Evaluating genome-wide DNA methylation changes in mice by Methylation Specific Digital Karyotyping

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Maruoka Shuichiro

2008-12-01

Full Text Available Abstract Background The study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK, a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci. Results Genomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05 after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes. Conclusion The genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models

Recent advances in the genome-wide study of DNA replication origins in yeast

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Chong ePeng

2015-02-01

Full Text Available DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs. Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genome. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some nonconventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve the replication origins prediction.

Recent advances in the genome-wide study of DNA replication origins in yeast

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Peng, Chong; Luo, Hao; Zhang, Xi; Gao, Feng

2015-01-01

DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs). Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genomes. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some non-conventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve yeast replication origins prediction. PMID:25745419

Genome-wide DNA methylation maps in follicular lymphoma cells determined by methylation-enriched bisulfite sequencing.

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Jeong-Hyeon Choi

Full Text Available BACKGROUND: Follicular lymphoma (FL is a form of non-Hodgkin's lymphoma (NHL that arises from germinal center (GC B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. CONCLUSIONS/SIGNIFICANCE: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

Epigenetic Variation in Monozygotic Twins: A Genome-Wide Analysis of DNA Methylation in Buccal Cells

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Jenny van Dongen

2014-05-01

Full Text Available DNA methylation is one of the most extensively studied epigenetic marks in humans. Yet, it is largely unknown what causes variation in DNA methylation between individuals. The comparison of DNA methylation profiles of monozygotic (MZ twins offers a unique experimental design to examine the extent to which such variation is related to individual-specific environmental influences and stochastic events or to familial factors (DNA sequence and shared environment. We measured genome-wide DNA methylation in buccal samples from ten MZ pairs (age 8–19 using the Illumina 450k array and examined twin correlations for methylation level at 420,921 CpGs after QC. After selecting CpGs showing the most variation in the methylation level between subjects, the mean genome-wide correlation (rho was 0.54. The correlation was higher, on average, for CpGs within CpG islands (CGIs, compared to CGI shores, shelves and non-CGI regions, particularly at hypomethylated CpGs. This finding suggests that individual-specific environmental and stochastic influences account for more variation in DNA methylation in CpG-poor regions. Our findings also indicate that it is worthwhile to examine heritable and shared environmental influences on buccal DNA methylation in larger studies that also include dizygotic twins.

Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

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Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna

2016-04-07

DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. Copyright © 2016 Vrljicak et al.

Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

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Raghav, Sunil Kumar; Deplancke, Bart

2012-01-01

Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.

Genome-Wide DNA Methylation Indicates Silencing of Tumor Suppressor Genes in Uterine Leiomyoma

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Navarro, Antonia; Yin, Ping; Monsivais, Diana; Lin, Simon M.; Du, Pan; Wei, Jian-Jun; Bulun, Serdar E.

2012-01-01

Background Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown. Principal Findings We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels. Conclusions These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women. PMID:22428009

Genome-Wide Expression of MicroRNAs Is Regulated by DNA Methylation in Hepatocarcinogenesis

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Jing Shen

2015-01-01

Full Text Available Background. Previous studies, including ours, have examined the regulation of microRNAs (miRNAs by DNA methylation, but whether this regulation occurs at a genome-wide level in hepatocellular carcinoma (HCC is unclear. Subjects/Methods. Using a two-phase study design, we conducted genome-wide screening for DNA methylation and miRNA expression to explore the potential role of methylation alterations in miRNAs regulation. Results. We found that expressions of 25 miRNAs were statistically significantly different between tumor and nontumor tissues and perfectly differentiated HCC tumor from nontumor. Six miRNAs were overexpressed, and 19 were repressed in tumors. Among 133 miRNAs with inverse correlations between methylation and expression, 8 miRNAs (6% showed statistically significant differences in expression between tumor and nontumor tissues. Six miRNAs were validated in 56 additional paired HCC tissues, and significant inverse correlations were observed for miR-125b and miR-199a, which is consistent with the inactive chromatin pattern found in HepG2 cells. Conclusion. These data suggest that the expressions of miR-125b and miR-199a are dramatically regulated by DNA hypermethylation that plays a key role in hepatocarcinogenesis.

DNA immunoprecipitation semiconductor sequencing (DIP-SC-seq) as a rapid method to generate genome wide epigenetic signatures

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Thomson, John P.; Fawkes, Angie; Ottaviano, Raffaele; Hunter, Jennifer M.; Shukla, Ruchi; Mjoseng, Heidi K.; Clark, Richard; Coutts, Audrey; Murphy, Lee; Meehan, Richard R.

2015-01-01

Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially ...

Genome-wide DNA methylation profiling in cultured eutopic and ectopic endometrial stromal cells.

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Yoshiaki Yamagata

Full Text Available The objective of this study was to characterize the genome-wide DNA methylation profiles of isolated endometrial stromal cells obtained from eutopic endometria with (euESCa and without endometriosis (euESCb and ovarian endometrial cysts (choESC. Three samples were analyzed in each group. The infinium methylation array identified more hypermethylated and hypomethylated CpGs in choESC than in euESCa, and only a few genes were methylated differently in euESCa and euESCb. A functional analysis revealed that signal transduction, developmental processes, immunity, etc. were different in choESC and euESCa. A clustering analysis and a principal component analysis performed based on the methylation levels segregated choESC from euESC, while euESCa and euESCb were identical. A transcriptome analysis was then conducted and the results were compared with those of the DNA methylation analysis. Interestingly, the hierarchical clustering and principal component analyses showed that choESC were segregated from euESCa and euESCb in the DNA methylation analysis, while no segregation was recognized in the transcriptome analysis. The mRNA expression levels of the epigenetic modification enzymes, including DNA methyltransferases, obtained from the specimens were not significantly different between the groups. Some of the differentially methylated and/or expressed genes (NR5A1, STAR, STRA6 and HSD17B2, which are related with steroidogenesis, were validated by independent methods in a larger number of samples. Our findings indicate that different DNA methylation profiles exist in ectopic ESC, highlighting the benefits of genome wide DNA methylation analyses over transcriptome analyses in clarifying the development and characterization of endometriosis.

Epigenomics of Total Acute Sleep Deprivation in Relation to Genome-Wide DNA Methylation Profiles and RNA Expression

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Nilsson, Emil K.; Bostr?m, Adrian E.; Mwinyi, Jessica; Schi?th, Helgi B.

2016-01-01

Abstract Despite an established link between sleep deprivation and epigenetic processes in humans, it remains unclear to what extent sleep deprivation modulates DNA methylation. We performed a within-subject randomized blinded study with 16 healthy subjects to examine the effect of one night of total sleep deprivation (TSD) on the genome-wide methylation profile in blood compared with that in normal sleep. Genome-wide differences in methylation between both conditions were assessed by applyin...

Genome-Wide Methylated DNA Immunoprecipitation Analysis of Patients with Polycystic Ovary Syndrome

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Shen, Hao-ran; Qiu, Li-hua; Zhang, Zhi-qing; Qin, Yuan-yuan; Cao, Cong; Di, Wen

2013-01-01

Polycystic ovary syndrome (PCOS) is a complex, heterogeneous disorder of uncertain etiology. Recent studies suggested that insulin resistance (IR) plays an important role in the development of PCOS. In the current study, we aimed to investigate the molecular mechanism of IR in PCOS. We employed genome-wide methylated DNA immunoprecipitation (MeDIP) analysis to characterize genes that are differentially methylated in PCOS patients vs. healthy controls. Besides, we also identified the different...

Genome-wide signatures of differential DNA methylation in pediatric acute lymphoblastic leukemia

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Nordlund, Jessica; Bäcklin, Christofer L; Wahlberg, Per

2013-01-01

BACKGROUND: Although aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic...... background, drug resistance and relapse in ALL is poorly understood. RESULTS: We surveyed the DNA methylation levels of 435,941 CpG sites in samples from 764 children at diagnosis of ALL and from 27 children at relapse. This survey uncovered four characteristic methylation signatures. First, compared...... cells at relapse, compared with matched samples at diagnosis. Analysis of relapse-free survival identified CpG sites with subtype-specific differential methylation that divided the patients into different risk groups, depending on their methylation status. CONCLUSIONS: Our results suggest an important...

Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents.

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2005-08-01

Full Text Available The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.

Tobacco smoking leads to extensive genome-wide changes in DNA methylation.

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Sonja Zeilinger

Full Text Available Environmental factors such as tobacco smoking may have long-lasting effects on DNA methylation patterns, which might lead to changes in gene expression and in a broader context to the development or progression of various diseases. We conducted an epigenome-wide association study (EWAs comparing current, former and never smokers from 1793 participants of the population-based KORA F4 panel, with replication in 479 participants from the KORA F3 panel, carried out by the 450K BeadChip with genomic DNA obtained from whole blood. We observed wide-spread differences in the degree of site-specific methylation (with p-values ranging from 9.31E-08 to 2.54E-182 as a function of tobacco smoking in each of the 22 autosomes, with the percent of variance explained by smoking ranging from 1.31 to 41.02. Depending on cessation time and pack-years, methylation levels in former smokers were found to be close to the ones seen in never smokers. In addition, methylation-specific protein binding patterns were observed for cg05575921 within AHRR, which had the highest level of detectable changes in DNA methylation associated with tobacco smoking (-24.40% methylation; p = 2.54E-182, suggesting a regulatory role for gene expression. The results of our study confirm the broad effect of tobacco smoking on the human organism, but also show that quitting tobacco smoking presumably allows regaining the DNA methylation state of never smokers.

Tobacco smoking leads to extensive genome-wide changes in DNA methylation.

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Zeilinger, Sonja; Kühnel, Brigitte; Klopp, Norman; Baurecht, Hansjörg; Kleinschmidt, Anja; Gieger, Christian; Weidinger, Stephan; Lattka, Eva; Adamski, Jerzy; Peters, Annette; Strauch, Konstantin; Waldenberger, Melanie; Illig, Thomas

2013-01-01

Environmental factors such as tobacco smoking may have long-lasting effects on DNA methylation patterns, which might lead to changes in gene expression and in a broader context to the development or progression of various diseases. We conducted an epigenome-wide association study (EWAs) comparing current, former and never smokers from 1793 participants of the population-based KORA F4 panel, with replication in 479 participants from the KORA F3 panel, carried out by the 450K BeadChip with genomic DNA obtained from whole blood. We observed wide-spread differences in the degree of site-specific methylation (with p-values ranging from 9.31E-08 to 2.54E-182) as a function of tobacco smoking in each of the 22 autosomes, with the percent of variance explained by smoking ranging from 1.31 to 41.02. Depending on cessation time and pack-years, methylation levels in former smokers were found to be close to the ones seen in never smokers. In addition, methylation-specific protein binding patterns were observed for cg05575921 within AHRR, which had the highest level of detectable changes in DNA methylation associated with tobacco smoking (-24.40% methylation; p = 2.54E-182), suggesting a regulatory role for gene expression. The results of our study confirm the broad effect of tobacco smoking on the human organism, but also show that quitting tobacco smoking presumably allows regaining the DNA methylation state of never smokers.

p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

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Hattori, Hiroyoshi; Janky, Rekin's; Nietfeld, Wilfried; Aerts, Stein; Madan Babu, M; Venkitaraman, Ashok R

2014-01-01

The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients.

Genome-Wide DNA Methylation in Mixed Ancestry Individuals with Diabetes and Prediabetes from South Africa

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Pheiffer, Carmen; Humphries, Stephen E.; Gamieldien, Junaid; Erasmus, Rajiv T.

2016-01-01

Aims. To conduct a genome-wide DNA methylation in individuals with type 2 diabetes, individuals with prediabetes, and control mixed ancestry individuals from South Africa. Methods. We used peripheral blood to perform genome-wide DNA methylation analysis in 3 individuals with screen detected diabetes, 3 individuals with prediabetes, and 3 individuals with normoglycaemia from the Bellville South Community, Cape Town, South Africa, who were age-, gender-, body mass index-, and duration of residency-matched. Methylated DNA immunoprecipitation (MeDIP) was performed by Arraystar Inc. (Rockville, MD, USA). Results. Hypermethylated DMRs were 1160 (81.97%) and 124 (43.20%), respectively, in individuals with diabetes and prediabetes when both were compared to subjects with normoglycaemia. Our data shows that genes related to the immune system, signal transduction, glucose transport, and pancreas development have altered DNA methylation in subjects with prediabetes and diabetes. Pathway analysis based on the functional analysis mapping of genes to KEGG pathways suggested that the linoleic acid metabolism and arachidonic acid metabolism pathways are hypomethylated in prediabetes and diabetes. Conclusions. Our study suggests that epigenetic changes are likely to be an early process that occurs before the onset of overt diabetes. Detailed analysis of DMRs that shows gradual methylation differences from control versus prediabetes to prediabetes versus diabetes in a larger sample size is required to confirm these findings. PMID:27555869

EG-13GENOME-WIDE METHYLATION ANALYSIS IDENTIFIES GENOMIC DNA DEMETHYLATION DURING MALIGNANT PROGRESSION OF GLIOMAS

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Saito, Kuniaki; Mukasa, Akitake; Nagae, Genta; Aihara, Koki; Otani, Ryohei; Takayanagi, Shunsaku; Omata, Mayu; Tanaka, Shota; Shibahara, Junji; Takahashi, Miwako; Momose, Toshimitsu; Shimamura, Teppei; Miyano, Satoru; Narita, Yoshitaka; Ueki, Keisuke; Nishikawa, Ryo; Nagane, Motoo; Aburatani, Hiroyuki; Saito, Nobuhito

2014-01-01

Low-grade gliomas often undergo malignant progression, and these transformations are a leading cause of death in patients with low-grade gliomas. However, the molecular mechanisms underlying malignant tumor progression are still not well understood. Recent evidence indicates that epigenetic deregulation is an important cause of gliomagenesis; therefore, we examined the impact of epigenetic changes during malignant progression of low-grade gliomas. Specifically, we used the Illumina Infinium Human Methylation 450K BeadChip to perform genome-wide DNA methylation analysis of 120 gliomas and four normal brains. This study sample included 25 matched-pairs of initial low-grade gliomas and recurrent tumors (temporal heterogeneity) and 20 of the 25 recurring tumors recurred as malignant progressions, and one matched-pair of newly emerging malignant lesions and pre-existing lesions (spatial heterogeneity). Analyses of methylation profiles demonstrated that most low-grade gliomas in our sample (43/51; 84%) had a CpG island methylator phenotype (G-CIMP). Remarkably, approximately 50% of secondary glioblastomas that had progressed from low-grade tumors with the G-CIMP status exhibited a characteristic partial demethylation of genomic DNA during malignant progression, but other recurrent gliomas showed no apparent change in DNA methylation pattern. Interestingly, we found that most loci that were demethylated during malignant progression were located outside of CpG islands. The information of histone modifications patterns in normal human astrocytes and embryonal stem cells also showed that the ratio of active marks at the site corresponding to DNA demethylated loci in G-CIMP-demethylated tumors was significantly lower; this finding indicated that most demethylated loci in G-CIMP-demethylated tumors were likely transcriptionally inactive. A small number of the genes that were upregulated and had demethylated CpG islands were associated with cell cycle-related pathway. In

Genome-wide screen of ovary-specific DNA methylation in polycystic ovary syndrome.

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Yu, Ying-Ying; Sun, Cui-Xiang; Liu, Yin-Kun; Li, Yan; Wang, Li; Zhang, Wei

2015-07-01

To compare genome-wide DNA methylation profiles in ovary tissue from women with polycystic ovary syndrome (PCOS) and healthy controls. Case-control study matched for age and body mass index. University-affiliated hospital. Ten women with PCOS who underwent ovarian drilling to induce ovulation and 10 healthy women who were undergoing laparoscopic sterilization, hysterectomy for benign conditions, diagnostic laparoscopy for pelvic pain, or oophorectomy for nonovarian indications. None. Genome-wide DNA methylation patterns determined by immunoprecipitation and microarray (MeDIP-chip) analysis. The methylation levels were statistically significantly higher in CpG island shores (CGI shores), which lie outside of core promoter regions, and lower within gene bodies in women with PCOS relative to the controls. In addition, high CpG content promoters were the most frequently hypermethylated promoters in PCOS ovaries but were more often hypomethylated in controls. Second, 872 CGIs, specifically methylated in PCOS, represented 342 genes that could be associated with various molecular functions, including protein binding, hormone activity, and transcription regulator activity. Finally, methylation differences were validated in seven genes by methylation-specific polymerase chain reaction. These genes correlated to several functional families related to the pathogenesis of PCOS and may be potential biomarkers for this disease. Our results demonstrated that epigenetic modification differs between PCOS and normal ovaries, which may help to further understand the pathophysiology of this disease. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

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Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

2015-08-01

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. © 2015 Polstein et al.; Published by Cold Spring Harbor Laboratory Press.

Genome-wide DNA methylation profiling with MeDIP-seq using archived dried blood spots

DEFF Research Database (Denmark)

Staunstrup, Nicklas H; Starnawska, Anna; Nyegaard, Mette

2016-01-01

BACKGROUND: In utero and early-life experienced environmental exposures are suggested to play an important role in many multifactorial diseases potentially mediated through lasting effects on the epigenome. As the epigenome in addition remains modifiable throughout life, identifying specific...... biobanks. However, availability of this biological material is highly limited as each DBS is made only from a few droplets of blood and storage conditions may be suboptimal for epigenetic studies. Furthermore, as relevant markers may reside outside gene bodies, epigenome-wide interrogation is needed....... RESULTS: Here we demonstrate, as a proof of principle, that genome-wide interrogation of the methylome based on methylated DNA immunoprecipitation coupled with next-generation sequencing (MeDIP-seq) is feasible using a single 3.2 mm DBS punch (60 ng DNA) from filter cards archived for up to 16 years...

Epigenomics of Total Acute Sleep Deprivation in Relation to Genome-Wide DNA Methylation Profiles and RNA Expression.

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Nilsson, Emil K; Boström, Adrian E; Mwinyi, Jessica; Schiöth, Helgi B

2016-06-01

Despite an established link between sleep deprivation and epigenetic processes in humans, it remains unclear to what extent sleep deprivation modulates DNA methylation. We performed a within-subject randomized blinded study with 16 healthy subjects to examine the effect of one night of total sleep deprivation (TSD) on the genome-wide methylation profile in blood compared with that in normal sleep. Genome-wide differences in methylation between both conditions were assessed by applying a paired regression model that corrected for monocyte subpopulations. In addition, the correlations between the methylation of genes detected to be modulated by TSD and gene expression were examined in a separate, publicly available cohort of 10 healthy male donors (E-GEOD-49065). Sleep deprivation significantly affected the DNA methylation profile both independently and in dependency of shifts in monocyte composition. Our study detected differential methylation of 269 probes. Notably, one CpG site was located 69 bp upstream of ING5, which has been shown to be differentially expressed after sleep deprivation. Gene set enrichment analysis detected the Notch and Wnt signaling pathways to be enriched among the differentially methylated genes. These results provide evidence that total acute sleep deprivation alters the methylation profile in healthy human subjects. This is, to our knowledge, the first study that systematically investigated the impact of total acute sleep deprivation on genome-wide DNA methylation profiles in blood and related the epigenomic findings to the expression data.

Genome-wide DNA methylation patterns in wild samples of two morphotypes of threespine stickleback (Gasterosteus aculeatus).

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Smith, Gilbert; Smith, Carl; Kenny, John G; Chaudhuri, Roy R; Ritchie, Michael G

2015-04-01

Epigenetic marks such as DNA methylation play important biological roles in gene expression regulation and cellular differentiation during development. To examine whether DNA methylation patterns are potentially associated with naturally occurring phenotypic differences, we examined genome-wide DNA methylation within Gasterosteus aculeatus, using reduced representation bisulfite sequencing. First, we identified highly methylated regions of the stickleback genome, finding such regions to be located predominantly within genes, and associated with genes functioning in metabolism and biosynthetic processes, cell adhesion, signaling pathways, and blood vessel development. Next, we identified putative differentially methylated regions (DMRs) of the genome between complete and low lateral plate morphs of G. aculeatus. We detected 77 DMRs that were mainly located in intergenic regions. Annotations of genes associated with these DMRs revealed potential functions in a number of known divergent adaptive phenotypes between G. aculeatus ecotypes, including cardiovascular development, growth, and neuromuscular development. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

DMS-Seq for In Vivo Genome-wide Mapping of Protein-DNA Interactions and Nucleosome Centers.

Science.gov (United States)

Umeyama, Taichi; Ito, Takashi

2017-10-03

Protein-DNA interactions provide the basis for chromatin structure and gene regulation. Comprehensive identification of protein-occupied sites is thus vital to an in-depth understanding of genome function. Dimethyl sulfate (DMS) is a chemical probe that has long been used to detect footprints of DNA-bound proteins in vitro and in vivo. Here, we describe a genomic footprinting method, dimethyl sulfate sequencing (DMS-seq), which exploits the cell-permeable nature of DMS to obviate the need for nuclear isolation. This feature makes DMS-seq simple in practice and removes the potential risk of protein re-localization during nuclear isolation. DMS-seq successfully detects transcription factors bound to cis-regulatory elements and non-canonical chromatin particles in nucleosome-free regions. Furthermore, an unexpected preference of DMS confers on DMS-seq a unique potential to directly detect nucleosome centers without using genetic manipulation. We expect that DMS-seq will serve as a characteristic method for genome-wide interrogation of in vivo protein-DNA interactions. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Genome-wide nucleosome occupancy and DNA methylation profiling of four human cell lines

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Aaron L. Statham

2015-03-01

Full Text Available DNA methylation and nucleosome positioning are two key mechanisms that contribute to the epigenetic control of gene expression. During carcinogenesis, the expression of many genes is altered alongside extensive changes in the epigenome, with repressed genes often being associated with local DNA hypermethylation and gain of nucleosomes at their promoters. However the spectrum of alterations that occur at distal regulatory regions has not been extensively studied. To address this we used Nucleosome Occupancy and Methylation sequencing (NOMe-seq to compare the genome-wide DNA methylation and nucleosome occupancy profiles between normal and cancer cell line models of the breast and prostate. Here we describe the bioinformatic pipeline and methods that we developed for the processing and analysis of the NOMe-seq data published by (Taberlay et al., 2014 [1] and deposited in the Gene Expression Omnibus with accession GSE57498.

