G.; Ragone, Diane; Wickett, Norman J.; Zerega, Nyree J. C.

    2016-01-01

    Premise of the study: We used moderately low-coverage (17×) whole-genome sequencing of Artocarpus camansi (Moraceae) to develop genomic resources for Artocarpus and Moraceae. Methods and Results: A de novo assembly of Illumina short reads (251,378,536 pairs, 2 × 100 bp) accounted for 93% of the predicted genome size. Predicted coding regions were used in a three-way orthology search with published genomes of Morus notabilis and Cannabis sativa. Phylogenetic markers for Moraceae were developed from 333 inferred single-copy exons. Ninety-eight putative MADS-box genes were identified. Analysis of all predicted coding regions resulted in preliminary annotation of 49,089 genes. An analysis of synonymous substitutions for pairs of orthologs (Ks analysis) in M. notabilis and A. camansi strongly suggested a lineage-specific whole-genome duplication in Artocarpus. Conclusions: This study substantially increases the genomic resources available for Artocarpus and Moraceae and demonstrates the value of low-coverage de novo assemblies for nonmodel organisms with moderately large genomes.

  10. Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing.

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    Alexander C Outhred

    Full Text Available Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways.We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants.Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade.Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.

  11. Efficient Haplotype Inference Algorithms in One Whole Genome Scan for Pedigree Data with Non-genotyped Founders

    Institute of Scientific and Technical Information of China (English)

    Yongxi Cheng; Hadi Sabaa; Zhipeng Cai; Randy Goebel; Guohui Lin

    2009-01-01

    An efficient rule-based algorithm is presented for haplotype inference from general pedigree genotype data, with the assumption of no recombination. This algorithm generalizes previous algorithms to handle the cases where some pedigree founders are not genotyped, provided that for each nuclear family at least one parent is genotyped and each non-genotyped founder appears in exactly one nuclear family. The importance of this generalization lies in that such cases frequently happen in real data, because some founders may have passed away and their genotype data can no longer be collected. The algorithm runs in O(m3n3) time, where m is the number of single nucleotide polymorphism (SNP) loci under consideration and n is the number of genotyped members in the pedigree. This zero-recombination haplotyping algorithm is extended to a maximum parsimoniously haplotyping algorithm in one whole genome scan to minimize the total number of breakpoint sites, or equivalently, the number of maximal zero-recombination chromosomal regions. We show that such a whole genome scan haplotyping algorithm can be implemented in O(m3n3) time in a novel incremental fashion,here m denotes the total number of SNP loci along the chromosome.

  12. Construction of whole genome radiation hybrid panels and map of chromosome 5A of wheat using asymmetric somatic hybridization.

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    Chuanen Zhou

    Full Text Available To explore the feasibility of constructing a whole genome radiation hybrid (WGRH map in plant species with large genomes, asymmetric somatic hybridization between wheat (Triticum aestivum L. and Bupleurum scorzonerifolium Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV and gamma-ray and rescued by protoplast fusion using B. scorzonerifolium as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ∼501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.

  13. Contamination-controlled high-throughput whole genome sequencing for influenza A viruses using the MiSeq sequencer.

    Science.gov (United States)

    Lee, Hong Kai; Lee, Chun Kiat; Tang, Julian Wei-Tze; Loh, Tze Ping; Koay, Evelyn Siew-Chuan

    2016-01-01

    Accurate full-length genomic sequences are important for viral phylogenetic studies. We developed a targeted high-throughput whole genome sequencing (HT-WGS) method for influenza A viruses, which utilized an enzymatic cleavage-based approach, the Nextera XT DNA library preparation kit, for library preparation. The entire library preparation workflow was adapted for the Sentosa SX101, a liquid handling platform, to automate this labor-intensive step. As the enzymatic cleavage-based approach generates low coverage reads at both ends of the cleaved products, we corrected this loss of sequencing coverage at the termini by introducing modified primers during the targeted amplification step to generate full-length influenza A sequences with even coverage across the whole genome. Another challenge of targeted HTS is the risk of specimen-to-specimen cross-contamination during the library preparation step that results in the calling of false-positive minority variants. We included an in-run, negative system control to capture contamination reads that may be generated during the liquid handling procedures. The upper limits of 99.99% prediction intervals of the contamination rate were adopted as cut-off values of contamination reads. Here, 148 influenza A/H3N2 samples were sequenced using the HTS protocol and were compared against a Sanger-based sequencing method. Our data showed that the rate of specimen-to-specimen cross-contamination was highly significant in HTS. PMID:27624998

  14. Evaluation ofA Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

    Institute of Scientific and Technical Information of China (English)

    ZOU Xiao Hui; CHEN Wen Bing; ZHAO Xiang; ZHU Wen Fei; YANG Lei; WANG Da Yan; SHU Yue Long

    2016-01-01

    ObjectiveTo evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform. MethodsVirus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. ResultsThe M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus. ConclusionThe M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

  15. Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing.

    Science.gov (United States)

    Crampton, Mollee; Sripathi, Venkateswara R; Hossain, Khwaja; Kalavacharla, Venu

    2016-01-01

    Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar ("Sierra") using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation. PMID:27199997

  16. Gene expression and pathway analysis of ovarian cancer cells selected for resistance to cisplatin, paclitaxel, or doxorubicin

    Directory of Open Access Journals (Sweden)

    Sherman-Baust Cheryl A

    2011-12-01

    Full Text Available Abstract Background Resistance to current chemotherapeutic agents is a major cause of therapy failure in ovarian cancer patients, but the exact mechanisms leading to the development of drug resistance remain unclear. Methods To better understand mechanisms of drug resistance, and possibly identify novel targets for therapy, we generated a series of drug resistant ovarian cancer cell lines through repeated exposure to three chemotherapeutic drugs (cisplatin, doxorubicin, or paclitaxel, and identified changes in gene expression patterns using Illumina whole-genome expression microarrays. Validation of selected genes was performed by RT-PCR and immunoblotting. Pathway enrichment analysis using the KEGG, GO, and Reactome databases was performed to identify pathways that may be important in each drug resistance phenotype. Results A total of 845 genes (p Conclusions Ovarian cancer cells develop drug resistance through different pathways depending on the drug used in the generation of chemoresistance. A better understanding of these mechanisms may lead to the development of novel strategies to circumvent the problem of drug resistance.

  17. Different responsiveness to a high-fat/cholesterol diet in two inbred mice and underlying genetic factors: a whole genome microarray analysis

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    Jin Gang

    2009-10-01

    Full Text Available Abstract Background To investigate different responses to a high-fat/cholesterol diet and uncover their underlying genetic factors between C57BL/6J (B6 and DBA/2J (D2 inbred mice. Methods B6 and D2 mice were fed a high-fat/cholesterol diet for a series of time-points. Serum and bile lipid profiles, bile acid yields, hepatic apoptosis, gallstones and atherosclerosis formation were measured. Furthermore, a whole genome microarray was performed to screen hepatic genes expression profile. Quantitative real-time PCR, western blot and TUNEL assay were conducted to validate microarray data. Results After fed the high-fat/cholesterol diet, serum and bile total cholesterol, serum cholesterol esters, HDL cholesterol and Non-HDL cholesterol levels were altered in B6 but not significantly changed in D2; meanwhile, biliary bile acid was decreased in B6 but increased in D2. At the same time, hepatic apoptosis, gallstones and atherosclerotic lesions occurred in B6 but not in D2. The hepatic microarray analysis revealed distinctly different genes expression patterns between B6 and D2 mice. Their functional pathway groups included lipid metabolism, oxidative stress, immune/inflammation response and apoptosis. Quantitative real time PCR, TUNEL assay and western-blot results were consistent with microarray analysis. Conclusion Different genes expression patterns between B6 and D2 mice might provide a genetic basis for their distinctive responses to a high-fat/cholesterol diet, and give us an opportunity to identify novel pharmaceutical targets in related diseases in the future.

  18. An evaluation of alternative methods for constructing phylogenies from whole genome sequence data: a case study with Salmonella

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    James B. Pettengill

    2014-10-01

    Full Text Available Comparative genomics based on whole genome sequencing (WGS is increasingly being applied to investigate questions within evolutionary and molecular biology, as well as questions concerning public health (e.g., pathogen outbreaks. Given the impact that conclusions derived from such analyses may have, we have evaluated the robustness of clustering individuals based on WGS data to three key factors: (1 next-generation sequencing (NGS platform (HiSeq, MiSeq, IonTorrent, 454, and SOLiD, (2 algorithms used to construct a SNP (single nucleotide polymorphism matrix (reference-based and reference-free, and (3 phylogenetic inference method (FastTreeMP, GARLI, and RAxML. We carried out these analyses on 194 whole genome sequences representing 107 unique Salmonella enterica subsp. enterica ser. Montevideo strains. Reference-based approaches for identifying SNPs produced trees that were significantly more similar to one another than those produced under the reference-free approach. Topologies inferred using a core matrix (i.e., no missing data were significantly more discordant than those inferred using a non-core matrix that allows for some missing data. However, allowing for too much missing data likely results in a high false discovery rate of SNPs. When analyzing the same SNP matrix, we observed that the more thorough inference methods implemented in GARLI and RAxML produced more similar topologies than FastTreeMP. Our results also confirm that reproducibility varies among NGS platforms where the MiSeq had the lowest number of pairwise differences among replicate runs. Our investigation into the robustness of clustering patterns illustrates the importance of carefully considering how data from different platforms are combined and analyzed. We found clear differences in the topologies inferred, and certain methods performed significantly better than others for discriminating between the highly clonal organisms investigated here. The methods supported by

  19. SOX4 expression in bladder carcinoma: clinical aspects and in vitro functional characterization

    DEFF Research Database (Denmark)

    Jensen, Mads Aaboe; Birkenkamp-Demtröder, Karin; Wiuf, Carsten;

    2006-01-01

    The human transcription factor SOX4 was 5-fold up-regulated in bladder tumors compared with normal tissue based on whole-genome expression profiling of 166 clinical bladder tumor samples and 27 normal urothelium samples. Using a SOX4-specific antibody, we found that the cancer cells expressed the...... strongly impaired cell viability and promoted apoptosis. To characterize downstream target genes and SOX4-induced pathways, we used a time-course global expression study of the overexpressed SOX4. Analysis of the microarray data showed 130 novel SOX4-related genes, some involved in signal transduction (MAP...

  20. Chemokine receptor expression by mast cells.

    Science.gov (United States)

    Juremalm, Mikael; Nilsson, Gunnar

    2005-01-01

    There is a growing interest in the role of chemokines and their receptors in the determination of mast cell tissue localization and how chemokines regulate mast cell function. At least nine chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CX3CR1, CCR1, CCR3, CCR4 and CCR5) have been described to be expressed by human mast cells of different origins. Seven chemokines (CXCL1, CXCL5, CXCL8, CXCL14, CX3CL1, CCL5 and CCL11) have been shown to act on some of these receptors and to induce mast cell migration. Mast cells have a unique expression pattern of CCR3, CXCR1 and CXCR2. These receptors are mainly expressed intracellularly on cytoplasmic membranes. Upon an allergic activation, CCR3 expression is increased on the cell surface and the cell becomes vulnerable for CCL11 treatment. Chemokines do not induce mast cell degranulation but CXCL14 causes secretion of de novo synthesized CXCL8. Because of the expression of CCR3, CCR5 and CXCR4 on mast cell progenitors, these cells are susceptible to HIV infection and mast cells might therefore be a persistent HIV reservoir in AIDS. In this review, we summarize the knowledge about chemokine receptor expression and function on mast cells. PMID:16107768

  1. Diversification and evolution of the SDG gene family in Brassica rapa after the whole genome triplication

    OpenAIRE

    Heng Dong; Dandan Liu; Tianyu Han; Yuxue Zhao; Ji Sun; Sue Lin; Jiashu Cao; Zhong-Hua Chen; Li Huang

    2015-01-01

    Histone lysine methylation, controlled by the SET Domain Group (SDG) gene family, is part of the histone code that regulates chromatin function and epigenetic control of gene expression. Analyzing the SDG gene family in Brassica rapa for their gene structure, domain architecture, subcellular localization, rate of molecular evolution and gene expression pattern revealed common occurrences of subfunctionalization and neofunctionalization in BrSDGs. In comparison with Arabidopsis thaliana, the B...

  2. Whole-genome sequences of influenza A(H3N2 viruses isolated from Brazilian patients with mild illness during the 2014 season

    Directory of Open Access Journals (Sweden)

    Paola Cristina Resende

    2015-02-01

    Full Text Available The influenza A(H3N2 virus has circulated worldwide for almost five decades and is the dominant subtype in most seasonal influenza epidemics, as occurred in the 2014 season in South America. In this study we evaluate five whole genome sequences of influenza A(H3N2 viruses detected in patients with mild illness collected from January-March 2014. To sequence the genomes, a new generation sequencing (NGS protocol was performed using the Ion Torrent PGM platform. In addition to analysing the common genes, haemagglutinin, neuraminidase and matrix, our work also comprised internal genes. This was the first report of a whole genome analysis with Brazilian influenza A(H3N2 samples. Considerable amino acid variability was encountered in all gene segments, demonstrating the importance of studying the internal genes. NGS of whole genomes in this study will facilitate deeper virus characterisation, contributing to the improvement of influenza strain surveillance in Brazil.

  3. Application of whole genome shotgun sequencing for detection and characterization of genetically modified organisms and derived products.

    Science.gov (United States)

    Holst-Jensen, Arne; Spilsberg, Bjørn; Arulandhu, Alfred J; Kok, Esther; Shi, Jianxin; Zel, Jana

    2016-07-01

    The emergence of high-throughput, massive or next-generation sequencing technologies has created a completely new foundation for molecular analyses. Various selective enrichment processes are commonly applied to facilitate detection of predefined (known) targets. Such approaches, however, inevitably introduce a bias and are prone to miss unknown targets. Here we review the application of high-throughput sequencing technologies and the preparation of fit-for-purpose whole genome shotgun sequencing libraries for the detection and characterization of genetically modified and derived products. The potential impact of these new sequencing technologies for the characterization, breeding selection, risk assessment, and traceability of genetically modified organisms and genetically modified products is yet to be fully acknowledged. The published literature is reviewed, and the prospects for future developments and use of the new sequencing technologies for these purposes are discussed. PMID:27100228

  4. Rapid and Easy In Silico Serotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data

    DEFF Research Database (Denmark)

    Joensen, Katrine Grimstrup; Tetzschner, Anna M. M.; Iguchi, Atsushi;

    2015-01-01

    typing and surveillance. The aim of this study was to establish a valid and publicly available tool for WGS-based in silico serotyping of E. coli applicable for routine typing and surveillance. A FASTA database of specific O-antigen processing system genes for O typing and flagellin genes for H typing...... was created as a component of the publicly available Web tools hosted by the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org). All E. coli isolates available with WGS data and conventional serotype information were subjected to WGS-based serotyping employing this specific Serotype......Finder CGE tool. SerotypeFinder was evaluated on 682 E. coli genomes, 108 of which were sequenced for this study, where both the whole genome and the serotype were available. In total, 601 and 509 isolates were included for O and H typing, respectively. The O-antigen genes wzx, wzy, wzm, and wzt and the...