Genome-wide DNA methylation analysis of pseudohypoparathyroidism patients with GNAS imprinting defects.

Science.gov (United States)

Rochtus, Anne; Martin-Trujillo, Alejandro; Izzi, Benedetta; Elli, Francesca; Garin, Intza; Linglart, Agnes; Mantovani, Giovanna; Perez de Nanclares, Guiomar; Thiele, Suzanne; Decallonne, Brigitte; Van Geet, Chris; Monk, David; Freson, Kathleen

2016-01-01

Pseudohypoparathyroidism (PHP) is caused by (epi)genetic defects in the imprinted GNAS cluster. Current classification of PHP patients is hampered by clinical and molecular diagnostic overlaps. The European Consortium for the study of PHP designed a genome-wide methylation study to improve molecular diagnosis. The HumanMethylation 450K BeadChip was used to analyze genome-wide methylation in 24 PHP patients with parathyroid hormone resistance and 20 age- and gender-matched controls. Patients were previously diagnosed with GNAS-specific differentially methylated regions (DMRs) and include 6 patients with known STX16 deletion (PHP(Δstx16)) and 18 without deletion (PHP(neg)). The array demonstrated that PHP patients do not show DNA methylation differences at the whole-genome level. Unsupervised clustering of GNAS-specific DMRs divides PHP(Δstx16) versus PHP(neg) patients. Interestingly, in contrast to the notion that all PHP patients share methylation defects in the A/B DMR while only PHP(Δstx16) patients have normal NESP, GNAS-AS1 and XL methylation, we found a novel DMR (named GNAS-AS2) in the GNAS-AS1 region that is significantly different in both PHP(Δstx16) and PHP(neg), as validated by Sequenom EpiTYPER in a larger PHP cohort. The analysis of 58 DMRs revealed that 8/18 PHP(neg) and 1/6 PHP(Δstx16) patients have multi-locus methylation defects. Validation was performed for FANCC and SVOPL DMRs. This is the first genome-wide methylation study for PHP patients that confirmed that GNAS is the most significant DMR, and the presence of STX16 deletion divides PHP patients in two groups. Moreover, a novel GNAS-AS2 DMR affects all PHP patients, and PHP patients seem sensitive to multi-locus methylation defects.

Insights into the Pathogenesis of Anaplastic Large-Cell Lymphoma through Genome-wide DNA Methylation Profiling

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Melanie R. Hassler

2016-10-01

Full Text Available Aberrant DNA methylation patterns in malignant cells allow insight into tumor evolution and development and can be used for disease classification. Here, we describe the genome-wide DNA methylation signatures of NPM-ALK-positive (ALK+ and NPM-ALK-negative (ALK− anaplastic large-cell lymphoma (ALCL. We find that ALK+ and ALK− ALCL share common DNA methylation changes for genes involved in T cell differentiation and immune response, including TCR and CTLA-4, without an ALK-specific impact on tumor DNA methylation in gene promoters. Furthermore, we uncover a close relationship between global ALCL DNA methylation patterns and those in distinct thymic developmental stages and observe tumor-specific DNA hypomethylation in regulatory regions that are enriched for conserved transcription factor binding motifs such as AP1. Our results indicate similarity between ALCL tumor cells and thymic T cell subsets and a direct relationship between ALCL oncogenic signaling and DNA methylation through transcription factor induction and occupancy.

Genome-wide, Single-Cell DNA Methylomics Reveals Increased Non-CpG Methylation during Human Oocyte Maturation

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Bo Yu

2017-07-01

Full Text Available The establishment of DNA methylation patterns in oocytes is a highly dynamic process marking gene-regulatory events during fertilization, embryonic development, and adulthood. However, after epigenetic reprogramming in primordial germ cells, how and when DNA methylation is re-established in developing human oocytes remains to be characterized. Here, using single-cell whole-genome bisulfite sequencing, we describe DNA methylation patterns in three different maturation stages of human oocytes. We found that while broad-scale patterns of CpG methylation have been largely established by the immature germinal vesicle stage, localized changes continue into later development. Non-CpG methylation, on the other hand, undergoes a large-scale, generalized remodeling through the final stage of maturation, with the net overall result being the accumulation of methylation as oocytes mature. The role of the genome-wide, non-CpG methylation remodeling in the final stage of oocyte maturation deserves further investigation.

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).

Science.gov (United States)

Langley, Alexander R; Gräf, Stefan; Smith, James C; Krude, Torsten

2016-12-01

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome

DEFF Research Database (Denmark)

Pedersen, Jakob Skou; Valen, Eivind; Velazquez, Amhed Missael Vargas

2014-01-01

Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence...... data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery...

Genome-wide Differences in DNA Methylation Changes in Two Contrasting Rice Genotypes in Response to Drought Conditions

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Wensheng Wang

2016-11-01

Full Text Available Differences in drought stress tolerance within diverse rice genotypes have been attributed to genetic diversity and epigenetic alterations. DNA methylation is an important epigenetic modification that influences diverse biological processes, but its effects on rice drought stress tolerance are poorly understood. In this study, methylated DNA immunoprecipitation sequencing and an Affymetrix GeneChip rice genome array were used to profile the DNA methylation patterns and transcriptomes of the drought-tolerant introgression line DK151 and its drought-sensitive recurrent parent IR64 under drought and control conditions. The introgression of donor genomic DNA induced genome-wide DNA methylation changes in DK151 plants. A total of 1190 differentially methylated regions (DMRs were detected between the two genotypes under normal growth conditions, and the DMR-associated genes in DK151 plants were mainly related to stress response, programmed cell death, and nutrient reservoir activity, which are implicated to constitutive drought stress tolerance. A comparison of the DNA methylation changes in the two genotypes under drought conditions indicated that DK151 plants have a more stable methylome, with only 92 drought-induced DMRs, than IR64 plants with 506 DMRs. Gene ontology analyses of the DMR-associated genes in drought-stressed plants revealed that changes to the DNA methylation status of genotype-specific genes are associated with the epigenetic regulation of drought stress responses. Transcriptome analysis further helped to identify a set of 12 and 23 DMR-associated genes that were differentially expressed in DK151 and IR64, respectively, under drought stress compared with respective controls. Correlation analysis indicated that DNA methylation has various effects on gene expression, implying that it affects gene expression directly or indirectly through diverse regulatory pathways. Our results indicate that drought-induced alterations to DNA

Differential DNA Methylation Analysis without a Reference Genome

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Johanna Klughammer

2015-12-01

Full Text Available Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS, which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish. Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org. The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome.

Genome-wide survey of repetitive DNA elements in the button mushroom Agaricus bisporus

NARCIS (Netherlands)

Foulongne-Oriol, M.; Murat, C.; Castanera, R.; Ramírez, L.; Sonnenberg, A.S.M.

2013-01-01

Repetitive DNA elements are ubiquitous constituents of eukaryotic genomes. The biological roles of these repetitive elements, supposed to impact genome organization and evolution, are not completely elucidated yet. The availability of whole genome sequence offers the opportunity to draw a picture of

Young men with low birthweight exhibit decreased plasticity of genome-wide muscle DNA methylation by high-fat overfeeding

DEFF Research Database (Denmark)

Jacobsen, Stine C; Gillberg, Linn; Bork-Jensen, Jette

2014-01-01

The association between low birthweight (LBW) and risk of developing type 2 diabetes may involve epigenetic mechanisms, with skeletal muscle being a prime target tissue. Differential DNA methylation patterns have been observed in single genes in muscle tissue from type 2 diabetic and LBW...... individuals, and we recently showed multiple DNA methylation changes during short-term high-fat overfeeding in muscle of healthy people. In a randomised crossover study, we analysed genome-wide DNA promoter methylation in skeletal muscle of 17 young LBW men and 23 matched normal birthweight (NBW) men after...... a control and a 5 day high-fat overfeeding diet....

Pervasive, Genome-Wide Transcription in the Organelle Genomes of Diverse Plastid-Bearing Protists.

Science.gov (United States)

Sanitá Lima, Matheus; Smith, David Roy

2017-11-06

Organelle genomes are among the most sequenced kinds of chromosome. This is largely because they are small and widely used in molecular studies, but also because next-generation sequencing technologies made sequencing easier, faster, and cheaper. However, studies of organelle RNA have not kept pace with those of DNA, despite huge amounts of freely available eukaryotic RNA-sequencing (RNA-seq) data. Little is known about organelle transcription in nonmodel species, and most of the available eukaryotic RNA-seq data have not been mined for organelle transcripts. Here, we use publicly available RNA-seq experiments to investigate organelle transcription in 30 diverse plastid-bearing protists with varying organelle genomic architectures. Mapping RNA-seq data to organelle genomes revealed pervasive, genome-wide transcription, regardless of the taxonomic grouping, gene organization, or noncoding content. For every species analyzed, transcripts covered ≥85% of the mitochondrial and/or plastid genomes (all of which were ≤105 kb), indicating that most of the organelle DNA-coding and noncoding-is transcriptionally active. These results follow earlier studies of model species showing that organellar transcription is coupled and ubiquitous across the genome, requiring significant downstream processing of polycistronic transcripts. Our findings suggest that noncoding organelle DNA can be transcriptionally active, raising questions about the underlying function of these transcripts and underscoring the utility of publicly available RNA-seq data for recovering complete genome sequences. If pervasive transcription is also found in bigger organelle genomes (>105 kb) and across a broader range of eukaryotes, this could indicate that noncoding organelle RNAs are regulating fundamental processes within eukaryotic cells. Copyright © 2017 Sanitá Lima and Smith.

Effects of short-term high-fat overfeeding on genome-wide DNA methylation in the skeletal muscle of healthy young men

DEFF Research Database (Denmark)

Jacobsen, S C; Brøns, Charlotte; Bork-Jensen, Jette

2012-01-01

Energy-dense diets that are high in fat are associated with a risk of metabolic diseases. The underlying molecular mechanisms could involve epigenetics, as recent data show altered DNA methylation of putative type 2 diabetes candidate genes in response to high-fat diets. We examined the effect...... of a short-term high-fat overfeeding (HFO) diet on genome-wide DNA methylation patterns in human skeletal muscle....

Genome-wide DNA methylation profiling in the superior temporal gyrus reveals epigenetic signatures associated with Alzheimer's disease.

Science.gov (United States)

Watson, Corey T; Roussos, Panos; Garg, Paras; Ho, Daniel J; Azam, Nidha; Katsel, Pavel L; Haroutunian, Vahram; Sharp, Andrew J

2016-01-19

Alzheimer's disease affects ~13% of people in the United States 65 years and older, making it the most common neurodegenerative disorder. Recent work has identified roles for environmental, genetic, and epigenetic factors in Alzheimer's disease risk. We performed a genome-wide screen of DNA methylation using the Illumina Infinium HumanMethylation450 platform on bulk tissue samples from the superior temporal gyrus of patients with Alzheimer's disease and non-demented controls. We paired a sliding window approach with multivariate linear regression to characterize Alzheimer's disease-associated differentially methylated regions (DMRs). We identified 479 DMRs exhibiting a strong bias for hypermethylated changes, a subset of which were independently associated with aging. DMR intervals overlapped 475 RefSeq genes enriched for gene ontology categories with relevant roles in neuron function and development, as well as cellular metabolism, and included genes reported in Alzheimer's disease genome-wide and epigenome-wide association studies. DMRs were enriched for brain-specific histone signatures and for binding motifs of transcription factors with roles in the brain and Alzheimer's disease pathology. Notably, hypermethylated DMRs preferentially overlapped poised promoter regions, marked by H3K27me3 and H3K4me3, previously shown to co-localize with aging-associated hypermethylation. Finally, the integration of DMR-associated single nucleotide polymorphisms with Alzheimer's disease genome-wide association study risk loci and brain expression quantitative trait loci highlights multiple potential DMRs of interest for further functional analysis. We have characterized changes in DNA methylation in the superior temporal gyrus of patients with Alzheimer's disease, highlighting novel loci that facilitate better characterization of pathways and mechanisms underlying Alzheimer's disease pathogenesis, and improve our understanding of epigenetic signatures that may contribute to the

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.

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Traverse, Charles C; Ochman, Howard

2017-08-29

Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola , which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. IMPORTANCE The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions-errors that potentially have more dire effects on protein function-is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions

Genome-wide association study for ovarian cancer susceptibility using pooled DNA

DEFF Research Database (Denmark)

Lu, Yi; Chen, Xiaoqing; Beesley, Jonathan

2012-01-01

stage 1 GWAS rather than due to problems with the pooling approach. We conclude that there are unlikely to be any moderate or large effects on ovarian cancer risk untagged by less dense arrays. However, our study lacked power to make clear statements on the existence of hitherto untagged small......Recent Genome-Wide Association Studies (GWAS) have identified four low-penetrance ovarian cancer susceptibility loci. We hypothesized that further moderate- or low-penetrance variants exist among the subset of single-nucleotide polymorphisms (SNPs) not well tagged by the genotyping arrays used...... in the previous studies, which would account for some of the remaining risk. We therefore conducted a time- and cost-effective stage 1 GWAS on 342 invasive serous cases and 643 controls genotyped on pooled DNA using the high-density Illumina 1M-Duo array. We followed up 20 of the most significantly associated...

The Genome-Wide DNA Methylation Profile of Peripheral Blood Is Not Systematically Changed by Short-Time Storage at Room Temperature

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Nicklas Heine Staunstrup

2017-12-01

Full Text Available Background: Epigenetic epidemiology has proven an important research discipline in the delineation of diseases of complex etiology. The approach, in such studies, is often to use bio-banked clinical material, however, many such samples were collected for purposes other than epigenetic studies and, thus, potentially not processed and stored appropriately. The Danish National Birth Cohort (DNBC includes more than 100,000 peripheral and umbilical cord blood samples shipped from maternity wards by ordinary mail in EDTA tubes. While this and other similar cohorts hold great promises for DNA methylation studies the potential systematic changes prompted by storage at ambient temperatures have never been assessed on a genome-wide level. Methods and Results: In this study, matched EDTA whole blood samples were stored up to three days at room temperature prior to DNA extraction and methylated DNA immunoprecipitation coupled with deep sequencing (MeDIP-seq. We established that the quality of the MeDIP-seq libraries was high and comparable across samples; and that the methylation profiles did not change systematically during the short-time storage at room temperature. Conclusion: The global DNA methylation profile is stable in whole blood samples stored for up to three days at room temperature in EDTA tubes making genome-wide methylation studies on such material feasible.

A Genome-Wide Methylation Study of Severe Vitamin D Deficiency in African American Adolescents

NARCIS (Netherlands)

Zhu, Haidong; Wang, Xiaoling; Shi, Huidong; Su, Shaoyong; Harshfield, Gregory A.; Gutin, Bernard; Snieder, Harold; Dong, Yanbin

Objectives To test the hypothesis that changes in DNA methylation are involved in vitamin D deficiency-related immune cell regulation using an unbiased genome-wide approach combined with a genomic and epigenomic integrative approach. Study design We performed a genome-wide methylation scan using the

Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain

International Nuclear Information System (INIS)

Maeda, Toyoki; Chijiiwa, Yoshiharu; Tsuji, Hideo; Sakoda, Saburo; Tani, Kenzaburo; Suzuki, Tomokazu

2004-01-01

In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis

Whole genome DNA methylation: beyond genes silencing

OpenAIRE

Tirado-Magallanes, Roberto; Rebbani, Khadija; Lim, Ricky; Pradhan, Sriharsa; Benoukraf, Touati

2016-01-01

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the ...

Pervasive, Genome-Wide Transcription in the Organelle Genomes of Diverse Plastid-Bearing Protists

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Matheus Sanitá Lima

2017-11-01

Full Text Available Organelle genomes are among the most sequenced kinds of chromosome. This is largely because they are small and widely used in molecular studies, but also because next-generation sequencing technologies made sequencing easier, faster, and cheaper. However, studies of organelle RNA have not kept pace with those of DNA, despite huge amounts of freely available eukaryotic RNA-sequencing (RNA-seq data. Little is known about organelle transcription in nonmodel species, and most of the available eukaryotic RNA-seq data have not been mined for organelle transcripts. Here, we use publicly available RNA-seq experiments to investigate organelle transcription in 30 diverse plastid-bearing protists with varying organelle genomic architectures. Mapping RNA-seq data to organelle genomes revealed pervasive, genome-wide transcription, regardless of the taxonomic grouping, gene organization, or noncoding content. For every species analyzed, transcripts covered ≥85% of the mitochondrial and/or plastid genomes (all of which were ≤105 kb, indicating that most of the organelle DNA—coding and noncoding—is transcriptionally active. These results follow earlier studies of model species showing that organellar transcription is coupled and ubiquitous across the genome, requiring significant downstream processing of polycistronic transcripts. Our findings suggest that noncoding organelle DNA can be transcriptionally active, raising questions about the underlying function of these transcripts and underscoring the utility of publicly available RNA-seq data for recovering complete genome sequences. If pervasive transcription is also found in bigger organelle genomes (>105 kb and across a broader range of eukaryotes, this could indicate that noncoding organelle RNAs are regulating fundamental processes within eukaryotic cells.

Genome-wide DNA methylation reprogramming in response to inorganic arsenic links inhibition of CTCF binding, DNMT expression and cellular transformation

Science.gov (United States)

Rea, Matthew; Eckstein, Meredith; Eleazer, Rebekah; Smith, Caroline; Fondufe-Mittendorf, Yvonne N.

2017-02-01

Chronic low dose inorganic arsenic (iAs) exposure leads to changes in gene expression and epithelial-to-mesenchymal transformation. During this transformation, cells adopt a fibroblast-like phenotype accompanied by profound gene expression changes. While many mechanisms have been implicated in this transformation, studies that focus on the role of epigenetic alterations in this process are just emerging. DNA methylation controls gene expression in physiologic and pathologic states. Several studies show alterations in DNA methylation patterns in iAs-mediated pathogenesis, but these studies focused on single genes. We present a comprehensive genome-wide DNA methylation analysis using methyl-sequencing to measure changes between normal and iAs-transformed cells. Additionally, these differential methylation changes correlated positively with changes in gene expression and alternative splicing. Interestingly, most of these differentially methylated genes function in cell adhesion and communication pathways. To gain insight into how genomic DNA methylation patterns are regulated during iAs-mediated carcinogenesis, we show that iAs probably targets CTCF binding at the promoter of DNA methyltransferases, regulating their expression. These findings reveal how CTCF binding regulates DNA methyltransferase to reprogram the methylome in response to an environmental toxin.

From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

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Sebastian Weiterer

Full Text Available Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner.The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.

Characterization and functional inferences of a genome-wide DNA methylation profile in the loin ( muscle of swine

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Woonsu Kim

2018-01-01

Full Text Available Objective DNA methylation plays a major role in regulating the expression of genes related to traits of economic interest (e.g., weight gain in livestock animals. This study characterized and investigated the functional inferences of genome-wide DNA methylome in the loin (longissimus dorsi muscle (LDM of swine. Methods A total of 8.99 Gb methylated DNA immunoprecipitation sequence data were obtained from LDM samples of eight Duroc pigs (four pairs of littermates. The reference pig genome was annotated with 78.5% of the raw reads. A total of 33,506 putative methylated regions (PMR were identified from methylated regions that overlapped at least two samples. Results Of these, only 3.1% were commonly observed in all eight samples. DNA methylation patterns between two littermates were as diverse as between unrelated individuals (p = 0.47, indicating that maternal genetic effects have little influence on the variation in DNA methylation of porcine LDM. The highest density of PMR was observed on chromosome 10. A major proportion (47.7% of PMR was present in the repeat regions, followed by introns (21.5%. The highest conservation of PMR was found in CpG islands (12.1%. These results show an important role for DNA methylation in species- and tissue-specific regulation of gene expression. PMR were also significantly related to muscular cell development, cell-cell communication, cellular integrity and transport, and nutrient metabolism. Conclusion This study indicated the biased distribution and functional role of DNA methylation in gene expression of porcine LDM. DNA methylation was related to cell development, cell-cell communication, cellular integrity and transport, and nutrient metabolism (e.g., insulin signaling pathways. Nutritional and environmental management may have a significant impact on the variation in DNA methylation of porcine LDM.

Dynamic DNA binding, junction recognition and G4 melting activity underlie the telomeric and genome-wide roles of human CST.

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Bhattacharjee, Anukana; Wang, Yongyao; Diao, Jiajie; Price, Carolyn M

2017-12-01

Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that helps resolve replication problems both at telomeres and genome-wide. CST resembles Replication Protein A (RPA) in that the two complexes harbor comparable arrays of OB-folds and have structurally similar small subunits. However, the overall architecture and functions of CST and RPA are distinct. Currently, the mechanism underlying CST action at diverse replication issues remains unclear. To clarify CST mechanism, we examined the capacity of CST to bind and resolve DNA structures found at sites of CST activity. We show that CST binds preferentially to ss-dsDNA junctions, an activity that can explain the incremental nature of telomeric C-strand synthesis following telomerase action. We also show that CST unfolds G-quadruplex structures, thus providing a mechanism for CST to facilitate replication through telomeres and other GC-rich regions. Finally, smFRET analysis indicates that CST binding to ssDNA is dynamic with CST complexes undergoing concentration-dependent self-displacement. These findings support an RPA-based model where dissociation and re-association of individual OB-folds allow CST to mediate loading and unloading of partner proteins to facilitate various aspects of telomere replication and genome-wide resolution of replication stress. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

GST-PRIME: an algorithm for genome-wide primer design.