  5. Genetic Diversity and Fingerprint Profiles of Commercial Lentinula edodes Cultivars Based on SSR Markers Developed from the Whole Genome Sequence

    Institute of Scientific and Technical Information of China (English)

    ZHANG Dan; SONG Chunyan; ZHANG Lujun; WU Ping; BAO Dapeng; SHANG Xiaodong; TAN Qi

    2014-01-01

    Lentinula edodes is an important cultivated mushroom in China, and accurate and reliable identification of individual cultivars is a prerequisite for successful cultivation and variety protection.In this study,the whole genome sequence of L.edodes was used to generate 200 simple sequence repeat (SSR) markers for delineating 25 commercial cultivars and for determining their genetic diversity.Our data revealed a relatively high level of genetic similarity among the cultivars,with average,minimum and maximum genetic similarity coefficient values of 0.776,0.567 and 1.000,respectively.Seven SSR primer pairs delineated eleven of the cultivars (Cr-02,Minfeng-1,Xianggu 241-4,Senyuan-1,Senyuan-8404,Xiang-9,Guangxiang-51,Huaxiang-5,L952,L9319 and L808)based on their unique multilocus SSR fingerprint profiles.

  6. Genotyping using whole-genome sequencing is a realistic alternative to surveillance based on phenotypic antimicrobial susceptibility testing

    DEFF Research Database (Denmark)

    Zankari, Ea; Hasman, Henrik; Kaas, Rolf Sommer;

    2013-01-01

    200 isolates originating from Danish pigs, covering four bacterial species. Genomic DNA was purified from all isolates and sequenced as paired-end reads on the Illumina platform. The web servers ResFinder and MLST (www.genomicepidemiology.org) were used to identify acquired antimicrobial resistance......Objectives: Antimicrobial susceptibility testing of bacterial isolates is essential for clinical diagnosis, to detect emerging problems and to guide empirical treatment. Current phenotypic procedures are sometimes associated with mistakes and may require further genetic testing. Whole......-genome sequencing (WGS) may soon be within reach even for routine surveillance and clinical diagnostics. The aim of this study was to evaluate WGS as a routine tool for surveillance of antimicrobial resistance compared with current phenotypic procedures. Methods: Antimicrobial susceptibility tests were performed on...

  7. Transmission of methicillin-resistant Staphylococcus aureus infection through solid organ transplantation: confirmation via whole genome sequencing.

    Science.gov (United States)

    Wendt, J M; Kaul, D; Limbago, B M; Ramesh, M; Cohle, S; Denison, A M; Driebe, E M; Rasheed, J K; Zaki, S R; Blau, D M; Paddock, C D; McDougal, L K; Engelthaler, D M; Keim, P S; Roe, C C; Akselrod, H; Kuehnert, M J; Basavaraju, S V

    2014-11-01

    We describe two cases of donor-derived methicillin-resistant Staphylococcus aureus (MRSA) bacteremia that developed after transplantation of organs from a common donor who died from acute MRSA endocarditis. Both recipients developed recurrent MRSA infection despite appropriate antibiotic therapy, and required prolonged hospitalization and hospital readmission. Comparison of S. aureus whole genome sequence of DNA extracted from fixed donor tissue and recipients' isolates confirmed donor-derived transmission. Current guidelines emphasize the risk posed by donors with bacteremia from multidrug-resistant organisms. This investigation suggests that, particularly in the setting of donor endocarditis, even a standard course of prophylactic antibiotics may not be sufficient to prevent donor-derived infection. PMID:25250717

  8. Genome-wide association analyses based on whole-genome sequencing in Sardinia provide insights into regulation of hemoglobin levels.

    Science.gov (United States)

    Danjou, Fabrice; Zoledziewska, Magdalena; Sidore, Carlo; Steri, Maristella; Busonero, Fabio; Maschio, Andrea; Mulas, Antonella; Perseu, Lucia; Barella, Susanna; Porcu, Eleonora; Pistis, Giorgio; Pitzalis, Maristella; Pala, Mauro; Menzel, Stephan; Metrustry, Sarah; Spector, Timothy D; Leoni, Lidia; Angius, Andrea; Uda, Manuela; Moi, Paolo; Thein, Swee Lay; Galanello, Renzo; Abecasis, Gonçalo R; Schlessinger, David; Sanna, Serena; Cucca, Francesco

    2015-11-01

    We report genome-wide association study results for the levels of A1, A2 and fetal hemoglobins, analyzed for the first time concurrently. Integrating high-density array genotyping and whole-genome sequencing in a large general population cohort from Sardinia, we detected 23 associations at 10 loci. Five signals are due to variants at previously undetected loci: MPHOSPH9, PLTP-PCIF1, ZFPM1 (FOG1), NFIX and CCND3. Among the signals at known loci, ten are new lead variants and four are new independent signals. Half of all variants also showed pleiotropic associations with different hemoglobins, which further corroborated some of the detected associations and identified features of coordinated hemoglobin species production. PMID:26366553

  9. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  10. Evidence and evolutionary analysis of ancient whole-genome duplication in barley predating the divergence from rice

    Directory of Open Access Journals (Sweden)

    Grosse Ivo

    2009-08-01

    Full Text Available Abstract Background Well preserved genomic colinearity among agronomically important grass species such as rice, maize, Sorghum, wheat and barley provides access to whole-genome structure information even in species lacking a reference genome sequence. We investigated footprints of whole-genome duplication (WGD in barley that shaped the cereal ancestor genome by analyzing shared synteny with rice using a ~2000 gene-based barley genetic map and the rice genome reference sequence. Results Based on a recent annotation of the rice genome, we reviewed the WGD in rice and identified 24 pairs of duplicated genomic segments involving 70% of the rice genome. Using 968 putative orthologous gene pairs, synteny covered 89% of the barley genetic map and 63% of the rice genome. We found strong evidence for seven shared segmental genome duplications, corresponding to more than 50% of the segmental genome duplications previously determined in rice. Analysis of synonymous substitution rates (Ks suggested that shared duplications originated before the divergence of these two species. While major genome rearrangements affected the ancestral genome of both species, small paracentric inversions were found to be species specific. Conclusion We provide a thorough analysis of comparative genome evolution between barley and rice. A barley genetic map of approximately 2000 non-redundant EST sequences provided sufficient density to allow a detailed view of shared synteny with the rice genome. Using an indirect approach that included the localization of WGD-derived duplicated genome segments in the rice genome, we determined the current extent of shared WGD-derived genome duplications that occurred prior to species divergence.

  11. Whole-genome SNP association in the horse: identification of a deletion in myosin Va responsible for Lavender Foal Syndrome.

    Directory of Open Access Journals (Sweden)

    Samantha A Brooks

    2010-04-01

    Full Text Available Lavender Foal Syndrome (LFS is a lethal inherited disease of horses with a suspected autosomal recessive mode of inheritance. LFS has been primarily diagnosed in a subgroup of the Arabian breed, the Egyptian Arabian horse. The condition is characterized by multiple neurological abnormalities and a dilute coat color. Candidate genes based on comparative phenotypes in mice and humans include the ras-associated protein RAB27a (RAB27A and myosin Va (MYO5A. Here we report mapping of the locus responsible for LFS using a small set of 36 horses segregating for LFS. These horses were genotyped using a newly available single nucleotide polymorphism (SNP chip containing 56,402 discriminatory elements. The whole genome scan identified an associated region containing these two functional candidate genes. Exon sequencing of the MYO5A gene from an affected foal revealed a single base deletion in exon 30 that changes the reading frame and introduces a premature stop codon. A PCR-based Restriction Fragment Length Polymorphism (PCR-RFLP assay was designed and used to investigate the frequency of the mutant gene. All affected horses tested were homozygous for this mutation. Heterozygous carriers were detected in high frequency in families segregating for this trait, and the frequency of carriers in unrelated Egyptian Arabians was 10.3%. The mapping and discovery of the LFS mutation represents the first successful use of whole-genome SNP scanning in the horse for any trait. The RFLP assay can be used to assist breeders in avoiding carrier-to-carrier matings and thus in preventing the birth of affected foals.

  12. Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance

    KAUST Repository

    Phelan, Jody

    2016-03-23

    Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. Methods To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. Results The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. Conclusions Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance

  13. Whole genome sequence of Treponema pallidum ssp. pallidum, strain Mexico A, suggests recombination between yaws and syphilis strains.

    Directory of Open Access Journals (Sweden)

    Helena Pětrošová

    Full Text Available BACKGROUND: Treponema pallidum ssp. pallidum (TPA, the causative agent of syphilis, and Treponema pallidum ssp. pertenue (TPE, the causative agent of yaws, are closely related spirochetes causing diseases with distinct clinical manifestations. The TPA Mexico A strain was isolated in 1953 from male, with primary syphilis, living in Mexico. Attempts to cultivate TPA Mexico A strain under in vitro conditions have revealed lower growth potential compared to other tested TPA strains. METHODOLOGY/PRINCIPAL FINDINGS: The complete genome sequence of the TPA Mexico A strain was determined using the Illumina sequencing technique. The genome sequence assembly was verified using the whole genome fingerprinting technique and the final sequence was annotated. The genome size of the Mexico A strain was determined to be 1,140,038 bp with 1,035 predicted ORFs. The Mexico A genome sequence was compared to the whole genome sequences of three TPA (Nichols, SS14 and Chicago and three TPE (CDC-2, Samoa D and Gauthier strains. No large rearrangements in the Mexico A genome were found and the identified nucleotide changes occurred most frequently in genes encoding putative virulence factors. Nevertheless, the genome of the Mexico A strain, revealed two genes (TPAMA_0326 (tp92 and TPAMA_0488 (mcp2-1 which combine TPA- and TPE- specific nucleotide sequences. Both genes were found to be under positive selection within TPA strains and also between TPA and TPE strains. CONCLUSIONS/SIGNIFICANCE: The observed mosaic character of the TPAMA_0326 and TPAMA_0488 loci is likely a result of inter-strain recombination between TPA and TPE strains during simultaneous infection of a single host suggesting horizontal gene transfer between treponemal subspecies.

  14. Whole Genome Re-Sequencing and Characterization of Powdery Mildew Disease-Associated Allelic Variation in Melon.

    Science.gov (United States)

    Natarajan, Sathishkumar; Kim, Hoy-Taek; Thamilarasan, Senthil Kumar; Veerappan, Karpagam; Park, Jong-In; Nou, Ill-Sup

    2016-01-01

    Powdery mildew is one of the most common fungal diseases in the world. This disease frequently affects melon (Cucumis melo L.) and other Cucurbitaceous family crops in both open field and greenhouse cultivation. One of the goals of genomics is to identify the polymorphic loci responsible for variation in phenotypic traits. In this study, powdery mildew disease assessment scores were calculated for four melon accessions, 'SCNU1154', 'Edisto47', 'MR-1', and 'PMR5'. To investigate the genetic variation of these accessions, whole genome re-sequencing using the Illumina HiSeq 2000 platform was performed. A total of 754,759,704 quality-filtered reads were generated, with an average of 82.64% coverage relative to the reference genome. Comparisons of the sequences for the melon accessions revealed around 7.4 million single nucleotide polymorphisms (SNPs), 1.9 million InDels, and 182,398 putative structural variations (SVs). Functional enrichment analysis of detected variations classified them into biological process, cellular component and molecular function categories. Further, a disease-associated QTL map was constructed for 390 SNPs and 45 InDels identified as related to defense-response genes. Among them 112 SNPs and 12 InDels were observed in powdery mildew responsive chromosomes. Accordingly, this whole genome re-sequencing study identified SNPs and InDels associated with defense genes that will serve as candidate polymorphisms in the search for sources of resistance against powdery mildew disease and could accelerate marker-assisted breeding in melon. PMID:27311063

  15. Whole Genome Re-Sequencing and Characterization of Powdery Mildew Disease-Associated Allelic Variation in Melon.

    Directory of Open Access Journals (Sweden)

    Sathishkumar Natarajan

    Full Text Available Powdery mildew is one of the most common fungal diseases in the world. This disease frequently affects melon (Cucumis melo L. and other Cucurbitaceous family crops in both open field and greenhouse cultivation. One of the goals of genomics is to identify the polymorphic loci responsible for variation in phenotypic traits. In this study, powdery mildew disease assessment scores were calculated for four melon accessions, 'SCNU1154', 'Edisto47', 'MR-1', and 'PMR5'. To investigate the genetic variation of these accessions, whole genome re-sequencing using the Illumina HiSeq 2000 platform was performed. A total of 754,759,704 quality-filtered reads were generated, with an average of 82.64% coverage relative to the reference genome. Comparisons of the sequences for the melon accessions revealed around 7.4 million single nucleotide polymorphisms (SNPs, 1.9 million InDels, and 182,398 putative structural variations (SVs. Functional enrichment analysis of detected variations classified them into biological process, cellular component and molecular function categories. Further, a disease-associated QTL map was constructed for 390 SNPs and 45 InDels identified as related to defense-response genes. Among them 112 SNPs and 12 InDels were observed in powdery mildew responsive chromosomes. Accordingly, this whole genome re-sequencing study identified SNPs and InDels associated with defense genes that will serve as candidate polymorphisms in the search for sources of resistance against powdery mildew disease and could accelerate marker-assisted breeding in melon.

  16. Transmission Clusters of Methicillin-Resistant Staphylococcus Aureus in Long-Term Care Facilities Based on Whole-Genome Sequencing.