Science.gov (United States)

Leister, Dario; Varotto, Claudio

2007-01-01

The profiling of mRNA expression based on DNA arrays has become a powerful tool to study genome-wide transcription of genes in a number of organisms. GST-PRIME is a software package created to facilitate large-scale primer design for the amplification of probes to be immobilized on arrays for transcriptome analyses, even though it can be also applied in low-throughput approaches. GST-PRIME allows highly efficient, direct amplification of gene-sequence tags (GSTs) from genomic DNA (gDNA), starting from annotated genome or transcript sequences. GST-PRIME provides a customer-friendly platform for automatic primer design, and despite the relative simplicity of the algorithm, experimental tests in the model plant species Arabidopsis thaliana confirmed the reliability of the software. This chapter describes the algorithm used for primer design, its input and output files, and the installation of the standalone package and its use.

A Genome-Wide mQTL Analysis in Human Adipose Tissue Identifies Genetic Variants Associated with DNA Methylation, Gene Expression and Metabolic Traits

DEFF Research Database (Denmark)

Volkov, Petr; Olsson, Anders H; Gillberg, Linn

2016-01-01

Little is known about the extent to which interactions between genetics and epigenetics may affect the risk of complex metabolic diseases and/or their intermediary phenotypes. We performed a genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human adipose tissue of 119 men, w...... and epigenetic variation in both cis and trans positions influencing gene expression in adipose tissue and in vivo (dys)metabolic traits associated with the development of obesity and diabetes.......Little is known about the extent to which interactions between genetics and epigenetics may affect the risk of complex metabolic diseases and/or their intermediary phenotypes. We performed a genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human adipose tissue of 119 men......, where 592,794 single nucleotide polymorphisms (SNPs) were related to DNA methylation of 477,891 CpG sites, covering 99% of RefSeq genes. SNPs in significant mQTLs were further related to gene expression in adipose tissue and obesity related traits. We found 101,911 SNP-CpG pairs (mQTLs) in cis and 5...

Genome-wide approaches towards identification of susceptibility genes in complex diseases

NARCIS (Netherlands)

Franke, L.H.

2008-01-01

Throughout the human genome millions of places exist where humans differ gentically. The aim of this PhD thesis was to systematically assess this genetic variation and its biological consequences in a genome-wide way, through the utilization of DNA oligonucleotide arrays that assess hundres of

Data analysis in the post-genome-wide association study era

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Qiao-Ling Wang

2016-12-01

Full Text Available Since the first report of a genome-wide association study (GWAS on human age-related macular degeneration, GWAS has successfully been used to discover genetic variants for a variety of complex human diseases and/or traits, and thousands of associated loci have been identified. However, the underlying mechanisms for these loci remain largely unknown. To make these GWAS findings more useful, it is necessary to perform in-depth data mining. The data analysis in the post-GWAS era will include the following aspects: fine-mapping of susceptibility regions to identify susceptibility genes for elucidating the biological mechanism of action; joint analysis of susceptibility genes in different diseases; integration of GWAS, transcriptome, and epigenetic data to analyze expression and methylation quantitative trait loci at the whole-genome level, and find single-nucleotide polymorphisms that influence gene expression and DNA methylation; genome-wide association analysis of disease-related DNA copy number variations. Applying these strategies and methods will serve to strengthen GWAS data to enhance the utility and significance of GWAS in improving understanding of the genetics of complex diseases or traits and translate these findings for clinical applications. Keywords: Genome-wide association study, Data mining, Integrative data analysis, Polymorphism, Copy number variation

Methyl-Analyzer--whole genome DNA methylation profiling.

Science.gov (United States)

Xin, Yurong; Ge, Yongchao; Haghighi, Fatemeh G

2011-08-15

Methyl-Analyzer is a python package that analyzes genome-wide DNA methylation data produced by the Methyl-MAPS (methylation mapping analysis by paired-end sequencing) method. Methyl-MAPS is an enzymatic-based method that uses both methylation-sensitive and -dependent enzymes covering >80% of CpG dinucleotides within mammalian genomes. It combines enzymatic-based approaches with high-throughput next-generation sequencing technology to provide whole genome DNA methylation profiles. Methyl-Analyzer processes and integrates sequencing reads from methylated and unmethylated compartments and estimates CpG methylation probabilities at single base resolution. Methyl-Analyzer is available at http://github.com/epigenomics/methylmaps. Sample dataset is available for download at http://epigenomicspub.columbia.edu/methylanalyzer_data.html. fgh3@columbia.edu Supplementary data are available at Bioinformatics online.

Defining functional DNA elements in the human genome

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Kellis, Manolis; Wold, Barbara; Snyder, Michael P.; Bernstein, Bradley E.; Kundaje, Anshul; Marinov, Georgi K.; Ward, Lucas D.; Birney, Ewan; Crawford, Gregory E.; Dekker, Job; Dunham, Ian; Elnitski, Laura L.; Farnham, Peggy J.; Feingold, Elise A.; Gerstein, Mark; Giddings, Morgan C.; Gilbert, David M.; Gingeras, Thomas R.; Green, Eric D.; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D.; Myers, Richard M.; Pazin, Michael J.; Ren, Bing; Stamatoyannopoulos, John A.; Weng, Zhiping; White, Kevin P.; Hardison, Ross C.

2014-01-01

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease. PMID:24753594

Genome-wide prediction of cis-regulatory regions using supervised deep learning methods.

Science.gov (United States)

Li, Yifeng; Shi, Wenqiang; Wasserman, Wyeth W

2018-05-31

In the human genome, 98% of DNA sequences are non-protein-coding regions that were previously disregarded as junk DNA. In fact, non-coding regions host a variety of cis-regulatory regions which precisely control the expression of genes. Thus, Identifying active cis-regulatory regions in the human genome is critical for understanding gene regulation and assessing the impact of genetic variation on phenotype. The developments of high-throughput sequencing and machine learning technologies make it possible to predict cis-regulatory regions genome wide. Based on rich data resources such as the Encyclopedia of DNA Elements (ENCODE) and the Functional Annotation of the Mammalian Genome (FANTOM) projects, we introduce DECRES based on supervised deep learning approaches for the identification of enhancer and promoter regions in the human genome. Due to their ability to discover patterns in large and complex data, the introduction of deep learning methods enables a significant advance in our knowledge of the genomic locations of cis-regulatory regions. Using models for well-characterized cell lines, we identify key experimental features that contribute to the predictive performance. Applying DECRES, we delineate locations of 300,000 candidate enhancers genome wide (6.8% of the genome, of which 40,000 are supported by bidirectional transcription data), and 26,000 candidate promoters (0.6% of the genome). The predicted annotations of cis-regulatory regions will provide broad utility for genome interpretation from functional genomics to clinical applications. The DECRES model demonstrates potentials of deep learning technologies when combined with high-throughput sequencing data, and inspires the development of other advanced neural network models for further improvement of genome annotations.

Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated

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Down Thomas A

2010-09-01

Full Text Available Abstract Background DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS" but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq not to be biological transcription factor binding sites ("empirical TFBS". We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. Results Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. Conclusions Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.

Genome-wide DNA methylation sequencing reveals miR-663a is a novel epimutation candidate in CIMP-high endometrial cancer.

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Yanokura, Megumi; Banno, Kouji; Adachi, Masataka; Aoki, Daisuke; Abe, Kuniya

2017-06-01

Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three genes, i.e. MLH1, CDH1 (E-cadherin) and APC: CIMP-high (CIMP-H, 2/25, 8.0%), CIMP-low (CIMP-L, 7/25, 28.0%) and CIMP-negative (CIMP(-), 16/25, 64.0%). We then selected two samples each from CIMP-H and CIMP(-) classes, and analyzed DNA methylation status of both normal (peripheral blood cells: PBCs) and cancer tissues by genome-wide, targeted bisulfite sequencing. Genomes of the CIMP-H cancer tissues were significantly hypermethylated compared to those of the CIMP(-). Surprisingly, in normal tissues of the CIMP-H patients, promoter region of the miR-663a locus is hypermethylated relative to CIMP(-) samples. Consistent with this finding, miR-663a expression was lower in the CIMP-H PBCs than in the CIMP(-) PBCs. The same region of the miR663a locus is found to be highly methylated in cancer tissues of both CIMP-H and CIMP(-) cases. This is the first report showing that aberrant DNA methylation of the miR-663a promoter can occur in normal tissue of the cancer patients, suggesting a possible link between this epigenetic abnormality and endometrial cancer. This raises the possibility that the hypermethylation of the miR-663a promoter represents an epimutation associated with the CIMP-H endometrial cancers. Based on these findings, relationship of the aberrant DNA methylation and CIMP-H phenotype is discussed.

Genetically contextual effects of smoking on genome wide DNA methylation.

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Dogan, Meeshanthini V; Beach, Steven R H; Philibert, Robert A

2017-09-01

Smoking is the leading cause of death in the United States. It exerts its effects by increasing susceptibility to a variety of complex disorders among those who smoke, and if pregnant, to their unborn children. In prior efforts to understand the epigenetic mechanisms through which this increased vulnerability is conveyed, a number of investigators have conducted genome wide methylation analyses. Unfortunately, secondary to methodological limitations, these studies were unable to examine methylation in gene regions with significant amounts of genetic variation. Using genome wide genetic and epigenetic data from the Framingham Heart Study, we re-examined the relationship of smoking status to genome wide methylation status. When only methylation status is considered, smoking was significantly associated with differential methylation in 310 genes that map to a variety of biological process and cellular differentiation pathways. However, when SNP effects on the magnitude of smoking associated methylation changes are also considered, cis and trans-interaction effects were noted at a total of 266 and 4353 genes with no marked enrichment for any biological pathways. Furthermore, the SNP variation participating in the significant interaction effects is enriched for loci previously associated with complex medical illnesses. The enlarged scope of the methylome shown to be affected by smoking may better explicate the mediational pathways linking smoking with a myriad of smoking related complex syndromes. Additionally, these results strongly suggest that combined epigenetic and genetic data analyses may be critical for a more complete understanding of the relationship between environmental variables, such as smoking, and pathophysiological outcomes. © 2017 Wiley Periodicals, Inc.

Genome-wide Association Study for Ovarian Cancer Susceptibility using Pooled DNA

Science.gov (United States)

Lu, Yi; Chen, Xiaoqing; Beesley, Jonathan; Johnatty, Sharon E.; deFazio, Anna; Lambrechts, Sandrina; Lambrechts, Diether; Despierre, Evelyn; Vergotes, Ignace; Chang-Claude, Jenny; Hein, Rebecca; Nickels, Stefan; Wang-Gohrke, Shan; Dörk, Thilo; Dürst, Matthias; Antonenkova, Natalia; Bogdanova, Natalia; Goodman, Marc T.; Lurie, Galina; Wilkens, Lynne R.; Carney, Michael E.; Butzow, Ralf; Nevanlinna, Heli; Heikkinen, Tuomas; Leminen, Arto; Kiemeney, Lambertus A.; Massuger, Leon F.A.G.; van Altena, Anne M.; Aben, Katja K.; Kjaer, Susanne Krüger; Høgdall, Estrid; Jensen, Allan; Brooks-Wilson, Angela; Le, Nhu; Cook, Linda; Earp, Madalene; Kelemen, Linda; Easton, Douglas; Pharoah, Paul; Song, Honglin; Tyrer, Jonathan; Ramus, Susan; Menon, Usha; Gentry-Maharaj, Alexandra; Gayther, Simon A.; Bandera, Elisa V.; Olson, Sara H.; Orlow, Irene; Rodriguez-Rodriguez, Lorna

2013-01-01

Recent genome-wide association studies (GWAS) have identified four low-penetrance ovarian cancer susceptibility loci. We hypothesized that further moderate or low penetrance variants exist among the subset of SNPs not well tagged by the genotyping arrays used in the previous studies which would account for some of the remaining risk. We therefore conducted a time- and cost-effective stage 1 GWAS on 342 invasive serous cases and 643 controls genotyped on pooled DNA using the high density Illumina 1M-Duo array. We followed up 20 of the most significantly associated SNPs, which are not well tagged by the lower density arrays used by the published GWAS, and genotyping them on individual DNA. Most of the top 20 SNPs were clearly validated by individually genotyping the samples used in the pools. However, none of the 20 SNPs replicated when tested for association in a much larger stage 2 set of 4,651 cases and 6,966 controls from the Ovarian Cancer Association Consortium. Given that most of the top 20 SNPs from pooling were validated in the same samples by individual genotyping, the lack of replication is likely to be due to the relatively small sample size in our stage 1 GWAS rather than due to problems with the pooling approach. We conclude that there are unlikely to be any moderate or large effects on ovarian cancer risk untagged by the less dense arrays. However our study lacked power to make clear statements on the existence of hitherto untagged small effect variants. PMID:22794196

Estimates of Continental Ancestry Vary Widely among Individuals with the Same mtDNA Haplogroup

Science.gov (United States)

Emery, Leslie S.; Magnaye, Kevin M.; Bigham, Abigail W.; Akey, Joshua M.; Bamshad, Michael J.

2015-01-01

The association between a geographical region and an mtDNA haplogroup(s) has provided the basis for using mtDNA haplogroups to infer an individual’s place of origin and genetic ancestry. Although it is well known that ancestry inferences using mtDNA haplogroups and those using genome-wide markers are frequently discrepant, little empirical information exists on the magnitude and scope of such discrepancies between multiple mtDNA haplogroups and worldwide populations. We compared genetic-ancestry inferences made by mtDNA-haplogroup membership to those made by autosomal SNPs in ∼940 samples of the Human Genome Diversity Panel and recently admixed populations from the 1000 Genomes Project. Continental-ancestry proportions often varied widely among individuals sharing the same mtDNA haplogroup. For only half of mtDNA haplogroups did the highest average continental-ancestry proportion match the highest continental-ancestry proportion of a majority of individuals with that haplogroup. Prediction of an individual’s mtDNA haplogroup from his or her continental-ancestry proportions was often incorrect. Collectively, these results indicate that for most individuals in the worldwide populations sampled, mtDNA-haplogroup membership provides limited information about either continental ancestry or continental region of origin. PMID:25620206

Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes.

Science.gov (United States)

Riechmann, J L; Heard, J; Martin, G; Reuber, L; Jiang, C; Keddie, J; Adam, L; Pineda, O; Ratcliffe, O J; Samaha, R R; Creelman, R; Pilgrim, M; Broun, P; Zhang, J Z; Ghandehari, D; Sherman, B K; Yu, G

2000-12-15

The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants. Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.

Critical threshold levels of DNA methyltransferase 1 are required to maintain DNA methylation across the genome in human cancer cells.

Science.gov (United States)

Cai, Yi; Tsai, Hsing-Chen; Yen, Ray-Whay Chiu; Zhang, Yang W; Kong, Xiangqian; Wang, Wei; Xia, Limin; Baylin, Stephen B

2017-04-01

Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential. © 2017 Cai et al.; Published by Cold Spring Harbor Laboratory Press.

Resource base influences genome-wide DNA methylation levels in wild baboons (Papio cynocephalus)

Science.gov (United States)

Lea, Amanda J.; Altmann, Jeanne; Alberts, Susan C.; Tung, Jenny

2015-01-01

Variation in resource availability commonly exerts strong effects on fitness-related traits in wild animals. However, we know little about the molecular mechanisms that mediate these effects, or about their persistence over time. To address these questions, we profiled genome-wide whole blood DNA methylation levels in two sets of wild baboons: (i) ‘wild-feeding’ baboons that foraged naturally in a savanna environment and (ii) ‘Lodge’ baboons that had ready access to spatially concentrated human food scraps, resulting in high feeding efficiency and low daily travel distances. We identified 1,014 sites (0.20% of sites tested) that were differentially methylated between wild-feeding and Lodge baboons, providing the first evidence that resource availability shapes the epigenome in a wild mammal. Differentially methylated sites tended to occur in contiguous stretches (i.e., in differentially methylated regions or DMRs), in promoters and enhancers, and near metabolism-related genes, supporting their functional importance in gene regulation. In agreement, reporter assay experiments confirmed that methylation at the largest identified DMR, located in the promoter of a key glycolysis-related gene, was sufficient to causally drive changes in gene expression. Intriguingly, all dispersing males carried a consistent epigenetic signature of their membership in a wild-feeding group, regardless of whether males dispersed into or out of this group as adults. Together, our findings support a role for DNA methylation in mediating ecological effects on phenotypic traits in the wild, and emphasize the dynamic environmental sensitivity of DNA methylation levels across the life course. PMID:26508127

A novel rat genomic simple repeat DNA with RNA-homology shows triplex (H-DNA)-like structure and tissue-specific RNA expression

International Nuclear Information System (INIS)

Dey, Indranil; Rath, Pramod C.

2005-01-01

Mammalian genome contains a wide variety of repetitive DNA sequences of relatively unknown function. We report a novel 227 bp simple repeat DNA (3.3 DNA) with a d {(GA) 7 A (AG) 7 } dinucleotide mirror repeat from the rat (Rattus norvegicus) genome. 3.3 DNA showed 75-85% homology with several eukaryotic mRNAs due to (GA/CU) n dinucleotide repeats by nBlast search and a dispersed distribution in the rat genome by Southern blot hybridization with [ 32 P]3.3 DNA. The d {(GA) 7 A (AG) 7 } mirror repeat formed a triplex (H-DNA)-like structure in vitro. Two large RNAs of 9.1 and 7.5 kb were detected by [ 32 P]3.3 DNA in rat brain by Northern blot hybridization indicating expression of such simple sequence repeats at RNA level in vivo. Further, several cDNAs were isolated from a rat cDNA library by [ 32 P]3.3 DNA probe. Three such cDNAs showed tissue-specific RNA expression in rat. pRT 4.1 cDNA showed strong expression of a 2.39 kb RNA in brain and spleen, pRT 5.5 cDNA showed strong expression of a 2.8 kb RNA in brain and a 3.9 kb RNA in lungs, and pRT 11.4 cDNA showed weak expression of a 2.4 kb RNA in lungs. Thus, genomic simple sequence repeats containing d (GA/CT) n dinucleotides are transcriptionally expressed and regulated in rat tissues. Such d (GA/CT) n dinucleotide repeats may form structural elements (e.g., triplex) which may be sites for functional regulation of genomic coding sequences as well as RNAs. This may be a general function of such transcriptionally active simple sequence repeats widely dispersed in mammalian genome

Genome-wide DNA binding pattern of two-component system response regulator RhpR in Pseudomonas syringae

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Tianhong Zhou

2015-06-01

Full Text Available Although Pseudomonas syringae uses the two-component system RhpRS to modulate the expression of type III secretion system (T3SS genes and pathogenicity, the molecular mechanisms and the regulon of RhpRS have yet to be fully demonstrated. We have performed a genome-wide analysis of RhpR binding to DNA prepared from P. syringae pv. phaseolicola in order to identify candidate direct targets of RhpR-mediated transcriptional regulation, as described in our recent article [1]. The data are available from NCBI Gene Expression Omnibus (GEO with the accession number GSE58533. Here we describe the detailed methods and data analyses of our RhpR ChIP-seq dataset.

Genome-wide association study for ovarian cancer susceptibility using pooled DNA.

NARCIS (Netherlands)

Lu, Y.; Chen, X.; Beesley, J.; Johnatty, S.E.; Defazio, A.; Lambrechts, S.; Lambrechts, D.; Despierre, E.; Vergotes, I.; Chang-Claude, J.; Hein, R.; Nickels, S.; Wang-Gohrke, S.; Dork, T.; Durst, M.; Antonenkova, N.; Bogdanova, N.; Goodman, M.T.; Lurie, G.; Wilkens, L.R.; Carney, M.E.; Butzow, R.; Nevanlinna, H.; Heikkinen, T.; Leminen, A.; Kiemeney, L.A.L.M.; Massuger, L.F.A.G.; Altena, A.M. van; Aben, K.K.H.; Kjaer, S.K.; Hogdall, E.; Jensen, A.; Brooks-Wilson, A.; Le, N.; Cook, L.; Earp, M.; Kelemen, L.; Easton, D.; Pharoah, P.; Song, H.; Tyrer, J.; Ramus, S.; Menon, U.; Gentry-Maharaj, A.; Gayther, S.A.; Bandera, E.V.; Olson, S.H.; Orlow, I.; Rodriguez-Rodriguez, L.; MacGregor, S.; Chenevix-Trench, G.

2012-01-01

Recent Genome-Wide Association Studies (GWAS) have identified four low-penetrance ovarian cancer susceptibility loci. We hypothesized that further moderate- or low-penetrance variants exist among the subset of single-nucleotide polymorphisms (SNPs) not well tagged by the genotyping arrays used in

Genome-Wide Analysis of DNA Methylation and Fine Particulate Matter Air Pollution in Three Study Populations: KORA F3, KORA F4, and the Normative Aging Study.