    Science.gov (United States)

    Stine, O Colin; Burrowes, Shana; David, Sophia; Johnson, J Kristie; Roghmann, Mary-Claire

    2016-06-01

    OBJECTIVE To define how often methicillin-resistant Staphylococcus aureus (MRSA) is spread from resident to resident in long-term care facilities using whole-genome sequencing DESIGN Prospective cohort study SETTING A long-term care facility PARTICIPANTS Elderly residents in a long-term care facility METHODS Cultures for MRSA were obtained weekly from multiple body sites from residents with known MRSA colonization over 12-week study periods. Simultaneously, cultures to detect MRSA acquisition were obtained weekly from 2 body sites in residents without known MRSA colonization. During the first 12-week cycle on a single unit, we sequenced 8 MRSA isolates per swab for 2 body sites from each of 6 residents. During the second 12-week cycle, we sequenced 30 MRSA isolates from 13 residents with known MRSA colonization and 3 residents who had acquired MRSA colonization. RESULTS MRSA isolates from the same swab showed little genetic variation between isolates with the exception of isolates from wounds. The genetic variation of isolates between body sites on an individual was greater than that within a single body site with the exception of 1 sample, which had 2 unrelated strains among the 8 isolates. In the second cycle, 10 of 16 residents colonized with MRSA (63%) shared 1 of 3 closely related strains. Of the 3 residents with newly acquired MRSA, 2 residents harbored isolates that were members of these clusters. CONCLUSIONS Point prevalence surveys with whole-genome sequencing of MRSA isolates may detect resident-to-resident transmission more accurately than routine surveillance cultures for MRSA in long-term care facilities. Infect Control Hosp Epidemiol 2016;37:685-691. PMID:26941060

  17. Whole-Genome Sequencing Allows for Improved Identification of Persistent Listeria monocytogenes in Food-Associated Environments.

    Science.gov (United States)

    Stasiewicz, Matthew J; Oliver, Haley F; Wiedmann, Martin; den Bakker, Henk C

    2015-09-01

    While the food-borne pathogen Listeria monocytogenes can persist in food associated environments, there are no whole-genome sequence (WGS) based methods to differentiate persistent from sporadic strains. Whole-genome sequencing of 188 isolates from a longitudinal study of L. monocytogenes in retail delis was used to (i) apply single-nucleotide polymorphism (SNP)-based phylogenetics for subtyping of L. monocytogenes, (ii) use SNP counts to differentiate persistent from repeatedly reintroduced strains, and (iii) identify genetic determinants of L. monocytogenes persistence. WGS analysis revealed three prophage regions that explained differences between three pairs of phylogenetically similar populations with pulsed-field gel electrophoresis types that differed by ≤3 bands. WGS-SNP-based phylogenetics found that putatively persistent L. monocytogenes represent SNP patterns (i) unique to a single retail deli, supporting persistence within the deli (11 clades), (ii) unique to a single state, supporting clonal spread within a state (7 clades), or (iii) spanning multiple states (5 clades). Isolates that formed one of 11 deli-specific clades differed by a median of 10 SNPs or fewer. Isolates from 12 putative persistence events had significantly fewer SNPs (median, 2 to 22 SNPs) than between isolates of the same subtype from other delis (median up to 77 SNPs), supporting persistence of the strain. In 13 events, nearly indistinguishable isolates (0 to 1 SNP) were found across multiple delis. No individual genes were enriched among persistent isolates compared to sporadic isolates. Our data show that WGS analysis improves food-borne pathogen subtyping and identification of persistent bacterial pathogens in food associated environments. PMID:26116683

  18. Whole-Genome Sequencing and Comparative Analysis of Mycobacterium brisbanense Reveals a Possible Soil Origin and Capability in Fertiliser Synthesis

    Science.gov (United States)

    Wee, Wei Yee; Tan, Tze King; Jakubovics, Nicholas S.; Choo, Siew Woh

    2016-01-01

    Mycobacterium brisbanense is a member of Mycobacterium fortuitum third biovariant complex, which includes rapidly growing Mycobacterium spp. that normally inhabit soil, dust and water, and can sometimes cause respiratory tract infections in humans. We present the first whole-genome analysis of M. brisbanense UM_WWY which was isolated from a 70-year-old Malaysian patient. Molecular phylogenetic analyses confirmed the identification of this strain as M. brisbanense and showed that it has an unusually large genome compared with related mycobacteria. The large genome size of M. brisbanense UM_WWY (~7.7Mbp) is consistent with further findings that this strain has a highly variable genome structure that contains many putative horizontally transferred genomic islands and prophage. Comparative analysis showed that M. brisbanense UM_WWY is the only Mycobacterium species that possesses a complete set of genes encoding enzymes involved in the urea cycle, suggesting that this soil bacterium is able to synthesize urea for use as plant fertilizers. It is likely that M. brisbanense UM_WWY is adapted to live in soil as its primary habitat since the genome contains many genes associated with nitrogen metabolism. Nevertheless, a large number of predicted virulence genes were identified in M. brisbanense UM_WWY that are mostly shared with well-studied mycobacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. These findings are consistent with the role of M. brisbanense as an opportunistic pathogen of humans. The whole-genome study of UM_WWY has provided the basis for future work of M. brisbanense. PMID:27031249

  19. Whole Genome Sequence of Treponema pallidum ssp. pallidum, Strain Mexico A, Suggests Recombination between Yaws and Syphilis Strains

    Science.gov (United States)

    Pětrošová, Helena; Zobaníková, Marie; Čejková, Darina; Mikalová, Lenka; Pospíšilová, Petra; Strouhal, Michal; Chen, Lei; Qin, Xiang; Muzny, Donna M.; Weinstock, George M.; Šmajs, David

    2012-01-01

    Background Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, and Treponema pallidum ssp. pertenue (TPE), the causative agent of yaws, are closely related spirochetes causing diseases with distinct clinical manifestations. The TPA Mexico A strain was isolated in 1953 from male, with primary syphilis, living in Mexico. Attempts to cultivate TPA Mexico A strain under in vitro conditions have revealed lower growth potential compared to other tested TPA strains. Methodology/Principal Findings The complete genome sequence of the TPA Mexico A strain was determined using the Illumina sequencing technique. The genome sequence assembly was verified using the whole genome fingerprinting technique and the final sequence was annotated. The genome size of the Mexico A strain was determined to be 1,140,038 bp with 1,035 predicted ORFs. The Mexico A genome sequence was compared to the whole genome sequences of three TPA (Nichols, SS14 and Chicago) and three TPE (CDC-2, Samoa D and Gauthier) strains. No large rearrangements in the Mexico A genome were found and the identified nucleotide changes occurred most frequently in genes encoding putative virulence factors. Nevertheless, the genome of the Mexico A strain, revealed two genes (TPAMA_0326 (tp92) and TPAMA_0488 (mcp2-1)) which combine TPA- and TPE- specific nucleotide sequences. Both genes were found to be under positive selection within TPA strains and also between TPA and TPE strains. Conclusions/Significance The observed mosaic character of the TPAMA_0326 and TPAMA_0488 loci is likely a result of inter-strain recombination between TPA and TPE strains during simultaneous infection of a single host suggesting horizontal gene transfer between treponemal subspecies. PMID:23029591

  20. Rapid identification of genetic modifications in Bacillus anthracis using whole genome draft sequences generated by 454 pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Peter E Chen

    Full Text Available BACKGROUND: The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e., within 3 days of appearance of symptoms. Microbiological detection methods are feasible only for organisms that can be cultured in vitro and cannot detect all genetic modifications with the exception of antibiotic resistance. Currently available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic resistant or virulence enhanced Bacillus anthracis, to advise on therapeutic treatments. METHODOLOGY/PRINCIPAL FINDINGS: We show here that the Roche 454-based pyrosequencing can generate whole genome draft sequences of deep and broad enough coverage of a bacterial genome in less than 24 hours. Furthermore, using the unfinished draft sequences, we demonstrate that unbiased identification of known as well as heretofore-unreported genetic modifications that include indels and single nucleotide polymorphisms conferring antibiotic and phage resistances is feasible within the next 12 hours. CONCLUSIONS/SIGNIFICANCE: Second generation sequencing technologies have paved the way for sequence-based rapid identification of both known and previously undocumented genetic modifications in cultured, conventional and newly emerging biothreat agents. Our findings have significant implications in