Science.gov (United States)

Panni, Tommaso; Mehta, Amar J; Schwartz, Joel D; Baccarelli, Andrea A; Just, Allan C; Wolf, Kathrin; Wahl, Simone; Cyrys, Josef; Kunze, Sonja; Strauch, Konstantin; Waldenberger, Melanie; Peters, Annette

2016-07-01

Epidemiological studies have reported associations between particulate matter (PM) concentrations and cancer and respiratory and cardiovascular diseases. DNA methylation has been identified as a possible link but so far it has only been analyzed in candidate sites. We studied the association between DNA methylation and short- and mid-term air pollution exposure using genome-wide data and identified potential biological pathways for additional investigation. We collected whole blood samples from three independent studies-KORA F3 (2004-2005) and F4 (2006-2008) in Germany, and the Normative Aging Study (1999-2007) in the United States-and measured genome-wide DNA methylation proportions with the Illumina 450k BeadChip. PM concentration was measured daily at fixed monitoring stations and three different trailing averages were considered and regressed against DNA methylation: 2-day, 7-day and 28-day. Meta-analysis was performed to pool the study-specific results. Random-effect meta-analysis revealed 12 CpG (cytosine-guanine dinucleotide) sites as associated with PM concentration (1 for 2-day average, 1 for 7-day, and 10 for 28-day) at a genome-wide Bonferroni significance level (p ≤ 7.5E-8); 9 out of these 12 sites expressed increased methylation. Through estimation of I2 for homogeneity assessment across the studies, 4 of these sites (annotated in NSMAF, C1orf212, MSGN1, NXN) showed p > 0.05 and I2 F4, and the Normative Aging Study. Environ Health Perspect 124:983-990; http://dx.doi.org/10.1289/ehp.1509966.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

Science.gov (United States)

Oldfield, Lauren M; Grzesik, Peter; Voorhies, Alexander A; Alperovich, Nina; MacMath, Derek; Najera, Claudia D; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N; Montague, Michael G; Friedman, Robert M; Desai, Prashant J; Vashee, Sanjay

2017-10-17

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS YA , replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq

Science.gov (United States)

Gim, Jeong-An; Hong, Chang Pyo; Kim, Dae-Soo; Moon, Jae-Woo; Choi, Yuri; Eo, Jungwoo; Kwon, Yun-Jeong; Lee, Ja-Rang; Jung, Yi-Deun; Bae, Jin-Han; Choi, Bong-Hwan; Ko, Junsu; Song, Sanghoon; Ahn, Kung; Ha, Hong-Seok; Yang, Young Mok; Lee, Hak-Kyo; Park, Kyung-Do; Do, Kyoung-Tag; Han, Kyudong; Yi, Joo Mi; Cha, Hee-Jae; Ayarpadikannan, Selvam; Cho, Byung-Wook; Bhak, Jong; Kim, Heui-Soo

2015-01-01

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits. PMID:25666347

Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

DEFF Research Database (Denmark)

Rodriguez, Jairo; Vives, Laura; Jordà, Mireia

2008-01-01

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family....

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.

Science.gov (United States)

Guillaud-Bataille, Marine; Valent, Alexander; Soularue, Pascal; Perot, Christine; Inda, Maria Mar; Receveur, Aline; Smaïli, Sadek; Roest Crollius, Hugues; Bénard, Jean; Bernheim, Alain; Gidrol, Xavier; Danglot, Gisèle

2004-07-29

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.

Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays.

Directory of Open Access Journals (Sweden)

Christine M Costello

2005-08-01

Full Text Available BACKGROUND: The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD, Crohn disease (CD, and ulcerative colitis (UC are only partially understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression. METHODS AND FINDINGS: High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11, CD patients (n = 10 and UC patients (n = 10. 33P-radiolabeled cDNA from purified poly(A+ RNA extracted from biopsies (unpooled was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome and CDH11 (cadherin 11, type 2. By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology. CONCLUSION: A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches.

Genome-wide identification and comparative analysis of cytosine-5 DNA methyltransferases and demethylase families in wild and cultivated peanut

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Pengfei eWang

2016-02-01

Full Text Available AbstractDNA methylation plays important roles in genome protection, regulation of gene expression and was associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferases and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequence, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases and demethylase in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in known MET, CMT and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 numbers didn’t contain UBA domain which was different from other plants such as Arabidopsis, maize, soybean. Five DNA demethylase were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTases gene mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferases and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or drought stress could influence the expression level of C5-MTases and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut.

DNA pooling base genome-wide association study identifies variants at NRXN3 associated with delayed encephalopathy after acute carbon monoxide poisoning.

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Wenqiang Li

Full Text Available Delayed encephalopathy after acute carbon monoxide poisoning (DEACMP is more characteristic of anoxic encephalopathy than of other types of anoxia. Those who have the same poisoning degree and are of similar age and gender have a greater risk of getting DEACMP. This has made it clear that there are obvious personal differences. Genetic factors may play a very important role. The authors performed a genome-wide association study involving pooling of DNA obtained from 175 patients and 244 matched acute carbon monoxide poisoning without delayed encephalopathy controls. The Illumina HumanHap 660 Chip array was used for DNA pools. Allele frequencies of all SNPs were compared between delayed encephalopathy after acute carbon monoxide poisoning and control groups and ranked. A total of 123 SNPs gave an OR >1.4. Of these, 46 mapped in or close to known genes. Forty-eight SNPs located in 19 genes were associated with DEACMP after correction for 5% FDR in the genome-wide association of pooled DNA. Two SNPs (rs11845632 and rs2196447 locate in the Neurexin 3 gene were selected for individual genotyping in all samples and another cohort consisted of 234 and 271 controls. There were significant differences in the genotype and allele frequencies of rs11845632 and rs2196447 between the DEACMP group and controls group (all P-values <0.05. This study describes a positive association between Neurexin 3 and controls in the Han Chinese population, and provides genetic evidence to support the susceptibility of DEACMP, which may be the resulting interaction of environmental and genetic factors.

DNA Oncogenic Virus-Induced Oxidative Stress, Genomic Damage, and Aberrant Epigenetic Alterations

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Mankgopo Magdeline Kgatle

2017-01-01

Full Text Available Approximately 20% of human cancers is attributable to DNA oncogenic viruses such as human papillomavirus (HPV, hepatitis B virus (HBV, and Epstein-Barr virus (EBV. Unrepaired DNA damage is the most common and overlapping feature of these DNA oncogenic viruses and a source of genomic instability and tumour development. Sustained DNA damage results from unceasing production of reactive oxygen species and activation of inflammasome cascades that trigger genomic changes and increased propensity of epigenetic alterations. Accumulation of epigenetic alterations may interfere with genome-wide cellular signalling machineries and promote malignant transformation leading to cancer development. Untangling and understanding the underlying mechanisms that promote these detrimental effects remain the major objectives for ongoing research and hope for effective virus-induced cancer therapy. Here, we review current literature with an emphasis on how DNA damage influences HPV, HVB, and EBV replication and epigenetic alterations that are associated with carcinogenesis.

Impacts of Genome-Wide Analyses on Our Understanding of Human Herpesvirus Diversity and Evolution.

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Renner, Daniel W; Szpara, Moriah L

2018-01-01

Until fairly recently, genome-wide evolutionary dynamics and within-host diversity were more commonly examined in the context of small viruses than in the context of large double-stranded DNA viruses such as herpesviruses. The high mutation rates and more compact genomes of RNA viruses have inspired the investigation of population dynamics for these species, and recent data now suggest that herpesviruses might also be considered candidates for population modeling. High-throughput sequencing (HTS) and bioinformatics have expanded our understanding of herpesviruses through genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures. Here we discuss recent data on the mechanisms that generate herpesvirus genomic diversity and underlie the evolution of these virus families. We focus on human herpesviruses, with key insights drawn from veterinary herpesviruses and other large DNA virus families. We consider the impacts of cell culture on herpesvirus genomes and how to accurately describe the viral populations under study. The need for a strong foundation of high-quality genomes is also discussed, since it underlies all secondary genomic analyses such as RNA sequencing (RNA-Seq), chromatin immunoprecipitation, and ribosome profiling. Areas where we foresee future progress, such as the linking of viral genetic differences to phenotypic or clinical outcomes, are highlighted as well. Copyright © 2017 Renner and Szpara.

Impacts of Genome-Wide Analyses on Our Understanding of Human Herpesvirus Diversity and Evolution

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Renner, Daniel W.

2017-01-01

ABSTRACT Until fairly recently, genome-wide evolutionary dynamics and within-host diversity were more commonly examined in the context of small viruses than in the context of large double-stranded DNA viruses such as herpesviruses. The high mutation rates and more compact genomes of RNA viruses have inspired the investigation of population dynamics for these species, and recent data now suggest that herpesviruses might also be considered candidates for population modeling. High-throughput sequencing (HTS) and bioinformatics have expanded our understanding of herpesviruses through genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures. Here we discuss recent data on the mechanisms that generate herpesvirus genomic diversity and underlie the evolution of these virus families. We focus on human herpesviruses, with key insights drawn from veterinary herpesviruses and other large DNA virus families. We consider the impacts of cell culture on herpesvirus genomes and how to accurately describe the viral populations under study. The need for a strong foundation of high-quality genomes is also discussed, since it underlies all secondary genomic analyses such as RNA sequencing (RNA-Seq), chromatin immunoprecipitation, and ribosome profiling. Areas where we foresee future progress, such as the linking of viral genetic differences to phenotypic or clinical outcomes, are highlighted as well. PMID:29046445

Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

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Wang, Chao; Lv, Yangyong; Wang, Bin; Yin, Chao; Lin, Ying; Pan, Li

2015-01-01

The genome-scale delineation of in vivo protein–DNA interactions is key to understanding genome function. Only ∼5% of transcription factors (TFs) in the Aspergillus genus have been identified using traditional methods. Although the Aspergillus oryzae genome contains >600 TFs, knowledge of the in vivo genome-wide TF-binding sites (TFBSs) in aspergilli remains limited because of the lack of high-quality antibodies. We investigated the landscape of in vivo protein–DNA interactions across the A. oryzae genome through coupling the DNase I digestion of intact nuclei with massively parallel sequencing and the analysis of cleavage patterns in protein–DNA interactions at single-nucleotide resolution. The resulting map identified overrepresented de novo TF-binding motifs from genomic footprints, and provided the detailed chromatin remodeling patterns and the distribution of digital footprints near transcription start sites. The TFBSs of 19 known Aspergillus TFs were also identified based on DNase I digestion data surrounding potential binding sites in conjunction with TF binding specificity information. We observed that the cleavage patterns of TFBSs were dependent on the orientation of TF motifs and independent of strand orientation, consistent with the DNA shape features of binding motifs with flanking sequences. PMID:25883143

Genome-wide identification of direct HBx genomic targets

KAUST Repository

Guerrieri, Francesca

2017-02-17

Background The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV. Results ChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication. Conclusions Our ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

Genomic and Molecular Landscape of DNA Damage Repair Deficiency across The Cancer Genome Atlas

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Theo A. Knijnenburg

2018-04-01

Full Text Available Summary: DNA damage repair (DDR pathways modulate cancer risk, progression, and therapeutic response. We systematically analyzed somatic alterations to provide a comprehensive view of DDR deficiency across 33 cancer types. Mutations with accompanying loss of heterozygosity were observed in over 1/3 of DDR genes, including TP53 and BRCA1/2. Other prevalent alterations included epigenetic silencing of the direct repair genes EXO5, MGMT, and ALKBH3 in ∼20% of samples. Homologous recombination deficiency (HRD was present at varying frequency in many cancer types, most notably ovarian cancer. However, in contrast to ovarian cancer, HRD was associated with worse outcomes in several other cancers. Protein structure-based analyses allowed us to predict functional consequences of rare, recurrent DDR mutations. A new machine-learning-based classifier developed from gene expression data allowed us to identify alterations that phenocopy deleterious TP53 mutations. These frequent DDR gene alterations in many human cancers have functional consequences that may determine cancer progression and guide therapy. : Knijnenburg et al. present The Cancer Genome Atlas (TCGA Pan-Cancer analysis of DNA damage repair (DDR deficiency in cancer. They use integrative genomic and molecular analyses to identify frequent DDR alterations across 33 cancer types, correlate gene- and pathway-level alterations with genome-wide measures of genome instability and impaired function, and demonstrate the prognostic utility of DDR deficiency scores. Keywords: The Cancer Genome Atlas PanCanAtlas project, DNA damage repair, somatic mutations, somatic copy-number alterations, epigenetic silencing, DNA damage footprints, mutational signatures, integrative statistical analysis, protein structure analysis

Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines.

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Noor, Dzul Azri Mohamed; Jeyapalan, Jennie N; Alhazmi, Safiah; Carr, Matthew; Squibb, Benjamin; Wallace, Claire; Tan, Christopher; Cusack, Martin; Hughes, Jaime; Reader, Tom; Shipley, Janet; Sheer, Denise; Scotting, Paul J

2016-01-01

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome-wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours' biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription-quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes.

Genome-Wide DNA Methylation Analysis and Epigenetic Variations Associated with Congenital Aortic Valve Stenosis (AVS.

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Uppala Radhakrishna

Full Text Available Congenital heart defect (CHD is the most common cause of death from congenital anomaly. Among several candidate epigenetic mechanisms, DNA methylation may play an important role in the etiology of CHDs. We conducted a genome-wide DNA methylation analysis using an Illumina Infinium 450k human methylation assay in a cohort of 24 newborns who had aortic valve stenosis (AVS, with gestational-age matched controls. The study identified significantly-altered CpG methylation at 59 sites in 52 genes in AVS subjects as compared to controls (either hypermethylated or demethylated. Gene Ontology analysis identified biological processes and functions for these genes including positive regulation of receptor-mediated endocytosis. Consistent with prior clinical data, the molecular function categories as determined using DAVID identified low-density lipoprotein receptor binding, lipoprotein receptor binding and identical protein binding to be over-represented in the AVS group. A significant epigenetic change in the APOA5 and PCSK9 genes known to be involved in AVS was also observed. A large number CpG methylation sites individually demonstrated good to excellent diagnostic accuracy for the prediction of AVS status, thus raising possibility of molecular screening markers for this disorder. Using epigenetic analysis we were able to identify genes significantly involved in the pathogenesis of AVS.

Genome-Wide DNA Methylation Analysis and Epigenetic Variations Associated with Congenital Aortic Valve Stenosis (AVS).

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Radhakrishna, Uppala; Albayrak, Samet; Alpay-Savasan, Zeynep; Zeb, Amna; Turkoglu, Onur; Sobolewski, Paul; Bahado-Singh, Ray O

2016-01-01

Congenital heart defect (CHD) is the most common cause of death from congenital anomaly. Among several candidate epigenetic mechanisms, DNA methylation may play an important role in the etiology of CHDs. We conducted a genome-wide DNA methylation analysis using an Illumina Infinium 450k human methylation assay in a cohort of 24 newborns who had aortic valve stenosis (AVS), with gestational-age matched controls. The study identified significantly-altered CpG methylation at 59 sites in 52 genes in AVS subjects as compared to controls (either hypermethylated or demethylated). Gene Ontology analysis identified biological processes and functions for these genes including positive regulation of receptor-mediated endocytosis. Consistent with prior clinical data, the molecular function categories as determined using DAVID identified low-density lipoprotein receptor binding, lipoprotein receptor binding and identical protein binding to be over-represented in the AVS group. A significant epigenetic change in the APOA5 and PCSK9 genes known to be involved in AVS was also observed. A large number CpG methylation sites individually demonstrated good to excellent diagnostic accuracy for the prediction of AVS status, thus raising possibility of molecular screening markers for this disorder. Using epigenetic analysis we were able to identify genes significantly involved in the pathogenesis of AVS.

SMRT Sequencing Revealed Mitogenome Characteristics and Mitogenome-Wide DNA Modification Pattern in Ophiocordyceps sinensis.

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Kang, Xincong; Hu, Liqin; Shen, Pengyuan; Li, Rui; Liu, Dongbo

2017-01-01

Single molecule, real-time (SMRT) sequencing was used to characterize mitochondrial (mt) genome of Ophiocordyceps sinensis and to analyze the mt genome-wide pattern of epigenetic DNA modification. The complete mt genome of O. sinensis , with a size of 157,539 bp, is the fourth largest Ascomycota mt genome sequenced to date. It contained 14 conserved protein-coding genes (PCGs), 1 intronic protein rps3 , 27 tRNAs and 2 rRNA subunits, which are common characteristics of the known mt genomes in Hypocreales. A phylogenetic tree inferred from 14 PCGs in Pezizomycotina fungi supports O. sinensis as most closely related to Hirsutella rhossiliensis in Ophiocordycipitaceae. A total of 36 sequence sites in rps3 were under positive selection, with dN/dS >1 in the 20 compared fungi. Among them, 16 sites were statistically significant. In addition, the mt genome-wide base modification pattern of O. sinensis was determined in this study, especially DNA methylation. The methylations were located in coding and uncoding regions of mt PCGs in O. sinensis , and might be closely related to the expression of PCGs or the binding affinity of transcription factor A to mtDNA. Consequently, these methylations may affect the enzymatic activity of oxidative phosphorylation and then the mt respiratory rate; or they may influence mt biogenesis. Therefore, methylations in the mitogenome of O. sinensis might be a genetic feature to adapt to the cold and low PO 2 environment at high altitude, where O. sinensis is endemic. This is the first report on epigenetic modifications in a fungal mt genome.

Imputation and quality control steps for combining multiple genome-wide datasets

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Shefali S Verma

2014-12-01

Full Text Available The electronic MEdical Records and GEnomics (eMERGE network brings together DNA biobanks linked to electronic health records (EHRs from multiple institutions. Approximately 52,000 DNA samples from distinct individuals have been genotyped using genome-wide SNP arrays across the nine sites of the network. The eMERGE Coordinating Center and the Genomics Workgroup developed a pipeline to impute and merge genomic data across the different SNP arrays to maximize sample size and power to detect associations with a variety of clinical endpoints. The 1000 Genomes cosmopolitan reference panel was used for imputation. Imputation results were evaluated using the following metrics: accuracy of imputation, allelic R2 (estimated correlation between the imputed and true genotypes, and the relationship between allelic R2 and minor allele frequency. Computation time and memory resources required by two different software packages (BEAGLE and IMPUTE2 were also evaluated. A number of challenges were encountered due to the complexity of using two different imputation software packages, multiple ancestral populations, and many different genotyping platforms. We present lessons learned and describe the pipeline implemented here to impute and merge genomic data sets. The eMERGE imputed dataset will serve as a valuable resource for discovery, leveraging the clinical data that can be mined from the EHR.

Nanobody®-based chromatin immunoprecipitation/micro-array analysis for genome-wide identification of transcription factor DNA binding sites

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Nguyen-Duc, Trong; Peeters, Eveline; Muyldermans, Serge; Charlier, Daniel; Hassanzadeh-Ghassabeh, Gholamreza

2013-01-01

Nanobodies® are single-domain antibody fragments derived from camelid heavy-chain antibodies. Because of their small size, straightforward production in Escherichia coli, easy tailoring, high affinity, specificity, stability and solubility, nanobodies® have been exploited in various biotechnological applications. A major challenge in the post-genomics and post-proteomics era is the identification of regulatory networks involving nucleic acid–protein and protein–protein interactions. Here, we apply a nanobody® in chromatin immunoprecipitation followed by DNA microarray hybridization (ChIP-chip) for genome-wide identification of DNA–protein interactions. The Lrp-like regulator Ss-LrpB, arguably one of the best-studied specific transcription factors of the hyperthermophilic archaeon Sulfolobus solfataricus, was chosen for this proof-of-principle nanobody®-assisted ChIP. Three distinct Ss-LrpB-specific nanobodies®, each interacting with a different epitope, were generated for ChIP. Genome-wide ChIP-chip with one of these nanobodies® identified the well-established Ss-LrpB binding sites and revealed several unknown target sequences. Furthermore, these ChIP-chip profiles revealed auxiliary operator sites in the open reading frame of Ss-lrpB. Our work introduces nanobodies® as a novel class of affinity reagents for ChIP. Taking into account the unique characteristics of nanobodies®, in particular, their short generation time, nanobody®-based ChIP is expected to further streamline ChIP-chip and ChIP-Seq experiments, especially in organisms with no (or limited) possibility of genetic manipulation. PMID:23275538

Quantification of trace-level DNA by real-time whole genome amplification.

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Kang, Min-Jung; Yu, Hannah; Kim, Sook-Kyung; Park, Sang-Ryoul; Yang, Inchul

2011-01-01

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.

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

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Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.

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Huang, Yun; Pastor, William A; Zepeda-Martínez, Jorge A; Rao, Anjana

2012-10-01

5-Hydroxymethylcytosine (5hmC) is a recently discovered base in the mammalian genome, produced upon oxidation of 5-methylcytosine (5mC) in a process catalyzed by TET proteins. The biological functions of 5hmC and further oxidation products of 5mC are under intense investigation, as they are likely intermediates in DNA demethylation pathways. Here we describe a novel protocol to profile 5hmC at a genome-wide scale. This approach is based on sodium bisulfite-mediated conversion of 5hmC to cytosine-5-methylenesulfonate (CMS); CMS-containing DNA fragments are then immunoprecipitated using a CMS-specific antiserum. The anti-CMS technique is highly specific with a low background, and is much less dependent on 5hmC density than anti-5hmC immunoprecipitation (IP). Moreover, it does not enrich for CA and CT repeats, as noted for 5hmC DNA IP using antibodies to 5hmC. The anti-CMS protocol takes 3 d to complete.

A method to evaluate genome-wide methylation in archival formalin-fixed, paraffin-embedded ovarian epithelial cells.

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Qiling Li

Full Text Available The use of DNA from archival formalin and paraffin embedded (FFPE tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue.Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E.Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample.We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples.

Genome-wide ancestry of 17th-century enslaved Africans from the Caribbean

DEFF Research Database (Denmark)

Schroeder, Hannes; Avila-Arcos, Maria C.; Malaspinas, Anna-Sapfo

2015-01-01

Between 1500 and 1850, more than 12 million enslaved Africans were transported to the New World. The vast majority were shipped from West and West-Central Africa, but their precise origins are largely unknown. We used genome-wide ancient DNA analyses to investigate the genetic origins of three en...

Quality assessment of buccal versus blood genomic DNA using the Affymetrix 500 K GeneChip

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Martin Lisa J

2007-11-01

Full Text Available Abstract Background With the advent of genome-wide genotyping, the utility of stored buccal brushes for DNA extraction and genotyping has been questioned. We sought to describe the genomic DNA yield and concordance between stored buccal brushes and blood samples from the same individuals in the context of Affymetrix 500 K Human GeneChip genotyping. Results Buccal cytobrushes stored for ~7 years at -80°C prior to extraction yielded sufficient double stranded DNA (dsDNA to be successfully genotyped on the Affymetrix ~262 K NspI chip, with yields between 536 and 1047 ng dsDNA. Using the BRLMM algorithm, genotyping call rates for blood samples averaged 98.4%, and for buccal samples averaged 97.8%. Matched blood samples exhibited 99.2% concordance, while matched blood and buccal samples exhibited 98.8% concordance. Conclusion Buccal cytobrushes stored long-term result in sufficient dsDNA concentrations to achieve high genotyping call rates and concordance with stored blood samples in the context of Affymetrix 500 K SNP genotyping. Thus, given high-quality collection and storage protocols, it is possible to use stored buccal cytobrush samples for genome-wide association studies.

Genome-Wide Analysis of DNA Methylation During Ovule Development of Female-Sterile Rice fsv1

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Helian Liu

2017-11-01

Full Text Available The regulation of female fertility is an important field of rice sexual reproduction research. DNA methylation is an essential epigenetic modification that dynamically regulates gene expression during development processes. However, few reports have described the methylation profiles of female-sterile rice during ovule development. In this study, ovules were continuously acquired from the beginning of megaspore mother cell meiosis until the mature female gametophyte formation period, and global DNA methylation patterns were compared in the ovules of a high-frequency female-sterile line (fsv1 and a wild-type rice line (Gui99 using whole-genome bisulfite sequencing (WGBS. Profiling of the global DNA methylation revealed hypo-methylation, and 3471 significantly differentially methylated regions (DMRs were observed in fsv1 ovules compared with Gui99. Based on functional annotation and Kyoto encyclopedia of genes and genomes (KEGG pathway analysis of differentially methylated genes (DMGs, we observed more DMGs enriched in cellular component, reproduction regulation, metabolic pathway, and other pathways. In particular, many ovule development genes and plant hormone-related genes showed significantly different methylation patterns in the two rice lines, and these differences may provide important clues for revealing the mechanism of female gametophyte abortion.

Genome-wide characterization of centromeric satellites from multiple mammalian genomes.

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Alkan, Can; Cardone, Maria Francesca; Catacchio, Claudia Rita; Antonacci, Francesca; O'Brien, Stephen J; Ryder, Oliver A; Purgato, Stefania; Zoli, Monica; Della Valle, Giuliano; Eichler, Evan E; Ventura, Mario

2011-01-01

Despite its importance in cell biology and evolution, the centromere has remained the final frontier in genome assembly and annotation due to its complex repeat structure. However, isolation and characterization of the centromeric repeats from newly sequenced species are necessary for a complete understanding of genome evolution and function. In recent years, various genomes have been sequenced, but the characterization of the corresponding centromeric DNA has lagged behind. Here, we present a computational method (RepeatNet) to systematically identify higher-order repeat structures from unassembled whole-genome shotgun sequence and test whether these sequence elements correspond to functional centromeric sequences. We analyzed genome datasets from six species of mammals representing the diversity of the mammalian lineage, namely, horse, dog, elephant, armadillo, opossum, and platypus. We define candidate monomer satellite repeats and demonstrate centromeric localization for five of the six genomes. Our analysis revealed the greatest diversity of centromeric sequences in horse and dog in contrast to elephant and armadillo, which showed high-centromeric sequence homogeneity. We could not isolate centromeric sequences within the platypus genome, suggesting that centromeres in platypus are not enriched in satellite DNA. Our method can be applied to the characterization of thousands of other vertebrate genomes anticipated for sequencing in the near future, providing an important tool for annotation of centromeres.

Genome-wide methylation study of diploid and triploid brown trout (Salmo trutta L.).

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Covelo-Soto, L; Leunda, P M; Pérez-Figueroa, A; Morán, P

2015-06-01

The induction of triploidization in fish is a very common practice in aquaculture. Although triploidization has been applied successfully in many salmonid species, little is known about the epigenetic mechanisms implicated in the maintenance of the normal functions of the new polyploid genome. By means of methylation-sensitive amplified polymorphism (MSAP) techniques, genome-wide methylation changes associated with triploidization were assessed in DNA samples obtained from diploid and triploid siblings of brown trout (Salmo trutta). Simple comparative body measurements showed that the triploid trout used in the study were statistically bigger, however, not heavier than their diploid counterparts. The statistical analysis of the MSAP data showed no significant differences between diploid and triploid brown trout in respect to brain, gill, heart, liver, kidney or muscle samples. Nonetheless, local analysis pointed to the possibility of differences in connection with concrete loci. This is the first study that has investigated DNA methylation alterations associated with triploidization in brown trout. Our results set the basis for new studies to be undertaken and provide a new approach concerning triploidization effects of the salmonid genome while also contributing to the better understanding of the genome-wide methylation processes. © 2015 Stichting International Foundation for Animal Genetics.

Meta-analysis of 32 genome-wide linkage studies of schizophrenia

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Ng, MYM; Levinson, DF; Faraone, SV; Suarez, BK; DeLisi, LE; Arinami, T; Riley, B; Paunio, T; Pulver, AE; Irmansyah; Holmans, PA; Escamilla, M; Wildenauer, DB; Williams, NM; Laurent, C; Mowry, BJ; Brzustowicz, LM; Maziade, M; Sklar, P; Garver, DL; Abecasis, GR; Lerer, B; Fallin, MD; Gurling, HMD; Gejman, PV; Lindholm, E; Moises, HW; Byerley, W; Wijsman, EM; Forabosco, P; Tsuang, MT; Hwu, H-G; Okazaki, Y; Kendler, KS; Wormley, B; Fanous, A; Walsh, D; O’Neill, FA; Peltonen, L; Nestadt, G; Lasseter, VK; Liang, KY; Papadimitriou, GM; Dikeos, DG; Schwab, SG; Owen, MJ; O’Donovan, MC; Norton, N; Hare, E; Raventos, H; Nicolini, H; Albus, M; Maier, W; Nimgaonkar, VL; Terenius, L; Mallet, J; Jay, M; Godard, S; Nertney, D; Alexander, M; Crowe, RR; Silverman, JM; Bassett, AS; Roy, M-A; Mérette, C; Pato, CN; Pato, MT; Roos, J Louw; Kohn, Y; Amann-Zalcenstein, D; Kalsi, G; McQuillin, A; Curtis, D; Brynjolfson, J; Sigmundsson, T; Petursson, H; Sanders, AR; Duan, J; Jazin, E; Myles-Worsley, M; Karayiorgou, M; Lewis, CM

2009-01-01

A genome scan meta-analysis (GSMA) was carried out on 32 independent genome-wide linkage scan analyses that included 3255 pedigrees with 7413 genotyped cases affected with schizophrenia (SCZ) or related disorders. The primary GSMA divided the autosomes into 120 bins, rank-ordered the bins within each study according to the most positive linkage result in each bin, summed these ranks (weighted for study size) for each bin across studies and determined the empirical probability of a given summed rank (PSR) by simulation. Suggestive evidence for linkage was observed in two single bins, on chromosomes 5q (142-168 Mb) and 2q (103-134 Mb). Genome-wide evidence for linkage was detected on chromosome 2q (119-152 Mb) when bin boundaries were shifted to the middle of the previous bins. The primary analysis met empirical criteria for ‘aggregate’ genome-wide significance, indicating that some or all of 10 bins are likely to contain loci linked to SCZ, including regions of chromosomes 1, 2q, 3q, 4q, 5q, 8p and 10q. In a secondary analysis of 22 studies of European-ancestry samples, suggestive evidence for linkage was observed on chromosome 8p (16-33 Mb). Although the newer genome-wide association methodology has greater power to detect weak associations to single common DNA sequence variants, linkage analysis can detect diverse genetic effects that segregate in families, including multiple rare variants within one locus or several weakly associated loci in the same region. Therefore, the regions supported by this meta-analysis deserve close attention in future studies. PMID:19349958

The DNA-encoded nucleosome organization of a eukaryotic genome.

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Kaplan, Noam; Moore, Irene K; Fondufe-Mittendorf, Yvonne; Gossett, Andrea J; Tillo, Desiree; Field, Yair; LeProust, Emily M; Hughes, Timothy R; Lieb, Jason D; Widom, Jonathan; Segal, Eran

2009-03-19

Nucleosome organization is critical for gene regulation. In living cells this organization is determined by multiple factors, including the action of chromatin remodellers, competition with site-specific DNA-binding proteins, and the DNA sequence preferences of the nucleosomes themselves. However, it has been difficult to estimate the relative importance of each of these mechanisms in vivo, because in vivo nucleosome maps reflect the combined action of all influencing factors. Here we determine the importance of nucleosome DNA sequence preferences experimentally by measuring the genome-wide occupancy of nucleosomes assembled on purified yeast genomic DNA. The resulting map, in which nucleosome occupancy is governed only by the intrinsic sequence preferences of nucleosomes, is similar to in vivo nucleosome maps generated in three different growth conditions. In vitro, nucleosome depletion is evident at many transcription factor binding sites and around gene start and end sites, indicating that nucleosome depletion at these sites in vivo is partly encoded in the genome. We confirm these results with a micrococcal nuclease-independent experiment that measures the relative affinity of nucleosomes for approximately 40,000 double-stranded 150-base-pair oligonucleotides. Using our in vitro data, we devise a computational model of nucleosome sequence preferences that is significantly correlated with in vivo nucleosome occupancy in Caenorhabditis elegans. Our results indicate that the intrinsic DNA sequence preferences of nucleosomes have a central role in determining the organization of nucleosomes in vivo.

Rapid Genome-wide Single Nucleotide Polymorphism Discovery in Soybean and Rice via Deep Resequencing of Reduced Representation Libraries with the Illumina Genome Analyzer

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Stéphane Deschamps

2010-07-01

Full Text Available Massively parallel sequencing platforms have allowed for the rapid discovery of single nucleotide polymorphisms (SNPs among related genotypes within a species. We describe the creation of reduced representation libraries (RRLs using an initial digestion of nuclear genomic DNA with a methylation-sensitive restriction endonuclease followed by a secondary digestion with the 4bp-restriction endonuclease This strategy allows for the enrichment of hypomethylated genomic DNA, which has been shown to be rich in genic sequences, and the digestion with serves to increase the number of common loci resequenced between individuals. Deep resequencing of these RRLs performed with the Illumina Genome Analyzer led to the identification of 2618 SNPs in rice and 1682 SNPs in soybean for two representative genotypes in each of the species. A subset of these SNPs was validated via Sanger sequencing, exhibiting validation rates of 96.4 and 97.0%, in rice ( and soybean (, respectively. Comparative analysis of the read distribution relative to annotated genes in the reference genome assemblies indicated that the RRL strategy was primarily sampling within genic regions for both species. The massively parallel sequencing of methylation-sensitive RRLs for genome-wide SNP discovery can be applied across a wide range of plant species having sufficient reference genomic sequence.

Impact of age, BMI and HbA1c levels on the genome-wide DNA methylation and mRNA expression patterns in human adipose tissue and identification of epigenetic biomarkers in blood

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Rönn, Tina; Volkov, Petr; Gillberg, Linn

2015-01-01

Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed the DNA methylation of ∼480 000 sites in human adipose tissue from 96 ma...

A genome-wide study of DNA methylation patterns and gene expression levels in multiple human and chimpanzee tissues.

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Athma A Pai

2011-02-01

Full Text Available The modification of DNA by methylation is an important epigenetic mechanism that affects the spatial and temporal regulation of gene expression. Methylation patterns have been described in many contexts within and across a range of species. However, the extent to which changes in methylation might underlie inter-species differences in gene regulation, in particular between humans and other primates, has not yet been studied. To this end, we studied DNA methylation patterns in livers, hearts, and kidneys from multiple humans and chimpanzees, using tissue samples for which genome-wide gene expression data were also available. Using the multi-species gene expression and methylation data for 7,723 genes, we were able to study the role of promoter DNA methylation in the evolution of gene regulation across tissues and species. We found that inter-tissue methylation patterns are often conserved between humans and chimpanzees. However, we also found a large number of gene expression differences between species that might be explained, at least in part, by corresponding differences in methylation levels. In particular, we estimate that, in the tissues we studied, inter-species differences in promoter methylation might underlie as much as 12%-18% of differences in gene expression levels between humans and chimpanzees.

Meristem micropropagation of cassava (Manihot esculenta) evokes genome-wide changes in DNA methylation.

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Kitimu, Shedrack R; Taylor, Julian; March, Timothy J; Tairo, Fred; Wilkinson, Mike J; Rodríguez López, Carlos M

2015-01-01

There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta) cultivars. Principal Co-ordinate Analysis of profiles generated by methylation-sensitive amplified polymorphism revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype by sequencing identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologs have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops.

Genome-wide DNA methylation profiling in infants born to gestational diabetes mellitus.

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Weng, Xiaoling; Liu, Fatao; Zhang, Hong; Kan, Mengyuan; Wang, Ting; Dong, Mingyue; Liu, Yun

2018-03-26

Offspring exposed to gestational diabetes mellitus (GDM) are at a high risk for metabolic diseases. The mechanisms behind the association between offspring exposed to GDM in utero and an increased risk of health consequences later in life remain unclear. The aim of this study was to clarify the changes in methylation levels in the foetuses of women with GDM and to explore the possible mechanisms linking maternal GDM with a high risk of metabolic diseases in offspring later in life. A genome-wide comparative methylome analysis on the umbilical cord blood of infants born to 30 women with GDM and 33 women with normal pregnancy was performed using Infinium HumanMethylation 450 BeadChip assays. A quantitative methylation analysis of 18 CpG dinucleotides was verified in the validation umbilical cord blood samples from 102 newborns exposed to GDM and 103 newborns who experienced normal pregnancy by MassARRAY EpiTYPER. A total of 4485 differentially methylated sites (DMSs), including 2150 hypermethylated sites and 2335 hypomethylated sites, with a mean β-value difference of >0.05, were identified by the 450k array. Good agreement was observed between the massarray validation data and the 450k array data (R 2 > 0.99; P 0.15 between the GDM and healthy groups were identified and showed potential as clinical biomarkers for GDM. "hsa04940: Type I diabetes mellitus" was the most significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, with a P-value = 3.20E-07 and 1.36E-02 in the hypermethylated and hypomethylated genepathway enrichment analyses, respectively. In the Gene Ontology (GO) pathway analyses, immune MHC-related pathways and neuron development-related pathways were significantly enriched. Our results suggest that GDM has epigenetic effects on genes that are preferentially involved in the Type I diabetes mellitus pathway, immune MHC (major histocompatibility complex)-related pathways and neuron development-related pathways, with consequences on fetal growth

Ribosomal DNA sequence heterogeneity reflects intraspecies phylogenies and predicts genome structure in two contrasting yeast species.

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West, Claire; James, Stephen A; Davey, Robert P; Dicks, Jo; Roberts, Ian N

2014-07-01

The ribosomal RNA encapsulates a wealth of evolutionary information, including genetic variation that can be used to discriminate between organisms at a wide range of taxonomic levels. For example, the prokaryotic 16S rDNA sequence is very widely used both in phylogenetic studies and as a marker in metagenomic surveys and the internal transcribed spacer region, frequently used in plant phylogenetics, is now recognized as a fungal DNA barcode. However, this widespread use does not escape criticism, principally due to issues such as difficulties in classification of paralogous versus orthologous rDNA units and intragenomic variation, both of which may be significant barriers to accurate phylogenetic inference. We recently analyzed data sets from the Saccharomyces Genome Resequencing Project, characterizing rDNA sequence variation within multiple strains of the baker's yeast Saccharomyces cerevisiae and its nearest wild relative Saccharomyces paradoxus in unprecedented detail. Notably, both species possess single locus rDNA systems. Here, we use these new variation datasets to assess whether a more detailed characterization of the rDNA locus can alleviate the second of these phylogenetic issues, sequence heterogeneity, while controlling for the first. We demonstrate that a strong phylogenetic signal exists within both datasets and illustrate how they can be used, with existing methodology, to estimate intraspecies phylogenies of yeast strains consistent with those derived from whole-genome approaches. We also describe the use of partial Single Nucleotide Polymorphisms, a type of sequence variation found only in repetitive genomic regions, in identifying key evolutionary features such as genome hybridization events and show their consistency with whole-genome Structure analyses. We conclude that our approach can transform rDNA sequence heterogeneity from a problem to a useful source of evolutionary information, enabling the estimation of highly accurate phylogenies of

Genome instabilities arising from ribonucleotides in DNA.

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Klein, Hannah L

2017-08-01

Genomic DNA is transiently contaminated with ribonucleotide residues during the process of DNA replication through misincorporation by the replicative DNA polymerases α, δ and ε, and by the normal replication process on the lagging strand, which uses RNA primers. These ribonucleotides are efficiently removed during replication by RNase H enzymes and the lagging strand synthesis machinery. However, when ribonucleotides remain in DNA they can distort the DNA helix, affect machineries for DNA replication, transcription and repair, and can stimulate genomic instabilities which are manifest as increased mutation, recombination and chromosome alterations. The genomic instabilities associated with embedded ribonucleotides are considered here, along with a discussion of the origin of the lesions that stimulate particular classes of instabilities. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome-wide methylation analysis identified sexually dimorphic methylated regions in hybrid tilapia

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Wan, Zi Yi; Xia, Jun Hong; Lin, Grace; Wang, Le; Lin, Valerie C. L.; Yue, Gen Hua

2016-01-01

Sexual dimorphism is an interesting biological phenomenon. Previous studies showed that DNA methylation might play a role in sexual dimorphism. However, the overall picture of the genome-wide methylation landscape in sexually dimorphic species remains unclear. We analyzed the DNA methylation landscape and transcriptome in hybrid tilapia (Oreochromis spp.) using whole genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). We found 4,757 sexually dimorphic differentially methylated regions (DMRs), with significant clusters of DMRs located on chromosomal regions associated with sex determination. CpG methylation in promoter regions was negatively correlated with the gene expression level. MAPK/ERK pathway was upregulated in male tilapia. We also inferred active cis-regulatory regions (ACRs) in skeletal muscle tissues from WGBS datasets, revealing sexually dimorphic cis-regulatory regions. These results suggest that DNA methylation contribute to sex-specific phenotypes and serve as resources for further investigation to analyze the functions of these regions and their contributions towards sexual dimorphisms. PMID:27782217

Genome-Wide Association Study of Antiphospholipid Antibodies

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M. Ilyas Kamboh

2013-01-01

Full Text Available Background. The persistent presence of antiphospholipid antibodies (APA may lead to the development of primary or secondary antiphospholipid syndrome. Although the genetic basis of APA has been suggested, the identity of the underlying genes is largely unknown. In this study, we have performed a genome-wide association study (GWAS in an effort to identify susceptibility loci/genes for three main APA: anticardiolipin antibodies (ACL, lupus anticoagulant (LAC, and anti-β2 glycoprotein I antibodies (anti-β2GPI. Methods. DNA samples were genotyped using the Affymetrix 6.0 array containing 906,600 single-nucleotide polymorphisms (SNPs. Association of SNPs with the antibody status (positive/negative was tested using logistic regression under the additive model. Results. We have identified a number of suggestive novel loci with Pgenome-wide significance, many of the suggestive loci are potential candidates for the production of APA. We have replicated the previously reported associations of HLA genes and APOH with APA but these were not the top loci. Conclusions. We have identified a number of suggestive novel loci for APA that will stimulate follow-up studies in independent and larger samples to replicate our findings.

Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

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Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

2016-01-01

Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings

Genome-Wide Footprints of Pig Domestication and Selection Revealed through Massive Parallel Sequencing of Pooled DNA

NARCIS (Netherlands)

Amaral, A.J.; Ferretti, L.; Megens, H.J.W.C.; Crooijmans, R.P.M.A.; Nie, H.; Ramos-Onsins, S.E.; Perez-Enciso, M.; Schook, L.B.; Groenen, M.A.M.

2011-01-01

Background Artificial selection has caused rapid evolution in domesticated species. The identification of selection footprints across domesticated genomes can contribute to uncover the genetic basis of phenotypic diversity. Methodology/Main Findings Genome wide footprints of pig domestication and

Effects of in ovo electroporation on endogenous gene expression: genome-wide analysis

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Chambers David

2011-04-01

Full Text Available Abstract Background In ovo electroporation is a widely used technique to study gene function in developmental biology. Despite the widespread acceptance of this technique, no genome-wide analysis of the effects of in ovo electroporation, principally the current applied across the tissue and exogenous vector DNA introduced, on endogenous gene expression has been undertaken. Here, the effects of electric current and expression of a GFP-containing construct, via electroporation into the midbrain of Hamburger-Hamilton stage 10 chicken embryos, are analysed by microarray. Results Both current alone and in combination with exogenous DNA expression have a small but reproducible effect on endogenous gene expression, changing the expression of the genes represented on the array by less than 0.1% (current and less than 0.5% (current + DNA, respectively. The subset of genes regulated by electric current and exogenous DNA span a disparate set of cellular functions. However, no genes involved in the regional identity were affected. In sharp contrast to this, electroporation of a known transcription factor, Dmrt5, caused a much greater change in gene expression. Conclusions These findings represent the first systematic genome-wide analysis of the effects of in ovo electroporation on gene expression during embryonic development. The analysis reveals that this process has minimal impact on the genetic basis of cell fate specification. Thus, the study demonstrates the validity of the in ovo electroporation technique to study gene function and expression during development. Furthermore, the data presented here can be used as a resource to refine the set of transcriptional responders in future in ovo electroporation studies of specific gene function.