  1. Lessons learned from the application of whole-genome analysis to the treatment of patients with advanced cancers

    Science.gov (United States)

    Laskin, Janessa; Jones, Steven; Aparicio, Samuel; Chia, Stephen; Ch'ng, Carolyn; Deyell, Rebecca; Eirew, Peter; Fok, Alexandra; Gelmon, Karen; Ho, Cheryl; Huntsman, David; Jones, Martin; Kasaian, Katayoon; Karsan, Aly; Leelakumari, Sreeja; Li, Yvonne; Lim, Howard; Ma, Yussanne; Mar, Colin; Martin, Monty; Moore, Richard; Mungall, Andrew; Mungall, Karen; Pleasance, Erin; Rassekh, S. Rod; Renouf, Daniel; Shen, Yaoqing; Schein, Jacqueline; Schrader, Kasmintan; Sun, Sophie; Tinker, Anna; Zhao, Eric; Yip, Stephen; Marra, Marco A.

    2015-01-01

    Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted “panel” sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from whole-genome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate “drivers.” Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions. PMID:27148575

  2. Whole Genome Re-Sequencing and Characterization of Powdery Mildew Disease-Associated Allelic Variation in Melon

    Science.gov (United States)

    Natarajan, Sathishkumar; Kim, Hoy-Taek; Thamilarasan, Senthil Kumar; Veerappan, Karpagam; Park, Jong-In; Nou, Ill-Sup

    2016-01-01

    Powdery mildew is one of the most common fungal diseases in the world. This disease frequently affects melon (Cucumis melo L.) and other Cucurbitaceous family crops in both open field and greenhouse cultivation. One of the goals of genomics is to identify the polymorphic loci responsible for variation in phenotypic traits. In this study, powdery mildew disease assessment scores were calculated for four melon accessions, ‘SCNU1154’, ‘Edisto47’, ‘MR-1’, and ‘PMR5’. To investigate the genetic variation of these accessions, whole genome re-sequencing using the Illumina HiSeq 2000 platform was performed. A total of 754,759,704 quality-filtered reads were generated, with an average of 82.64% coverage relative to the reference genome. Comparisons of the sequences for the melon accessions revealed around 7.4 million single nucleotide polymorphisms (SNPs), 1.9 million InDels, and 182,398 putative structural variations (SVs). Functional enrichment analysis of detected variations classified them into biological process, cellular component and molecular function categories. Further, a disease-associated QTL map was constructed for 390 SNPs and 45 InDels identified as related to defense-response genes. Among them 112 SNPs and 12 InDels were observed in powdery mildew responsive chromosomes. Accordingly, this whole genome re-sequencing study identified SNPs and InDels associated with defense genes that will serve as candidate polymorphisms in the search for sources of resistance against powdery mildew disease and could accelerate marker-assisted breeding in melon. PMID:27311063

  3. Development and preliminary evaluation of an online educational video about whole-genome sequencing for research participants, patients, and the general public

    OpenAIRE

    Sanderson, Saskia C.; Suckiel, Sabrina A; Zweig, Micol; Bottinger, Erwin P.; Jabs, Ethylin Wang; Richardson, Lynne D.

    2015-01-01

    Background: As whole-genome sequencing (WGS) increases in availability, WGS educational aids are needed for research participants, patients, and the general public. Our aim was therefore to develop an accessible and scalable WGS educational aid. Genet Med 18 5, 501–512. Methods: We engaged multiple stakeholders in an iterative process over a 1-year period culminating in the production of a novel 10-minute WGS educational animated video, “Whole Genome Sequencing and You” (https://goo.gl/HV8ezJ...

  4. Analysis of the gene expression profile in response to human epididymis protein 4 in epithelial ovarian cancer cells.

    Science.gov (United States)

    Zhu, Liancheng; Guo, Qian; Jin, Shan; Feng, Huilin; Zhuang, Huiyu; Liu, Cong; Tan, Mingzi; Liu, Juanjuan; Li, Xiao; Lin, Bei

    2016-09-01

    Currently, there are emerging multiple studies on human epididymis protein 4 (HE4) in ovarian cancer. HE4 possesses higher sensitivity and specificity than CA125 in the confirmative early diagnosis for ovarian cancer. Although much attention has been given to explore its clinical application, research of the basic mechanisms of HE4 in ovarian cancer are still unclear. In the present study, we provide fundamental data to identify full-scale differentially expressed genes (DEGs) in response to HE4 by use of human whole-genome microarrays in human epithelial ovarian cancer cell line ES-2 following overexpression and silencing of HE4. We found that a total of 717 genes were upregulated and 898 genes were downregulated in the HE4-overexpressing cells vs. the HE4-Mock cells, and 166 genes were upregulated and 285 were downregulated in the HE4-silenced cells vs. the HE4-Mock cells. An overlap of 16 genes consistently upregulated and 8 genes downregulated in response to HE4 were noted. These DEGs were involved in MAPK, steroid biosynthesis, cell cycle, the p53 hypoxia pathway, and focal adhesion pathways. Interaction network analysis predicted that the genes participated in the regulatory connection. Highly differential expression of the FOXA2, SERPIND1, BDKRD1 and IL1A genes was verified by quantitative real-time PCR in 4 cell line samples. Finally, SERPIND1 (HCII) was validated at the protein level by immunohistochemistry in 107 paraffin-embedded ovarian tissues. We found that SERPIND1 may act as a potential oncogene in the development of ovarian cancer. The present study displayed the most fundamental and full-scale data to show DEGs in response to HE4. These identified genes may provide a theoretical basis for investigations of the underlying molecular mechanism of HE4 in ovarian cancer. PMID:27430660

  5. Deciphering Clostridium tyrobutyricum Metabolism Based on the Whole-Genome Sequence and Proteome Analyses

    Science.gov (United States)

    Lee, Joungmin; Jang, Yu-Sin; Han, Mee-Jung; Kim, Jin Young

    2016-01-01

    ABSTRACT Clostridium tyrobutyricum is a Gram-positive anaerobic bacterium that efficiently produces butyric acid and is considered a promising host for anaerobic production of bulk chemicals. Due to limited knowledge on the genetic and metabolic characteristics of this strain, however, little progress has been made in metabolic engineering of this strain. Here we report the complete genome sequence of C. tyrobutyricum KCTC 5387 (ATCC 25755), which consists of a 3.07-Mbp chromosome and a 63-kbp plasmid. The results of genomic analyses suggested that C. tyrobutyricum produces butyrate from butyryl-coenzyme A (butyryl-CoA) through acetate reassimilation by CoA transferase, differently from Clostridium acetobutylicum, which uses the phosphotransbutyrylase-butyrate kinase pathway; this was validated by reverse transcription-PCR (RT-PCR) of related genes, protein expression levels, in vitro CoA transferase assay, and fed-batch fermentation. In addition, the changes in protein expression levels during the course of batch fermentations on glucose were examined by shotgun proteomics. Unlike C. acetobutylicum, the expression levels of proteins involved in glycolytic and fermentative pathways in C. tyrobutyricum did not decrease even at the stationary phase. Proteins related to energy conservation mechanisms, including Rnf complex, NfnAB, and pyruvate-phosphate dikinase that are absent in C. acetobutylicum, were identified. Such features explain why this organism can produce butyric acid to a much higher titer and better tolerate toxic metabolites. This study presenting the complete genome sequence, global protein expression profiles, and genome-based metabolic characteristics during the batch fermentation of C. tyrobutyricum will be valuable in designing strategies for metabolic engineering of this strain. PMID:27302759