Genome-wide DNA methylation analyses in the brain reveal four differentially methylated regions between humans and non-human primates

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Wang Jinkai

2012-08-01

Full Text Available Abstract Background The highly improved cognitive function is the most significant change in human evolutionary history. Recently, several large-scale studies reported the evolutionary roles of DNA methylation; however, the role of DNA methylation on brain evolution is largely unknown. Results To test if DNA methylation has contributed to the evolution of human brain, with the use of MeDIP-Chip and SEQUENOM MassARRAY, we conducted a genome-wide analysis to identify differentially methylated regions (DMRs in the brain between humans and rhesus macaques. We first identified a total of 150 candidate DMRs by the MeDIP-Chip method, among which 4 DMRs were confirmed by the MassARRAY analysis. All 4 DMRs are within or close to the CpG islands, and a MIR3 repeat element was identified in one DMR, but no repeat sequence was observed in the other 3 DMRs. For the 4 DMR genes, their proteins tend to be conserved and two genes have neural related functions. Bisulfite sequencing and phylogenetic comparison among human, chimpanzee, rhesus macaque and rat suggested several regions of lineage specific DNA methylation, including a human specific hypomethylated region in the promoter of K6IRS2 gene. Conclusions Our study provides a new angle of studying human brain evolution and understanding the evolutionary role of DNA methylation in the central nervous system. The results suggest that the patterns of DNA methylation in the brain are in general similar between humans and non-human primates, and only a few DMRs were identified.

Genome-wide profiling of transcription factor binding and epigenetic marks in adipocytes by ChIP-seq

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Nielsen, Ronni; Mandrup, Susanne

2014-01-01

of the most widely used of these technologies. Using these methods, association of transcription factors, cofactors, and epigenetic marks can be mapped to DNA in a genome-wide manner. Here, we provide a detailed protocol for performing ChIP-seq analyses in preadipocytes and adipocytes. We have focused mainly...

A Genome-Wide mQTL Analysis in Human Adipose Tissue Identifies Genetic Variants Associated with DNA Methylation, Gene Expression and Metabolic Traits.

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Petr Volkov

Full Text Available Little is known about the extent to which interactions between genetics and epigenetics may affect the risk of complex metabolic diseases and/or their intermediary phenotypes. We performed a genome-wide DNA methylation quantitative trait locus (mQTL analysis in human adipose tissue of 119 men, where 592,794 single nucleotide polymorphisms (SNPs were related to DNA methylation of 477,891 CpG sites, covering 99% of RefSeq genes. SNPs in significant mQTLs were further related to gene expression in adipose tissue and obesity related traits. We found 101,911 SNP-CpG pairs (mQTLs in cis and 5,342 SNP-CpG pairs in trans showing significant associations between genotype and DNA methylation in adipose tissue after correction for multiple testing, where cis is defined as distance less than 500 kb between a SNP and CpG site. These mQTLs include reported obesity, lipid and type 2 diabetes loci, e.g. ADCY3/POMC, APOA5, CETP, FADS2, GCKR, SORT1 and LEPR. Significant mQTLs were overrepresented in intergenic regions meanwhile underrepresented in promoter regions and CpG islands. We further identified 635 SNPs in significant cis-mQTLs associated with expression of 86 genes in adipose tissue including CHRNA5, G6PC2, GPX7, RPL27A, THNSL2 and ZFP57. SNPs in significant mQTLs were also associated with body mass index (BMI, lipid traits and glucose and insulin levels in our study cohort and public available consortia data. Importantly, the Causal Inference Test (CIT demonstrates how genetic variants mediate their effects on metabolic traits (e.g. BMI, cholesterol, high-density lipoprotein (HDL, hemoglobin A1c (HbA1c and homeostatic model assessment of insulin resistance (HOMA-IR via altered DNA methylation in human adipose tissue. This study identifies genome-wide interactions between genetic and epigenetic variation in both cis and trans positions influencing gene expression in adipose tissue and in vivo (dysmetabolic traits associated with the development of

Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

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Dessie Salilew-Wondim

Full Text Available Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY, 4-cell (4C or 16-cell (16C were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP. Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic

StereoGene: rapid estimation of genome-wide correlation of continuous or interval feature data.

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Stavrovskaya, Elena D; Niranjan, Tejasvi; Fertig, Elana J; Wheelan, Sarah J; Favorov, Alexander V; Mironov, Andrey A

2017-10-15

Genomics features with similar genome-wide distributions are generally hypothesized to be functionally related, for example, colocalization of histones and transcription start sites indicate chromatin regulation of transcription factor activity. Therefore, statistical algorithms to perform spatial, genome-wide correlation among genomic features are required. Here, we propose a method, StereoGene, that rapidly estimates genome-wide correlation among pairs of genomic features. These features may represent high-throughput data mapped to reference genome or sets of genomic annotations in that reference genome. StereoGene enables correlation of continuous data directly, avoiding the data binarization and subsequent data loss. Correlations are computed among neighboring genomic positions using kernel correlation. Representing the correlation as a function of the genome position, StereoGene outputs the local correlation track as part of the analysis. StereoGene also accounts for confounders such as input DNA by partial correlation. We apply our method to numerous comparisons of ChIP-Seq datasets from the Human Epigenome Atlas and FANTOM CAGE to demonstrate its wide applicability. We observe the changes in the correlation between epigenomic features across developmental trajectories of several tissue types consistent with known biology and find a novel spatial correlation of CAGE clusters with donor splice sites and with poly(A) sites. These analyses provide examples for the broad applicability of StereoGene for regulatory genomics. The StereoGene C ++ source code, program documentation, Galaxy integration scripts and examples are available from the project homepage http://stereogene.bioinf.fbb.msu.ru/. favorov@sensi.org. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Hematopoietic transcriptional mechanisms: from locus-specific to genome-wide vantage points.

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DeVilbiss, Andrew W; Sanalkumar, Rajendran; Johnson, Kirby D; Keles, Sunduz; Bresnick, Emery H

2014-08-01

Hematopoiesis is an exquisitely regulated process in which stem cells in the developing embryo and the adult generate progenitor cells that give rise to all blood lineages. Master regulatory transcription factors control hematopoiesis by integrating signals from the microenvironment and dynamically establishing and maintaining genetic networks. One of the most rudimentary aspects of cell type-specific transcription factor function, how they occupy a highly restricted cohort of cis-elements in chromatin, remains poorly understood. Transformative technologic advances involving the coupling of next-generation DNA sequencing technology with the chromatin immunoprecipitation assay (ChIP-seq) have enabled genome-wide mapping of factor occupancy patterns. However, formidable problems remain; notably, ChIP-seq analysis yields hundreds to thousands of chromatin sites occupied by a given transcription factor, and only a fraction of the sites appear to be endowed with critical, non-redundant function. It has become en vogue to map transcription factor occupancy patterns genome-wide, while using powerful statistical tools to establish correlations to inform biology and mechanisms. With the advent of revolutionary genome editing technologies, one can now reach beyond correlations to conduct definitive hypothesis testing. This review focuses on key discoveries that have emerged during the path from single loci to genome-wide analyses, specifically in the context of hematopoietic transcriptional mechanisms. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Human Genome Research: Decoding DNA

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dropdown arrow Site Map A-Z Index Menu Synopsis Human Genome Research: Decoding DNA Resources with of the DNA double helix during April 2003. James D. Watson, Francis Crick, and Maurice Wilkins were company Celera announced the completion of a "working draft" reference DNA sequence of the human

Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

NARCIS (Netherlands)

Bellaoui, Mohammed; Chang, Michael; Ou, Jiongwen; Xu, Hong; Boone, Charles; Brown, Grant W

2003-01-01

Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA

GenoGAM: genome-wide generalized additive models for ChIP-Seq analysis.

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Stricker, Georg; Engelhardt, Alexander; Schulz, Daniel; Schmid, Matthias; Tresch, Achim; Gagneur, Julien

2017-08-01

Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is a widely used approach to study protein-DNA interactions. Often, the quantities of interest are the differential occupancies relative to controls, between genetic backgrounds, treatments, or combinations thereof. Current methods for differential occupancy of ChIP-Seq data rely however on binning or sliding window techniques, for which the choice of the window and bin sizes are subjective. Here, we present GenoGAM (Genome-wide Generalized Additive Model), which brings the well-established and flexible generalized additive models framework to genomic applications using a data parallelism strategy. We model ChIP-Seq read count frequencies as products of smooth functions along chromosomes. Smoothing parameters are objectively estimated from the data by cross-validation, eliminating ad hoc binning and windowing needed by current approaches. GenoGAM provides base-level and region-level significance testing for full factorial designs. Application to a ChIP-Seq dataset in yeast showed increased sensitivity over existing differential occupancy methods while controlling for type I error rate. By analyzing a set of DNA methylation data and illustrating an extension to a peak caller, we further demonstrate the potential of GenoGAM as a generic statistical modeling tool for genome-wide assays. Software is available from Bioconductor: https://www.bioconductor.org/packages/release/bioc/html/GenoGAM.html . gagneur@in.tum.de. Supplementary information is available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Meristem micropropagation of cassava (Manihot esculenta evokes genome-wide changes in DNA methylation

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Shedrack Reuben Kitimu

2015-08-01

Full Text Available There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta cultivars. Principal Co-ordinate Analysis of profiles generated by Methylation Sensitive Amplified Polymorphism (MSAP revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype By Sequencing (msGBS identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologues have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops.

Trans-ancestry genome-wide association study identifies 12 genetic loci influencing blood pressure and implicates a role for DNA methylation

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Drong, Alexander W; Abbott, James; Wahl, Simone; Tan, Sian-Tsung; Scott, William R; Campanella, Gianluca; Chadeau-Hyam, Marc; Afzal, Uzma; Ahluwalia, Tarunveer S; Bonder, Marc Jan; Chen, Peng; Dehghan, Abbas; Edwards, Todd L; Esko, Tõnu; Go, Min Jin; Harris, Sarah E; Hartiala, Jaana; Kasela, Silva; Kasturiratne, Anuradhani; Khor, Chiea-Chuen; Kleber, Marcus E; Li, Huaixing; Yu Mok, Zuan; Nakatochi, Masahiro; Sapari, Nur Sabrina; Saxena, Richa; Stewart, Alexandre F R; Stolk, Lisette; Tabara, Yasuharu; Teh, Ai Ling; Wu, Ying; Wu, Jer-Yuarn; Zhang, Yi; Aits, Imke; Da Silva Couto Alves, Alexessander; Das, Shikta; Dorajoo, Rajkumar; Hopewell, Jemma C; Kim, Yun Kyoung; Koivula, Robert W; Luan, Jian’an; Lyytikäinen, Leo-Pekka; Nguyen, Quang N; Pereira, Mark A; Postmus, Iris; Raitakari, Olli T; Bryan, Molly Scannell; Scott, Robert A; Sorice, Rossella; Tragante, Vinicius; Traglia, Michela; White, Jon; Yamamoto, Ken; Zhang, Yonghong; Adair, Linda S; Ahmed, Alauddin; Akiyama, Koichi; Asif, Rasheed; Aung, Tin; Barroso, Inês; Bjonnes, Andrew; Braun, Timothy R; Cai, Hui; Chang, Li-Ching; Chen, Chien-Hsiun; Cheng, Ching-Yu; Chong, Yap-Seng; Collins, Rory; Courtney, Regina; Davies, Gail; Delgado, Graciela; Do, Loi D; Doevendans, Pieter A; Gansevoort, Ron T; Gao, Yu-Tang; Grammer, Tanja B; Grarup, Niels; Grewal, Jagvir; Gu, Dongfeng; Wander, Gurpreet S; Hartikainen, Anna-Liisa; Hazen, Stanley L; He, Jing; Heng, Chew-Kiat; Hixson, James E; Hofman, Albert; Hsu, Chris; Huang, Wei; Husemoen, Lise L N; Hwang, Joo-Yeon; Ichihara, Sahoko; Igase, Michiya; Isono, Masato; Justesen, Johanne M; Katsuya, Tomohiro; Kibriya, Muhammad G; Kim, Young Jin; Kishimoto, Miyako; Koh, Woon-Puay; Kohara, Katsuhiko; Kumari, Meena; Kwek, Kenneth; Lee, Nanette R; Lee, Jeannette; Liao, Jiemin; Lieb, Wolfgang; Liewald, David C M; Matsubara, Tatsuaki; Matsushita, Yumi; Meitinger, Thomas; Mihailov, Evelin; Milani, Lili; Mills, Rebecca; Mononen, Nina; Müller-Nurasyid, Martina; Nabika, Toru; Nakashima, Eitaro; Ng, Hong Kiat; Nikus, Kjell; Nutile, Teresa; Ohkubo, Takayoshi; Ohnaka, Keizo; Parish, Sarah; Paternoster, Lavinia; Peng, Hao; Peters, Annette; Pham, Son T; Pinidiyapathirage, Mohitha J; Rahman, Mahfuzar; Rakugi, Hiromi; Rolandsson, Olov; Ann Rozario, Michelle; Ruggiero, Daniela; Sala, Cinzia F; Sarju, Ralhan; Shimokawa, Kazuro; Snieder, Harold; Sparsø, Thomas; Spiering, Wilko; Starr, John M; Stott, David J; Stram, Daniel O; Sugiyama, Takao; Szymczak, Silke; Tang, W H Wilson; Tong, Lin; Trompet, Stella; Turjanmaa, Väinö; Ueshima, Hirotsugu; Uitterlinden, André G; Umemura, Satoshi; Vaarasmaki, Marja; van Dam, Rob M; van Gilst, Wiek H; van Veldhuisen, Dirk J; Viikari, Jorma S; Waldenberger, Melanie; Wang, Yiqin; Wang, Aili; Wilson, Rory; Wong, Tien-Yin; Xiang, Yong-Bing; Yamaguchi, Shuhei; Ye, Xingwang; Young, Robin D; Young, Terri L; Yuan, Jian-Min; Zhou, Xueya; Asselbergs, Folkert W; Ciullo, Marina; Clarke, Robert; Deloukas, Panos; Franke, Andre; Franks, Paul W; Franks, Steve; Friedlander, Yechiel; Gross, Myron D; Guo, Zhirong; Hansen, Torben; Jarvelin, Marjo-Riitta; Jørgensen, Torben; Jukema, J Wouter; kähönen, Mika; Kajio, Hiroshi; Kivimaki, Mika; Lee, Jong-Young; Lehtimäki, Terho; Linneberg, Allan; Miki, Tetsuro; Pedersen, Oluf; Samani, Nilesh J; Sørensen, Thorkild I A; Takayanagi, Ryoichi; Toniolo, Daniela; Ahsan, Habibul; Allayee, Hooman; Chen, Yuan-Tsong; Danesh, John; Deary, Ian J; Franco, Oscar H; Franke, Lude; Heijman, Bastiaan T; Holbrook, Joanna D; Isaacs, Aaron; Kim, Bong-Jo; Lin, Xu; Liu, Jianjun; März, Winfried; Metspalu, Andres; Mohlke, Karen L; Sanghera, Dharambir K; Shu, Xiao-Ou; van Meurs, Joyce B J; Vithana, Eranga; Wickremasinghe, Ananda R; Wijmenga, Cisca; Wolffenbuttel, Bruce H W; Yokota, Mitsuhiro; Zheng, Wei; Zhu, Dingliang; Vineis, Paolo; Kyrtopoulos, Soterios A; Kleinjans, Jos C S; McCarthy, Mark I; Soong, Richie; Gieger, Christian; Scott, James

2016-01-01

We carried out a trans-ancestry genome-wide association and replication study of blood pressure phenotypes among up to 320,251 individuals of East Asian, European and South Asian ancestry. We find genetic variants at 12 new loci to be associated with blood pressure (P = 3.9 × 10−11 to 5.0 × 10−21). The sentinel blood pressure SNPs are enriched for association with DNA methylation at multiple nearby CpG sites, suggesting that, at some of the loci identified, DNA methylation may lie on the regulatory pathway linking sequence variation to blood pressure. The sentinel SNPs at the 12 new loci point to genes involved in vascular smooth muscle (IGFBP3, KCNK3, PDE3A and PRDM6) and renal (ARHGAP24, OSR1, SLC22A7 and TBX2) function. The new and known genetic variants predict increased left ventricular mass, circulating levels of NT-proBNP, and cardiovascular and all-cause mortality (P = 0.04 to 8.6 × 10−6). Our results provide new evidence for the role of DNA methylation in blood pressure regulation. PMID:26390057

RICD: A rice indica cDNA database resource for rice functional genomics

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Zhang Qifa

2008-11-01

Full Text Available Abstract Background The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones. Results Rice Indica cDNA Database (RICD is an online MySQL-PHP driven database with a user-friendly web interface. It allows investigators to query the cDNA clones by keyword, genome position, nucleotide or protein sequence, and putative function. It also provides a series of information, including sequences, protein domain annotations, similarity search results, SNPs and InDels information, and hyperlinks to gene annotation in both The Rice Annotation Project Database (RAP-DB and The TIGR Rice Genome Annotation Resource, expression atlas in RiceGE and variation report in Gramene of each cDNA. Conclusion The online rice indica cDNA database provides cDNA resource with comprehensive information to researchers for functional analysis of indica subspecies and for comparative genomics. The RICD database is available through our website http://www.ncgr.ac.cn/ricd.

Genome-Wide DNA Copy Number Analysis of Acute Lymphoblastic Leukemia Identifies New Genetic Markers Associated with Clinical Outcome.

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Maribel Forero-Castro

Full Text Available Identifying additional genetic alterations associated with poor prognosis in acute lymphoblastic leukemia (ALL is still a challenge.To characterize the presence of additional DNA copy number alterations (CNAs in children and adults with ALL by whole-genome oligonucleotide array (aCGH analysis, and to identify their associations with clinical features and outcome. Array-CGH was carried out in 265 newly diagnosed ALLs (142 children and 123 adults. The NimbleGen CGH 12x135K array (Roche was used to analyze genetic gains and losses. CNAs were analyzed with GISTIC and aCGHweb software. Clinical and biological variables were analyzed. Three of the patients showed chromothripsis (cth6, cth14q and cth15q. CNAs were associated with age, phenotype, genetic subtype and overall survival (OS. In the whole cohort of children, the losses on 14q32.33 (p = 0.019 and 15q13.2 (p = 0.04 were related to shorter OS. In the group of children without good- or poor-risk cytogenetics, the gain on 1p36.11 was a prognostic marker independently associated with shorter OS. In adults, the gains on 19q13.2 (p = 0.001 and Xp21.1 (p = 0.029, and the loss of 17p (p = 0.014 were independent markers of poor prognosis with respect to OS. In summary, CNAs are frequent in ALL and are associated with clinical parameters and survival. Genome-wide DNA copy number analysis allows the identification of genetic markers that predict clinical outcome, suggesting that detection of these genetic lesions will be useful in the management of patients newly diagnosed with ALL.

DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy.

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Benjamin D Pope

Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is linked to contraction of an array of tandem 3.3-kb repeats (D4Z4 at 4q35.2 from 11-100 copies to 1-10 copies. The extent to which D4Z4 contraction at 4q35.2 affects overall 4q35.2 chromatin organization remains unclear. Because DNA replication timing is highly predictive of long-range chromatin interactions, we generated genome-wide replication-timing profiles for FSHD and control myogenic precursor cells. We compared non-immortalized myoblasts from four FSHD patients and three control individuals to each other and to a variety of other human cell types. This study also represents the first genome-wide comparison of replication timing profiles in non-immortalized human cell cultures. Myoblasts from both control and FSHD individuals all shared a myoblast-specific replication profile. In contrast, male and female individuals were readily distinguished by monoallelic differences in replication timing at DXZ4 and other regions across the X chromosome affected by X inactivation. We conclude that replication timing is a robust cell-type specific feature that is unaffected by FSHD-related D4Z4 contraction.

A Pooled Genome-Wide Association Study of Asperger Syndrome.

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Varun Warrier

Full Text Available Asperger Syndrome (AS is a neurodevelopmental condition characterized by impairments in social interaction and communication, alongside the presence of unusually repetitive, restricted interests and stereotyped behaviour. Individuals with AS have no delay in cognitive and language development. It is a subset of Autism Spectrum Conditions (ASC, which are highly heritable and has a population prevalence of approximately 1%. Few studies have investigated the genetic basis of AS. To address this gap in the literature, we performed a genome-wide pooled DNA association study to identify candidate loci in 612 individuals (294 cases and 318 controls of Caucasian ancestry, using the Affymetrix GeneChip Human Mapping version 6.0 array. We identified 11 SNPs that had a p-value below 1x10-5. These SNPs were independently genotyped in the same sample. Three of the SNPs (rs1268055, rs7785891 and rs2782448 were nominally significant, though none remained significant after Bonferroni correction. Two of our top three SNPs (rs7785891 and rs2782448 lie in loci previously implicated in ASC. However, investigation of the three SNPs in the ASC genome-wide association dataset from the Psychiatric Genomics Consortium indicated that these three SNPs were not significantly associated with ASC. The effect sizes of the variants were modest, indicating that our study was not sufficiently powered to identify causal variants with precision.

Genome-wide mapping of autonomous promoter activity in human cells.

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van Arensbergen, Joris; FitzPatrick, Vincent D; de Haas, Marcel; Pagie, Ludo; Sluimer, Jasper; Bussemaker, Harmen J; van Steensel, Bas

2017-02-01

Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of the sequences that could be tested. Here we present 'survey of regulatory elements' (SuRE), a method that assays more than 10 8 DNA fragments, each 0.2-2 kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library of random genomic fragments upstream of a 20-bp barcode is constructed, and decoded by paired-end sequencing. This library is used to transfect cells, and barcodes in transcribed RNA are quantified by high-throughput sequencing. When applied to the human genome, we achieve 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide in K562 cells. By computational modeling we delineate subregions within promoters that are relevant for their activity. We show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites.

Genome-wide analysis of intraspecific DNA polymorphism in 'Micro-Tom', a model cultivar of tomato (Solanum lycopersicum).

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Kobayashi, Masaaki; Nagasaki, Hideki; Garcia, Virginie; Just, Daniel; Bres, Cécile; Mauxion, Jean-Philippe; Le Paslier, Marie-Christine; Brunel, Dominique; Suda, Kunihiro; Minakuchi, Yohei; Toyoda, Atsushi; Fujiyama, Asao; Toyoshima, Hiromi; Suzuki, Takayuki; Igarashi, Kaori; Rothan, Christophe; Kaminuma, Eli; Nakamura, Yasukazu; Yano, Kentaro; Aoki, Koh

2014-02-01

Tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. The genome sequencing of the tomato cultivar 'Heinz 1706' was recently completed. To accelerate the progress of tomato genomics studies, systematic bioresources, such as mutagenized lines and full-length cDNA libraries, have been established for the cultivar 'Micro-Tom'. However, these resources cannot be utilized to their full potential without the completion of the genome sequencing of 'Micro-Tom'. We undertook the genome sequencing of 'Micro-Tom' and here report the identification of single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) between 'Micro-Tom' and 'Heinz 1706'. The analysis demonstrated the presence of 1.23 million SNPs and 0.19 million indels between the two cultivars. The density of SNPs and indels was high in chromosomes 2, 5 and 11, but was low in chromosomes 6, 8 and 10. Three known mutations of 'Micro-Tom' were localized on chromosomal regions where the density of SNPs and indels was low, which was consistent with the fact that these mutations were relatively new and introgressed into 'Micro-Tom' during the breeding of this cultivar. We also report SNP analysis for two 'Micro-Tom' varieties that have been maintained independently in Japan and France, both of which have served as standard lines for 'Micro-Tom' mutant collections. Approximately 28,000 SNPs were identified between these two 'Micro-Tom' lines. These results provide high-resolution DNA polymorphic information on 'Micro-Tom' and represent a valuable contribution to the 'Micro-Tom'-based genomics resources.

Incidence of genome structure, DNA asymmetry, and cell physiology on T-DNA integration in chromosomes of the phytopathogenic fungus Leptosphaeria maculans.

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Bourras, Salim; Meyer, Michel; Grandaubert, Jonathan; Lapalu, Nicolas; Fudal, Isabelle; Linglin, Juliette; Ollivier, Benedicte; Blaise, Françoise; Balesdent, Marie-Hélène; Rouxel, Thierry

2012-08-01

The ever-increasing generation of sequence data is accompanied by unsatisfactory functional annotation, and complex genomes, such as those of plants and filamentous fungi, show a large number of genes with no predicted or known function. For functional annotation of unknown or hypothetical genes, the production of collections of mutants using Agrobacterium tumefaciens-mediated transformation (ATMT) associated with genotyping and phenotyping has gained wide acceptance. ATMT is also widely used to identify pathogenicity determinants in pathogenic fungi. A systematic analysis of T-DNA borders was performed in an ATMT-mutagenized collection of the phytopathogenic fungus Leptosphaeria maculans to evaluate the features of T-DNA integration in its particular transposable element-rich compartmentalized genome. A total of 318 T-DNA tags were recovered and analyzed for biases in chromosome and genic compartments, existence of CG/AT skews at the insertion site, and occurrence of microhomologies between the T-DNA left border (LB) and the target sequence. Functional annotation of targeted genes was done using the Gene Ontology annotation. The T-DNA integration mainly targeted gene-rich, transcriptionally active regions, and it favored biological processes consistent with the physiological status of a germinating spore. T-DNA integration was strongly biased toward regulatory regions, and mainly promoters. Consistent with the T-DNA intranuclear-targeting model, the density of T-DNA insertion correlated with CG skew near the transcription initiation site. The existence of microhomologies between promoter sequences and the T-DNA LB flanking sequence was also consistent with T-DNA integration to host DNA mediated by homologous recombination based on the microhomology-mediated end-joining pathway.

Using rice genome-wide association studies to identify DNA markers for marker-assisted selection

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Rice association mapping panels are collections of rice (Oryza sativa L.) accessions developed for genome-wide association (GWA) studies. One of these panels, the Rice Diversity Panel 1 (RDP1) was phenotyped by various research groups for several traits of interest, and more recently, genotyped with...

Genome-wide DNA-(de)methylation is associated with Noninfectious Bud-failure exhibition in Almond (Prunus dulcis [Mill.] D.A.Webb).

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Fresnedo-Ramírez, Jonathan; Chan, Helen M; Parfitt, Dan E; Crisosto, Carlos H; Gradziel, Thomas M

2017-02-16

Noninfectious bud-failure (BF) remains a major threat to almond production in California, particularly with the recent rapid expansion of acreage and as more intensive cultural practices and modern cultivars are adopted. BF has been shown to be inherited in both vegetative and sexual progeny, with exhibition related to the age and propagation history of scion clonal sources. These characteristics suggest an epigenetic influence, such as the loss of juvenility mediated by DNA-(de)methylation. Various degrees of BF have been reported among cultivars as well as within sources of clonal propagation of the same cultivar. Genome-wide methylation profiles for different clones within almond genotypes were developed to examine their association with BF levels and association with the chronological time from initial propagation. The degree of BF exhibition was found to be associated with DNA-(de)methylation and clonal age, which suggests that epigenetic changes associated with ageing may be involved in the differential exhibition of BF within and among almond clones. Research is needed to investigate the potential of DNA-(de)methylation status as a predictor for BF as well as for effective strategies to improve clonal selection against age related deterioration. This is the first report of an epigenetic-related disorder threatening a major tree crop.

Genomic selection: genome-wide prediction in plant improvement.

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Desta, Zeratsion Abera; Ortiz, Rodomiro

2014-09-01

Association analysis is used to measure relations between markers and quantitative trait loci (QTL). Their estimation ignores genes with small effects that trigger underpinning quantitative traits. By contrast, genome-wide selection estimates marker effects across the whole genome on the target population based on a prediction model developed in the training population (TP). Whole-genome prediction models estimate all marker effects in all loci and capture small QTL effects. Here, we review several genomic selection (GS) models with respect to both the prediction accuracy and genetic gain from selection. Phenotypic selection or marker-assisted breeding protocols can be replaced by selection, based on whole-genome predictions in which phenotyping updates the model to build up the prediction accuracy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genome-wide identification of estrogen receptor alpha-binding sites in mouse liver

DEFF Research Database (Denmark)

Gao, Hui; Fält, Susann; Sandelin, Albin

2007-01-01

We report the genome-wide identification of estrogen receptor alpha (ERalpha)-binding regions in mouse liver using a combination of chromatin immunoprecipitation and tiled microarrays that cover all nonrepetitive sequences in the mouse genome. This analysis identified 5568 ERalpha-binding regions...... genes. The majority of ERalpha-binding regions lie in regions that are evolutionarily conserved between human and mouse. Motif-finding algorithms identified the estrogen response element, and variants thereof, together with binding sites for activator protein 1, basic-helix-loop-helix proteins, ETS...... signaling in mouse liver, by characterizing the first step in this signaling cascade, the binding of ERalpha to DNA in intact chromatin....

Genome-wide DNA methylation study in human placenta identifies novel loci associated with maternal smoking during pregnancy.

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Morales, Eva; Vilahur, Nadia; Salas, Lucas A; Motta, Valeria; Fernandez, Mariana F; Murcia, Mario; Llop, Sabrina; Tardon, Adonina; Fernandez-Tardon, Guillermo; Santa-Marina, Loreto; Gallastegui, Mara; Bollati, Valentina; Estivill, Xavier; Olea, Nicolas; Sunyer, Jordi; Bustamante, Mariona

2016-10-01

We conducted an epigenome-wide association study (EWAS) of DNA methylation in placenta in relation to maternal tobacco smoking during pregnancy and examined whether smoking-induced changes lead to low birthweight. DNA methylation in placenta was measured using the Illumina HumanMethylation450 BeadChip in 179 participants from the INfancia y Medio Ambiente (INMA) birth cohort. Methylation levels across 431 311 CpGs were tested for differential methylation between smokers and non-smokers in pregnancy. We took forward three top-ranking loci for further validation and replication by bisulfite pyrosequencing using data of 248 additional participants of the INMA cohort. We examined the association of methylation at smoking-associated loci with birthweight by applying a mediation analysis and a two-sample Mendelian randomization approach. Fifty CpGs were differentially methylated in placenta between smokers and non-smokers during pregnancy [false discovery rate (FDR) < 0.05]. We validated and replicated differential methylation at three top-ranking loci: cg27402634 located between LINC00086 and LEKR1, a gene previously related to birthweight in genome-wide association studies; cg20340720 (WBP1L); and cg25585967 and cg12294026 (TRIO). Dose-response relationships with maternal urine cotinine concentration during pregnancy were confirmed. Differential methylation at cg27402634 explained up to 36% of the lower birthweight in the offspring of smokers (Sobel P-value < 0.05). A two-sample Mendelian randomization analysis provided evidence that decreases in methylation levels at cg27402634 lead to decreases in birthweight. We identified novel loci differentially methylated in placenta in relation to maternal smoking during pregnancy. Adverse effects of maternal smoking on birthweight of the offspring may be mediated by alterations in the placental methylome. © The Author 2016; all rights reserved. Published by Oxford University Press on behalf of the International

High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot.

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Aberg, Karolina A; Xie, Lin Y; Nerella, Srilaxmi; Copeland, William E; Costello, E Jane; van den Oord, Edwin J C G

2013-05-01

The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study, we apply a methyl-CpG binding domain (MBD) protein enrichment-based technique in combination with next generation sequencing (MBD-seq) to assess the methylation status of the ~27 million CpGs in the human autosomal reference genome. We investigate eight methylomes using DNA from blood spots. This data are compared with 1,500 methylomes previously assayed with the same MBD-seq approach using DNA from whole blood. When investigating the sequence quality and the enrichment profile across biological features, we find that DNA extracted from blood spots gives comparable results with DNA extracted from whole blood. Only if the amount of starting material is ≤ 0.5µg DNA we observe a slight decrease in the assay performance. In conclusion, we show that high quality methylome-wide investigations using MBD-seq can be conducted in DNA extracted from archived dry blood spots without sacrificing quality and without bias in enrichment profile as long as the amount of starting material is sufficient. In general, the amount of DNA extracted from a single blood spot is sufficient for methylome-wide investigations with the MBD-seq approach.

Sequencing intractable DNA to close microbial genomes.

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Richard A Hurt

Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

Sequencing Intractable DNA to Close Microbial Genomes

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Hurt, Jr., Richard Ashley [ORNL; Brown, Steven D [ORNL; Podar, Mircea [ORNL; Palumbo, Anthony Vito [ORNL; Elias, Dwayne A [ORNL

2012-01-01

Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled intractable resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such difficult regions in the non-contiguous finished Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. These developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

Genome-Wide Screening of Genes Showing Altered Expression in Liver Metastases of Human Colorectal Cancers by cDNA Microarray

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Rempei Yanagawa

2001-01-01

Full Text Available In spite of intensive and increasingly successful attempts to determine the multiple steps involved in colorectal carcinogenesis, the mechanisms responsible for metastasis of colorectal tumors to the liver remain to be clarified. To identify genes that are candidates for involvement in the metastatic process, we analyzed genome-wide expression profiles of 10 primary colorectal cancers and their corresponding metastatic lesions by means of a cDNA microarray consisting of 9121 human genes. This analysis identified 40 genes whose expression was commonly upregulated in metastatic lesions, and 7 that were commonly downregulated. The upregulated genes encoded proteins involved in cell adhesion, or remodeling of the actin cytoskeleton. Investigation of the functions of more of the altered genes should improve our understanding of metastasis and may identify diagnostic markers and/or novel molecular targets for prevention or therapy of metastatic lesions.

Genome-wide DNA polymorphisms in Kavuni, a traditional rice cultivar with nutritional and therapeutic properties.

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Rathinasabapathi, Pasupathi; Purushothaman, Natarajan; Parani, Madasamy

2016-05-01

Although rice genome was sequenced in the year 2002, efforts in resequencing the large number of available accessions, landraces, traditional cultivars, and improved varieties of this important food crop are limited. We have initiated resequencing of the traditional cultivars from India. Kavuni is an important traditional rice cultivar from South India that attracts premium price for its nutritional and therapeutic properties. Whole-genome sequencing of Kavuni using Illumina platform and SNPs analysis using Nipponbare reference genome identified 1 150 711 SNPs of which 377 381 SNPs were located in the genic regions. Non-synonymous SNPs (62 708) were distributed in 19 251 genes, and their number varied between 1 and 115 per gene. Large-effect DNA polymorphisms (7769) were present in 3475 genes. Pathway mapping of these polymorphisms revealed the involvement of genes related to carbohydrate metabolism, translation, protein-folding, and cell death. Analysis of the starch biosynthesis related genes revealed that the granule-bound starch synthase I gene had T/G SNPs at the first intron/exon junction and a two-nucleotide combination, which were reported to favour high amylose content and low glycemic index. The present study provided a valuable genomics resource to study the rice varieties with nutritional and medicinal properties.

Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57.

Science.gov (United States)

Bak, Mads; Boonen, Susanne E; Dahl, Christina; Hahnemann, Johanne M D; Mackay, Deborah J D G; Tümer, Zeynep; Grønskov, Karen; Temple, I Karen; Guldberg, Per; Tommerup, Niels

2016-04-14

Transient neonatal diabetes mellitus 1 (TNDM1) is a rare imprinting disorder characterized by intrautering growth retardation and diabetes mellitus usually presenting within the first six weeks of life and resolves by the age of 18 months. However, patients have an increased risk of developing diabetes mellitus type 2 later in life. Transient neonatal diabetes mellitus 1 is caused by overexpression of the maternally imprinted genes PLAGL1 and HYMAI on chromosome 6q24. One of the mechanisms leading to overexpression of the locus is hypomethylation of the maternal allele of PLAGL1 and HYMAI. A subset of patients with maternal hypomethylation at PLAGL1 have hypomethylation at additional imprinted loci throughout the genome, including GRB10, ZIM2 (PEG3), MEST (PEG1), KCNQ1OT1 and NESPAS (GNAS-AS1). About half of the TNDM1 patients carry mutations in ZFP57, a transcription factor involved in establishment and maintenance of methylation of imprinted loci. Our objective was to investigate whether additional regions are aberrantly methylated in ZFP57 mutation carriers. Genome-wide DNA methylation analysis was performed on four individuals with homozygous or compound heterozygous ZFP57 mutations, three relatives with heterozygous ZFP57 mutations and five controls. Methylation status of selected regions showing aberrant methylation in the patients was verified using bisulfite-sequencing. We found large variability among the patients concerning the number and identity of the differentially methylated regions, but more than 60 regions were aberrantly methylated in two or more patients and a novel region within PPP1R13L was found to be hypomethylated in all the patients. The hypomethylated regions in common between the patients are enriched for the ZFP57 DNA binding motif. We have expanded the epimutational spectrum of TNDM1 associated with ZFP57 mutations and found one novel region within PPP1R13L which is hypomethylated in all TNDM1 patients included in this study. Functional

G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae.

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John A Capra

2010-07-01

Full Text Available G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs. Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

Genome wide binding (ChIP-Seq of murine Bapx1 and Sox9 proteins in vivo and in vitro

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Sumantra Chatterjee

2016-12-01

Full Text Available This work pertains to GEO submission GSE36672, in vivo and in vitro genome wide binding (ChIP-Seq of Bapx1/Nkx3.2 and Sox9 proteins. We have previously shown that data from a genome wide binding assay combined with transcriptional profiling is an insightful means to divulge the mechanisms directing cell type specification and the generation of tissues and subsequent organs [1]. Our earlier work identified the role of the DNA-binding homeodomain containing protein Bapx1/Nkx3.2 in midgestation murine embryos. Microarray analysis of EGFP-tagged cells (both wildtype and null was integrated using ChIP-Seq analysis of Bapx1/Nkx3.2 and Sox9 DNA-binding proteins in living tissue.

ChIP on SNP-chip for genome-wide analysis of human histone H4 hyperacetylation

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Porter Christopher J

2007-09-01

Full Text Available Abstract Background SNP microarrays are designed to genotype Single Nucleotide Polymorphisms (SNPs. These microarrays report hybridization of DNA fragments and therefore can be used for the purpose of detecting genomic fragments. Results Here, we demonstrate that a SNP microarray can be effectively used in this way to perform chromatin immunoprecipitation (ChIP on chip as an alternative to tiling microarrays. We illustrate this novel application by mapping whole genome histone H4 hyperacetylation in human myoblasts and myotubes. We detect clusters of hyperacetylated histone H4, often spanning across up to 300 kilobases of genomic sequence. Using complementary genome-wide analyses of gene expression by DNA microarray we demonstrate that these clusters of hyperacetylated histone H4 tend to be associated with expressed genes. Conclusion The use of a SNP array for a ChIP-on-chip application (ChIP on SNP-chip will be of great value to laboratories whose interest is the determination of general rules regarding the relationship of specific chromatin modifications to transcriptional status throughout the genome and to examine the asymmetric modification of chromatin at heterozygous loci.

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

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Bekim Sadikovic

Full Text Available Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.

Extensive variation in the density and distribution of DNA polymorphism in sorghum genomes.

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Joseph Evans

Full Text Available Sorghum genotypes currently used for grain production in the United States were developed from African landraces that were imported starting in the mid-to-late 19(th century. Farmers and plant breeders selected genotypes for grain production with reduced plant height, early flowering, increased grain yield, adaptation to drought, and improved resistance to lodging, diseases and pests. DNA polymorphisms that distinguish three historically important grain sorghum genotypes, BTx623, BTx642 and Tx7000, were characterized by genome sequencing, genotyping by sequencing, genetic mapping, and pedigree-based haplotype analysis. The distribution and density of DNA polymorphisms in the sequenced genomes varied widely, in part because the lines were derived through breeding and selection from diverse Kafir, Durra, and Caudatum race accessions. Genomic DNA spanning dw1 (SBI-09 and dw3 (SBI-07 had identical haplotypes due to selection for reduced height. Lower SNP density in genes located in pericentromeric regions compared with genes located in euchromatic regions is consistent with background selection in these regions of low recombination. SNP density was higher in euchromatic DNA and varied >100-fold in contiguous intervals that spanned up to 300 Kbp. The localized variation in DNA polymorphism density occurred throughout euchromatic regions where recombination is elevated, however, polymorphism density was not correlated with gene density or DNA methylation. Overall, sorghum chromosomes contain distal euchromatic regions characterized by extensive, localized variation in DNA polymorphism density, and large pericentromeric regions of low gene density, diversity, and recombination.

A CTAB Procedure Of Total Genomic DNA Extraction For Medicinal Mushrooms

International Nuclear Information System (INIS)

Azhar Mohamad; Muhammad Hussaini Mohd Mustafa; Muhammad Hanif Azhari Noor; Rosnani Abdul Rashid; Hasan Hamdani Hasan Mutaat; Meswan Meskom; Mat Rasol Awang

2014-01-01

Medicinal mushroom is defined as mushrooms used in medicine or medical research. Isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including Polymerase Chain Reaction (PCR), endonuclease restriction digestion, Southern blot analysis, and genomic library construction. The most important and prerequisite towards reliable molecular biology work is the total genomic DNA of a sample must be in good quality. Five freshly samples of medicinal mushroom were used in this work known as Auriculariapolytricha, Lentinus edode, Pleurotus sayorcaju, Sczhizopyllum commune and Ganodermalucidum. 5 mg of each sample were used to extraction the DNA, prepared in 3 replications and repeated twice. PCR based technique by using ISSR markers were used in checking the amplification ability of the total genomic extraction. A standard Doyle and Doyle protocol for genomic DNA extraction was modified in optimizing the total genomic DNA from the medicinal mushroom.The modification parameters were percentage of CTAB, incubation period and temperature. The results reveal that each sample required a certain combinations of time and period of incubation. Besides, percentage of CTAB in the buffer was found significant in giving a high yielding of extracted total genomic DNA. The extracted total genomic DNA from the medicinal mushroom yielded from 39.7 ng/ μl to 919.1 ng/ μl. The different yield among the samples found to be corresponded to polysaccharide content in the medicinal mushrooms. The objective of this works is to optimize total genomic DNA extraction of medicinal mushrooms towards a high quality intact genomic DNA for molecular activities. (author)

GWAMA: software for genome-wide association meta-analysis

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Mägi Reedik

2010-05-01

Full Text Available Abstract Background Despite the recent success of genome-wide association studies in identifying novel loci contributing effects to complex human traits, such as type 2 diabetes and obesity, much of the genetic component of variation in these phenotypes remains unexplained. One way to improving power to detect further novel loci is through meta-analysis of studies from the same population, increasing the sample size over any individual study. Although statistical software analysis packages incorporate routines for meta-analysis, they are ill equipped to meet the challenges of the scale and complexity of data generated in genome-wide association studies. Results We have developed flexible, open-source software for the meta-analysis of genome-wide association studies. The software incorporates a variety of error trapping facilities, and provides a range of meta-analysis summary statistics. The software is distributed with scripts that allow simple formatting of files containing the results of each association study and generate graphical summaries of genome-wide meta-analysis results. Conclusions The GWAMA (Genome-Wide Association Meta-Analysis software has been developed to perform meta-analysis of summary statistics generated from genome-wide association studies of dichotomous phenotypes or quantitative traits. Software with source files, documentation and example data files are freely available online at http://www.well.ox.ac.uk/GWAMA.

Genome-wide comparison of medieval and modern Mycobacterium leprae.

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Schuenemann, Verena J; Singh, Pushpendra; Mendum, Thomas A; Krause-Kyora, Ben; Jäger, Günter; Bos, Kirsten I; Herbig, Alexander; Economou, Christos; Benjak, Andrej; Busso, Philippe; Nebel, Almut; Boldsen, Jesper L; Kjellström, Anna; Wu, Huihai; Stewart, Graham R; Taylor, G Michael; Bauer, Peter; Lee, Oona Y-C; Wu, Houdini H T; Minnikin, David E; Besra, Gurdyal S; Tucker, Katie; Roffey, Simon; Sow, Samba O; Cole, Stewart T; Nieselt, Kay; Krause, Johannes

2013-07-12

Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.

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

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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.