  6. Whole-genome bisulfite DNA sequencing of a DNMT3B mutant patient

    OpenAIRE

    Heyn, Holger; Vidal, Enrique; Sayols, Sergi; Sanchez-Mut, Jose V.; Moran, Sebastian; Medina, Ignacio; Sandoval, Juan; Simó-Riudalbas, Laia; Szczesna, Karolina; Huertas, Dori; Gatto, Sole; Matarazzo, Maria R.; Dopazo, Joaquin; Esteller, Manel

    2012-01-01

    The immunodeficiency, centromere instability and facial anomalies (ICF) syndrome is associated to mutations of the DNA methyl-transferase DNMT3B, resulting in a reduction of enzyme activity. Aberrant expression of immune system genes and hypomethylation of pericentromeric regions accompanied by chromosomal instability were determined as alterations driving the disease phenotype. However, so far only technologies capable to analyze single loci were applied to determine epigenetic alterations i...

  7. Boolean implication networks derived from large scale, whole genome microarray datasets

    OpenAIRE

    Sahoo, Debashis; Dill, David L.; Gentles, Andrew J.; Tibshirani, Robert; Plevritis, Sylvia K.

    2008-01-01

    We describe a method for extracting Boolean implications (if-then relationships) in very large amounts of gene expression microarray data. A meta-analysis of data from thousands of microarrays for humans, mice, and fruit flies finds millions of implication relationships between genes that would be missed by other methods. These relationships capture gender differences, tissue differences, development, and differentiation. New relationships are discovered that are preserved across all three sp...

  8. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    DEFF Research Database (Denmark)

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.;

    2015-01-01

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing...... interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV...

  9. Integrative genome-wide gene expression profiling of clear cell renal cell carcinoma in Czech Republic and in the United States.

    Directory of Open Access Journals (Sweden)

    Magdalena B Wozniak

    Full Text Available Gene expression microarray and next generation sequencing efforts on conventional, clear cell renal cell carcinoma (ccRCC have been mostly performed in North American and Western European populations, while the highest incidence rates are found in Central/Eastern Europe. We conducted whole-genome expression profiling on 101 pairs of ccRCC tumours and adjacent non-tumour renal tissue from Czech patients recruited within the "K2 Study", using the Illumina HumanHT-12 v4 Expression BeadChips to explore the molecular variations underlying the biological and clinical heterogeneity of this cancer. Differential expression analysis identified 1650 significant probes (fold change ≥2 and false discovery rate <0.05 mapping to 630 up- and 720 down-regulated unique genes. We performed similar statistical analysis on the RNA sequencing data of 65 ccRCC cases from the Cancer Genome Atlas (TCGA project and identified 60% (402 of the downregulated and 74% (469 of the upregulated genes found in the K2 series. The biological characterization of the significantly deregulated genes demonstrated involvement of downregulated genes in metabolic and catabolic processes, excretion, oxidation reduction, ion transport and response to chemical stimulus, while simultaneously upregulated genes were associated with immune and inflammatory responses, response to hypoxia, stress, wounding, vasculature development and cell activation. Furthermore, genome-wide DNA methylation analysis of 317 TCGA ccRCC/adjacent non-tumour renal tissue pairs indicated that deregulation of approximately 7% of genes could be explained by epigenetic changes. Finally, survival analysis conducted on 89 K2 and 464 TCGA cases identified 8 genes associated with differential prognostic outcomes. In conclusion, a large proportion of ccRCC molecular characteristics were common to the two populations and several may have clinical implications when validated further through large clinical cohorts.

  10. A flexible whole-genome microarray for transcriptomics in three-spine stickleback (Gasterosteus aculeatus

    Directory of Open Access Journals (Sweden)

    Primmer Craig R

    2009-09-01

    Full Text Available Abstract Background The use of microarray technology for describing changes in mRNA expression to address ecological and evolutionary questions is becoming increasingly popular. Since three-spine stickleback are an important ecological and evolutionary model-species as well as an emerging model for eco-toxicology, the ability to have a functional and flexible microarray platform for transcriptome studies will greatly enhance the research potential in these areas. Results We designed 43,392 unique oligonucleotide probes representing 19,274 genes (93% of the estimated total gene number, and tested the hybridization performance of both DNA and RNA from different populations to determine the efficacy of probe design for transcriptome analysis using the Agilent array platform. The majority of probes were functional as evidenced by the DNA hybridization success, and 30,946 probes (14,615 genes had a signal that was significantly above background for RNA isolated from liver tissue. Genes identified as being expressed in liver tissue were grouped into functional categories for each of the three Gene Ontology groups: biological process, molecular function, and cellular component. As expected, the highest proportions of functional categories belonged to those associated with metabolic functions: metabolic process, binding, catabolism, and organelles. Conclusion The probe and microarray design presented here provides an important step facilitating transcriptomics research for this important research organism by providing a set of over 43,000 probes whose hybridization success and specificity to liver expression has been demonstrated. Probes can easily be added or removed from the current design to tailor the array to specific experiments and additional flexibility lies in the ability to perform either one-color or two-color hybridizations.

  11. Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer

    Science.gov (United States)

    Morrison, Carl D.; Liu, Pengyuan; Woloszynska-Read, Anna; Zhang, Jianmin; Luo, Wei; Qin, Maochun; Bshara, Wiam; Conroy, Jeffrey M.; Sabatini, Linda; Vedell, Peter; Xiong, Donghai; Liu, Song; Wang, Jianmin; Shen, He; Li, Yinwei; Omilian, Angela R.; Hill, Annette; Head, Karen; Guru, Khurshid; Kunnev, Dimiter; Leach, Robert; Eng, Kevin H.; Darlak, Christopher; Hoeflich, Christopher; Veeranki, Srividya; Glenn, Sean; You, Ming; Pruitt, Steven C.; Johnson, Candace S.; Trump, Donald L.

    2014-01-01

    Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as “stitchers,” to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication–licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder carcinoma cell lines to study a unique SV (translocation and amplification) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer. PMID:24469795

  12. Implementation of whole genome massively parallel sequencing for noninvasive prenatal testing in laboratories

    NARCIS (Netherlands)

    Thung, G.W.D.T.; Beulen, L.; Hehir-Kwa, J.Y.; Faas, B.H.W.

    2015-01-01

    Noninvasive prenatal testing (NIPT) for fetal aneuploidies using cell-free fetal DNA in maternal plasma has revolutionized the field of prenatal care and methods using massively parallel sequencing are now being implemented almost worldwide. Substantial progress has been made from initially testing

  13. Gene expressions changes in bronchial epithelial cells

    DEFF Research Database (Denmark)

    Remy, S.; Verstraelen, S.; Van Den Heuvel, R.;

    2014-01-01

    cells were exposed during 6, 10, and 24 h to 4 respiratory sensitizers and 6 non-respiratory sensitizers (3 skin sensitizers and 3 respiratory irritants) at a concentration inducing 20% cell viability loss after 24 h. Changes in gene expression were evaluated using Agilent Whole Human Genome 4 x 44 K...... differentially expressed compared to vehicle control for each chemical. The results show that the NRF2-mediated oxidative stress response is activated in the cell line after stimulation with all of the chemicals that were selected in our study, and that - at the level of gene expression - this pathway shows no...

  14. Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced Multi-mutation Strains and Using the Sequence Kernel Association Test (SKAT).

    Science.gov (United States)

    Timbers, Tiffany A; Garland, Stephanie J; Mohan, Swetha; Flibotte, Stephane; Edgley, Mark; Muncaster, Quintin; Au, Vinci; Li-Leger, Erica; Rosell, Federico I; Cai, Jerry; Rademakers, Suzanne; Jansen, Gert; Moerman, Donald G; Leroux, Michel R

    2016-08-01

    Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using C. elegans as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT-1.1 in ciliated sensory neuron function and morphogenesis. BGNT-1.1 functions at the trans-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT-1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy. PMID:27508411

  15. Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced Multi-mutation Strains and Using the Sequence Kernel Association Test (SKAT)

    Science.gov (United States)

    Garland, Stephanie J.; Mohan, Swetha; Flibotte, Stephane; Muncaster, Quintin; Cai, Jerry; Rademakers, Suzanne; Moerman, Donald G.; Leroux, Michel R.

    2016-01-01

    Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using C. elegans as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT-1.1 in ciliated sensory neuron function and morphogenesis. BGNT-1.1 functions at the trans-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT-1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy. PMID:27508411

  16. Diversification and evolution of the SDG gene family in Brassica rapa after the whole genome triplication.

    Science.gov (United States)

    Dong, Heng; Liu, Dandan; Han, Tianyu; Zhao, Yuxue; Sun, Ji; Lin, Sue; Cao, Jiashu; Chen, Zhong-Hua; Huang, Li

    2015-01-01

    Histone lysine methylation, controlled by the SET Domain Group (SDG) gene family, is part of the histone code that regulates chromatin function and epigenetic control of gene expression. Analyzing the SDG gene family in Brassica rapa for their gene structure, domain architecture, subcellular localization, rate of molecular evolution and gene expression pattern revealed common occurrences of subfunctionalization and neofunctionalization in BrSDGs. In comparison with Arabidopsis thaliana, the BrSDG gene family was found to be more divergent than AtSDGs, which might partly explain the rich variety of morphotypes in B. rapa. In addition, a new evolutionary pattern of the four main groups of SDGs was presented, in which the Trx group and the SUVR subgroup evolved faster than the E(z), Ash groups and the SUVH subgroup. These differences in evolutionary rate among the four main groups of SDGs are perhaps due to the complexity and variability of the regions that bind with biomacromolecules, which guide SDGs to their target loci. PMID:26596461

  17. A whole-genome sequence and transcriptome perspective on HER2-positive breast cancers.

    Science.gov (United States)

    Ferrari, Anthony; Vincent-Salomon, Anne; Pivot, Xavier; Sertier, Anne-Sophie; Thomas, Emilie; Tonon, Laurie; Boyault, Sandrine; Mulugeta, Eskeatnaf; Treilleux, Isabelle; MacGrogan, Gaëtan; Arnould, Laurent; Kielbassa, Janice; Le Texier, Vincent; Blanché, Hélène; Deleuze, Jean-François; Jacquemier, Jocelyne; Mathieu, Marie-Christine; Penault-Llorca, Frédérique; Bibeau, Frédéric; Mariani, Odette; Mannina, Cécile; Pierga, Jean-Yves; Trédan, Olivier; Bachelot, Thomas; Bonnefoi, Hervé; Romieu, Gilles; Fumoleau, Pierre; Delaloge, Suzette; Rios, Maria; Ferrero, Jean-Marc; Tarpin, Carole; Bouteille, Catherine; Calvo, Fabien; Gut, Ivo Glynne; Gut, Marta; Martin, Sancha; Nik-Zainal, Serena; Stratton, Michael R; Pauporté, Iris; Saintigny, Pierre; Birnbaum, Daniel; Viari, Alain; Thomas, Gilles

    2016-01-01

    HER2-positive breast cancer has long proven to be a clinically distinct class of breast cancers for which several targeted therapies are now available. However, resistance to the treatment associated with specific gene expressions or mutations has been observed, revealing the underlying diversity of these cancers. Therefore, understanding the full extent of the HER2-positive disease heterogeneity still remains challenging. Here we carry out an in-depth genomic characterization of 64 HER2-positive breast tumour genomes that exhibit four subgroups, based on the expression data, with distinctive genomic features in terms of somatic mutations, copy-number changes or structural variations. The results suggest that, despite being clinically defined by a specific gene amplification, HER2-positive tumours melt into the whole luminal-basal breast cancer spectrum rather than standing apart. The results also lead to a refined ERBB2 amplicon of 106 kb and show that several cases of amplifications are compatible with a breakage-fusion-bridge mechanism. PMID:27406316

  18. Foxp3 expression in human cancer cells

    Directory of Open Access Journals (Sweden)

    Gourgoulianis Konstantinos I

    2008-04-01

    Full Text Available Abstract Objective Transcription factor forkhead box protein 3 (Foxp3 specifically characterizes the thymically derived naturally occurring regulatory T cells (Tregs. Limited evidence indicates that it is also expressed, albeit to a lesser extent, in tissues other than thymus and spleen, while, very recently, it was shown that Foxp3 is expressed by pancreatic carcinoma. This study was scheduled to investigate whether expression of Foxp3 transcripts and mature protein occurs constitutively in various tumor types. Materials and methods Twenty five tumor cell lines of different tissue origins (lung cancer, colon cancer, breast cancer, melanoma, erythroid leukemia, acute T-cell leukemia were studied. Detection of Foxp3 mRNA was performed using both conventional RT-PCR and quantitative real-time PCR while protein expression was assessed by immunocytochemistry and flow cytometry, using different antibody clones. Results Foxp3 mRNA as well as Foxp3 protein was detected in all tumor cell lines, albeit in variable levels, not related to the tissue of origin. This expression correlated with the expression levels of IL-10 and TGFb1. Conclusion We offer evidence that Foxp3 expression, characterizes tumor cells of various tissue origins. The biological significance of these findings warrants further investigation in the context of tumor immune escape, and especially under the light of current anti-cancer efforts interfering with Foxp3 expression.

  19. Whole-genome cartography of p53 response elements ranked on transactivation potential

    OpenAIRE

    Tebaldi, Toma; Zaccara, Sara; Alessandrini, Federica; Bisio, Alessandra; Ciribilli, Yari; Inga, Alberto

    2015-01-01

    Background Many recent studies using ChIP-seq approaches cross-referenced to trascriptome data and also to potentially unbiased in vitro DNA binding selection experiments are detailing with increasing precision the p53-directed gene regulatory network that, nevertheless, is still expanding. However, most experiments have been conducted in established cell lines subjected to specific p53-inducing stimuli, both factors potentially biasing the results. Results We developed p53retriever, a patter...

  20. Those other mammals: The immunoglobulins and T cell receptors of marsupials and monotremes

    OpenAIRE

    Miller, Robert D.

    2009-01-01

    This review summarizes analyses of marsupial and monotreme immunoglobulin and T cell receptor genetics and expression published over the past decade. Analyses of recently completed whole genome sequences from the opossum and the platypus have yielded insight into the evolution of the common antigen receptor systems, as well as discovery of novel receptors that appear to have been lost in eutherian mammals. These species are also useful for investigation of the development of the immune system...