The clinical application of genome-wide sequencing for monogenic diseases in Canada: Position Statement of the Canadian College of Medical Geneticists

OpenAIRE

Boycott, Kym; Hartley, Taila; Adam, Shelin; Bernier, Francois; Chong, Karen; Fernandez, Bridget A; Friedman, Jan M; Geraghty, Michael T; Hume, Stacey; Knoppers, Bartha M; Laberge, Anne-Marie; Majewski, Jacek; Mendoza-Londono, Roberto; Meyn, M Stephen; Michaud, Jacques L

2015-01-01

Purpose and scope The aim of this Position Statement is to provide recommendations for Canadian medical geneticists, clinical laboratory geneticists, genetic counsellors and other physicians regarding the use of genome-wide sequencing of germline DNA in the context of clinical genetic diagnosis. This statement has been developed to facilitate the clinical translation and development of best practices for clinical genome-wide sequencing for genetic diagnosis of monogenic diseases in Canada; it...

Integration of Genome-Wide TF Binding and Gene Expression Data to Characterize Gene Regulatory Networks in Plant Development.

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Chen, Dijun; Kaufmann, Kerstin

2017-01-01

Key transcription factors (TFs) controlling the morphogenesis of flowers and leaves have been identified in the model plant Arabidopsis thaliana. Recent genome-wide approaches based on chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) enable systematic identification of genome-wide TF binding sites (TFBSs) of these regulators. Here, we describe a computational pipeline for analyzing ChIP-seq data to identify TFBSs and to characterize gene regulatory networks (GRNs) with applications to the regulatory studies of flower development. In particular, we provide step-by-step instructions on how to download, analyze, visualize, and integrate genome-wide data in order to construct GRNs for beginners of bioinformatics. The practical guide presented here is ready to apply to other similar ChIP-seq datasets to characterize GRNs of interest.

Genomic relations among 31 species of Mammillaria haworth (Cactaceae) using random amplified polymorphic DNA.

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Mattagajasingh, Ilwola; Mukherjee, Arup Kumar; Das, Premananda

2006-01-01

Thirty-one species of Mammillaria were selected to study the molecular phylogeny using random amplified polymorphic DNA (RAPD) markers. High amount of mucilage (gelling polysaccharides) present in Mammillaria was a major obstacle in isolating good quality genomic DNA. The CTAB (cetyl trimethyl ammonium bromide) method was modified to obtain good quality genomic DNA. Twenty-two random decamer primers resulted in 621 bands, all of which were polymorphic. The similarity matrix value varied from 0.109 to 0.622 indicating wide variability among the studied species. The dendrogram obtained from the unweighted pair group method using arithmetic averages (UPGMA) analysis revealed that some of the species did not follow the conventional classification. The present work shows the usefulness of RAPD markers for genetic characterization to establish phylogenetic relations among Mammillaria species.

Genome-wide analysis of potential cross-reactive endogenous allergens in rice (Oryza sativa L.

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Fang Chao Zhu

2015-01-01

Full Text Available The proteins in the food are the source of common allergic components to certain patients. Current lists of plant endogenous allergens were based on the medical/clinical reports as well as laboratory results. Plant genome sequences made it possible to predict and characterize the genome-wide of putative endogenous allergens in rice (Oryza sativa L.. In this work, we identified and characterized 122 candidate rice allergens including the 22 allergens in present databases. Conserved domain analysis also revealed 37 domains among rice allergens including one novel domain (histidine kinase-, DNA gyrase B-, and HSP90-like ATPase, PF13589 adding to the allergen protein database. Phylogenetic analysis of the allergens revealed the diversity among the Prolamin superfamily and DnaK protein family, respectively. Additionally, some allergens proteins clustered on the rice chromosome might suggest the molecular function during the evolution.

A direct detection of Escherichia coli genomic DNA using gold nanoprobes

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Padmavathy

2012-02-01

Full Text Available Abstract Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to

Exploring repetitive DNA landscapes using REPCLASS, a tool that automates the classification of transposable elements in eukaryotic genomes.

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Feschotte, Cédric; Keswani, Umeshkumar; Ranganathan, Nirmal; Guibotsy, Marcel L; Levine, David

2009-07-23

Eukaryotic genomes contain large amount of repetitive DNA, most of which is derived from transposable elements (TEs). Progress has been made to develop computational tools for ab initio identification of repeat families, but there is an urgent need to develop tools to automate the annotation of TEs in genome sequences. Here we introduce REPCLASS, a tool that automates the classification of TE sequences. Using control repeat libraries, we show that the program can classify accurately virtually any known TE types. Combining REPCLASS to ab initio repeat finding in the genomes of Caenorhabditis elegans and Drosophila melanogaster allowed us to recover the contrasting TE landscape characteristic of these species. Unexpectedly, REPCLASS also uncovered several novel TE families in both genomes, augmenting the TE repertoire of these model species. When applied to the genomes of distant Caenorhabditis and Drosophila species, the approach revealed a remarkable conservation of TE composition profile within each genus, despite substantial interspecific covariations in genome size and in the number of TEs and TE families. Lastly, we applied REPCLASS to analyze 10 fungal genomes from a wide taxonomic range, most of which have not been analyzed for TE content previously. The results showed that TE diversity varies widely across the fungi "kingdom" and appears to positively correlate with genome size, in particular for DNA transposons. Together, these data validate REPCLASS as a powerful tool to explore the repetitive DNA landscapes of eukaryotes and to shed light onto the evolutionary forces shaping TE diversity and genome architecture.

Genome-wide DNA methylation alterations of Alternanthera philoxeroides in natural and manipulated habitats: implications for epigenetic regulation of rapid responses to environmental fluctuation and phenotypic variation.

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Gao, Lexuan; Geng, Yupeng; Li, Bo; Chen, Jiakuan; Yang, Ji

2010-11-01

Alternanthera philoxeroides (alligator weed) is an invasive weed that can colonize both aquatic and terrestrial habitats. Individuals growing in different habitats exhibit extensive phenotypic variation but little genetic differentiation in its introduced range. The mechanisms underpinning the wide range of phenotypic variation and rapid adaptation to novel and changing environments remain uncharacterized. In this study, we examined the epigenetic variation and its correlation with phenotypic variation in plants exposed to natural and manipulated environmental variability. Genome-wide methylation profiling using methylation-sensitive amplified fragment length polymorphism (MSAP) revealed considerable DNA methylation polymorphisms within and between natural populations. Plants of different source populations not only underwent significant morphological changes in common garden environments, but also underwent a genome-wide epigenetic reprogramming in response to different treatments. Methylation alterations associated with response to different water availability were detected in 78.2% (169/216) of common garden induced polymorphic sites, demonstrating the environmental sensitivity and flexibility of the epigenetic regulatory system. These data provide evidence of the correlation between epigenetic reprogramming and the reversible phenotypic response of alligator weed to particular environmental factors. © 2010 Blackwell Publishing Ltd.

DNA Repair and Genome Maintenance in Bacillus subtilis

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Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

2012-01-01

Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity.

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Mao, Peng; Brown, Alexander J; Malc, Ewa P; Mieczkowski, Piotr A; Smerdon, Michael J; Roberts, Steven A; Wyrick, John J

2017-10-01

DNA base damage is an important contributor to genome instability, but how the formation and repair of these lesions is affected by the genomic landscape and contributes to mutagenesis is unknown. Here, we describe genome-wide maps of DNA base damage, repair, and mutagenesis at single nucleotide resolution in yeast treated with the alkylating agent methyl methanesulfonate (MMS). Analysis of these maps revealed that base excision repair (BER) of alkylation damage is significantly modulated by chromatin, with faster repair in nucleosome-depleted regions, and slower repair and higher mutation density within strongly positioned nucleosomes. Both the translational and rotational settings of lesions within nucleosomes significantly influence BER efficiency; moreover, this effect is asymmetric relative to the nucleosome dyad axis and is regulated by histone modifications. Our data also indicate that MMS-induced mutations at adenine nucleotides are significantly enriched on the nontranscribed strand (NTS) of yeast genes, particularly in BER-deficient strains, due to higher damage formation on the NTS and transcription-coupled repair of the transcribed strand (TS). These findings reveal the influence of chromatin on repair and mutagenesis of base lesions on a genome-wide scale and suggest a novel mechanism for transcription-associated mutation asymmetry, which is frequently observed in human cancers. © 2017 Mao et al.; Published by Cold Spring Harbor Laboratory Press.

Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq.

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Marchal, Claire; Sasaki, Takayo; Vera, Daniel; Wilson, Korey; Sima, Jiao; Rivera-Mulia, Juan Carlos; Trevilla-García, Claudia; Nogues, Coralin; Nafie, Ebtesam; Gilbert, David M

2018-05-01

This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.

Direct detection of chicken genomic DNA for gender determination by thymine-DNA glycosylase.

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Porat, N; Bogdanov, K; Danielli, A; Arie, A; Samina, I; Hadani, A

2011-02-01

1. Birds, especially nestlings, are generally difficult to sex by morphology and early detection of chick gender in ovo in the hatchery would facilitate removal of unwanted chicks and diminish welfare objections regarding culling after hatch. 2. We describe a method to determine chicken gender without the need for PCR via use of Thymine-DNA Glycosylase (TDG). TDG restores thymine (T)/guanine (G) mismatches to cytosine (C)/G. We show here, that like DNA Polymerase, TDG can recognise, bind and function on a primer hybridised to chicken genomic DNA. 3. The primer contained a T to mismatch a G in a chicken genomic template and the T/G was cleaved with high fidelity by TDG. Thus, the chicken genomic DNA can be identified without PCR amplification via direct and linear detection. Sensitivity was increased using gender specific sequences from the chicken genome. 4. Currently, these are laboratory results, but we anticipate that further development will allow this method to be used in non-laboratory settings, where PCR cannot be employed.

High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot

OpenAIRE

Aberg, Karolina A.; Xie, Lin Y.; Nerella, Srilaxmi; Copeland, William E.; Costello, E. Jane; van den Oord, Edwin J.C.G.

2013-01-01

The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study,...

Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

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Oates, A C; Wollberg, P; Achen, M G; Wilks, A F

1998-08-28

The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

Genome-wide indel markers shared by diverse Asian rice cultivars compared to Japanese rice cultivar ?Koshihikari?

OpenAIRE

Yonemaru, Jun-ichi; Choi, Sun Hee; Sakai, Hiroaki; Ando, Tsuyu; Shomura, Ayahiko; Yano, Masahiro; Wu, Jianzhong; Fukuoka, Shuichi

2015-01-01

Insertion-deletion (indel) polymorphisms, such as simple sequence repeats, have been widely used as DNA markers to identify QTLs and genes and to facilitate rice breeding. Recently, next-generation sequencing has produced deep sequences that allow genome-wide detection of indels. These polymorphisms can potentially be used to develop high-accuracy polymerase chain reaction (PCR)-based markers. Here, re-sequencing of 5 indica, 2 aus, and 3 tropical japonica cultivars and Japanese elite cultiva...

Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation

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Takashi Kubota

2015-08-01

Full Text Available The sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication.

Genome-wide single nucleotide polymorphisms (SNPs) for a model invasive ascidian Botryllus schlosseri.

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Gao, Yangchun; Li, Shiguo; Zhan, Aibin

2018-04-01

Invasive species cause huge damages to ecology, environment and economy globally. The comprehensive understanding of invasion mechanisms, particularly genetic bases of micro-evolutionary processes responsible for invasion success, is essential for reducing potential damages caused by invasive species. The golden star tunicate, Botryllus schlosseri, has become a model species in invasion biology, mainly owing to its high invasiveness nature and small well-sequenced genome. However, the genome-wide genetic markers have not been well developed in this highly invasive species, thus limiting the comprehensive understanding of genetic mechanisms of invasion success. Using restriction site-associated DNA (RAD) tag sequencing, here we developed a high-quality resource of 14,119 out of 158,821 SNPs for B. schlosseri. These SNPs were relatively evenly distributed at each chromosome. SNP annotations showed that the majority of SNPs (63.20%) were located at intergenic regions, and 21.51% and 14.58% were located at introns and exons, respectively. In addition, the potential use of the developed SNPs for population genomics studies was primarily assessed, such as the estimate of observed heterozygosity (H O ), expected heterozygosity (H E ), nucleotide diversity (π), Wright's inbreeding coefficient (F IS ) and effective population size (Ne). Our developed SNP resource would provide future studies the genome-wide genetic markers for genetic and genomic investigations, such as genetic bases of micro-evolutionary processes responsible for invasion success.

A novel statistic for genome-wide interaction analysis.

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Xuesen Wu

2010-09-01

Full Text Available Although great progress in genome-wide association studies (GWAS has been made, the significant SNP associations identified by GWAS account for only a few percent of the genetic variance, leading many to question where and how we can find the missing heritability. There is increasing interest in genome-wide interaction analysis as a possible source of finding heritability unexplained by current GWAS. However, the existing statistics for testing interaction have low power for genome-wide interaction analysis. To meet challenges raised by genome-wide interactional analysis, we have developed a novel statistic for testing interaction between two loci (either linked or unlinked. The null distribution and the type I error rates of the new statistic for testing interaction are validated using simulations. Extensive power studies show that the developed statistic has much higher power to detect interaction than classical logistic regression. The results identified 44 and 211 pairs of SNPs showing significant evidence of interactions with FDR<0.001 and 0.001genome-wide interaction analysis is a valuable tool for finding remaining missing heritability unexplained by the current GWAS, and the developed novel statistic is able to search significant interaction between SNPs across the genome. Real data analysis showed that the results of genome-wide interaction analysis can be replicated in two independent studies.

Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi.

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Serna-Domínguez, María G; Andrade-Michel, Gilda Y; Arredondo-Bernal, Hugo C; Gallou, Adrien

2018-03-27

Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/β-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Genomic DNA extraction from sapwood of Pinus roxburghii for ...

African Journals Online (AJOL)

Ashish

2013-02-22

Feb 22, 2013 ... A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, .... DNA as a template. PCR was performed on a thermal cycler. (Biorad, Mycycler) incorporating 10 ng genomic DNA to a 25 µl reaction mix containing 1X Taq buffer, 3 mM MgCl2, 0.2 mM each of dNTPs ...

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

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Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

2016-01-01

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

The pathological consequences of impaired genome integrity in humans; disorders of the DNA replication machinery.

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O'Driscoll, Mark

2017-01-01

Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Rapid DNA extraction of bacterial genome using laundry detergents ...

African Journals Online (AJOL)

Genomic DNA extraction from bacterial cells involves processes normally performed in most biological laboratories. Therefore, various methods have been offered, manually and kit, but these methods may be time consuming and costly. In this paper, genomic DNA extraction of Pseudomonas aeruginosa was investigated ...

Rapid DNA extraction of bacterial genome using laundry detergents ...

African Journals Online (AJOL)

Yomi

2012-01-03

Jan 3, 2012 ... Genomic DNA extraction from bacterial cells involves processes normally performed in most biological laboratories. Therefore, various methods have been offered, manually and kit, but these methods may be time consuming and costly. In this paper, genomic DNA extraction of Pseudomonas aeruginosa ...

Genome-Wide Approaches to Drosophila Heart Development

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Manfred Frasch

2016-05-01

Full Text Available The development of the dorsal vessel in Drosophila is one of the first systems in which key mechanisms regulating cardiogenesis have been defined in great detail at the genetic and molecular level. Due to evolutionary conservation, these findings have also provided major inputs into studies of cardiogenesis in vertebrates. Many of the major components that control Drosophila cardiogenesis were discovered based on candidate gene approaches and their functions were defined by employing the outstanding genetic tools and molecular techniques available in this system. More recently, approaches have been taken that aim to interrogate the entire genome in order to identify novel components and describe genomic features that are pertinent to the regulation of heart development. Apart from classical forward genetic screens, the availability of the thoroughly annotated Drosophila genome sequence made new genome-wide approaches possible, which include the generation of massive numbers of RNA interference (RNAi reagents that were used in forward genetic screens, as well as studies of the transcriptomes and proteomes of the developing heart under normal and experimentally manipulated conditions. Moreover, genome-wide chromatin immunoprecipitation experiments have been performed with the aim to define the full set of genomic binding sites of the major cardiogenic transcription factors, their relevant target genes, and a more complete picture of the regulatory network that drives cardiogenesis. This review will give an overview on these genome-wide approaches to Drosophila heart development and on computational analyses of the obtained information that ultimately aim to provide a description of this process at the systems level.

Genome-wide DNA binding pattern of the homeodomain transcription factor Sine oculis (So in the developing eye of Drosophila melanogaster

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Barbara Jusiak

2014-12-01

Full Text Available The eye of the fruit fly Drosophila melanogaster provides a highly tractable genetic model system for the study of animal development, and many genes that regulate Drosophila eye formation have homologs implicated in human development and disease. Among these is the homeobox gene sine oculis (so, which encodes a homeodomain transcription factor (TF that is both necessary for eye development and sufficient to reprogram a subset of cells outside the normal eye field toward an eye fate. We have performed a genome-wide analysis of So binding to DNA prepared from developing Drosophila eye tissue in order to identify candidate direct targets of So-mediated transcriptional regulation, as described in our recent article [20]. The data are available from NCBI Gene Expression Omnibus (GEO with the accession number GSE52943. Here we describe the methods, data analysis, and quality control of our So ChIP-seq dataset.

A genome-wide scan reveals important roles of DNA methylation in human longevity by regulating age-related disease genes.

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Fu-Hui Xiao

Full Text Available It is recognized that genetic factors contribute to human longevity. Besides the hypothesis of existence of longevity genes, another suggests that a lower frequency of risk alleles decreases the incidence of age-related diseases in the long-lived people. However, the latter finds no support from recent genetic studies. Considering the crucial role of epigenetic modification in gene regulation, we then hypothesize that suppressing disease-related genes in longevity individuals is likely achieved by epigenetic modification, e.g. DNA methylation. To test this hypothesis, we investigated the genome-wide methylation profile in 4 Chinese female centenarians and 4 middle-aged controls using methyl-DNA immunoprecipitation sequencing. 626 differentially methylated regions (DMRs were observed between both groups. Interestingly, genes with these DMRs were enriched in age-related diseases, including type-2 diabetes, cardiovascular disease, stroke and Alzheimer's disease. This pattern remains rather stable after including methylomes of two white individuals. Further analyses suggest that the observed DMRs likely have functional roles in regulating disease-associated gene expressions, with some genes [e.g. caspase 3 (CASP3] being down-regulated whereas the others [i.e. interleukin 1 receptor, type 2 (IL1R2] up-regulated. Therefore, our study suggests that suppressing the disease-related genes via epigenetic modification is an important contributor to human longevity.

Genome-Wide Association of Copy Number Polymorphisms and Kidney Function.

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Man Li

Full Text Available Genome-wide association studies (GWAS using single nucleotide polymorphisms (SNPs have identified more than 50 loci associated with estimated glomerular filtration rate (eGFR, a measure of kidney function. However, significant SNPs account for a small proportion of eGFR variability. Other forms of genetic variation have not been comprehensively evaluated for association with eGFR. In this study, we assess whether changes in germline DNA copy number are associated with GFR estimated from serum creatinine, eGFRcrea. We used hidden Markov models (HMMs to identify copy number polymorphic regions (CNPs from high-throughput SNP arrays for 2,514 African (AA and 8,645 European ancestry (EA participants in the Atherosclerosis Risk in Communities (ARIC study. Separately for the EA and AA cohorts, we used Bayesian Gaussian mixture models to estimate copy number at regions identified by the HMM or previously reported in the HapMap Project. We identified 312 and 464 autosomal CNPs among individuals of EA and AA, respectively. Multivariate models adjusted for SNP-derived covariates of population structure identified one CNP in the EA cohort near genome-wide statistical significance (Bonferroni-adjusted p = 0.067 located on chromosome 5 (876-880kb. Overall, our findings suggest a limited role of CNPs in explaining eGFR variability.

Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe.

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Marques, Catarina A; Dickens, Nicholas J; Paape, Daniel; Campbell, Samantha J; McCulloch, Richard

2015-10-19

DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania. Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins. The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture.

Comparative chloroplast genomes of eleven Schima (Theaceae) species: Insights into DNA barcoding and phylogeny.

Science.gov (United States)

Yu, Xiang-Qin; Drew, Bryan T; Yang, Jun-Bo; Gao, Lian-Ming; Li, De-Zhu

2017-01-01

Schima is an ecologically and economically important woody genus in tea family (Theaceae). Unresolved species delimitations and phylogenetic relationships within Schima limit our understanding of the genus and hinder utilization of the genus for economic purposes. In the present study, we conducted comparative analysis among the complete chloroplast (cp) genomes of 11 Schima species. Our results indicate that Schima cp genomes possess a typical quadripartite structure, with conserved genomic structure and gene order. The size of the Schima cp genome is about 157 kilo base pairs (kb). They consistently encode 114 unique genes, including 80 protein-coding genes, 30 tRNAs, and 4 rRNAs, with 17 duplicated in the inverted repeat (IR). These cp genomes are highly conserved and do not show obvious expansion or contraction of the IR region. The percent variability of the 68 coding and 93 noncoding (>150 bp) fragments is consistently less than 3%. The seven most widely touted DNA barcode regions as well as one promising barcode candidate showed low sequence divergence. Eight mutational hotspots were identified from the 11 cp genomes. These hotspots may potentially be useful as specific DNA barcodes for species identification of Schima. The 58 cpSSR loci reported here are complementary to the microsatellite markers identified from the nuclear genome, and will be leveraged for further population-level studies. Phylogenetic relationships among the 11 Schima species were resolved with strong support based on the cp genome data set, which corresponds well with the species distribution pattern. The data presented here will serve as a foundation to facilitate species identification, DNA barcoding and phylogenetic reconstructions for future exploration of Schima.

Genome-Wide Mutagenesis in Borrelia burgdorferi.

Science.gov (United States)

Lin, Tao; Gao, Lihui

2018-01-01

Signature-tagged mutagenesis (STM) is a functional genomics approach to identify bacterial virulence determinants and virulence factors by simultaneously screening multiple mutants in a single host animal, and has been utilized extensively for the study of bacterial pathogenesis, host-pathogen interactions, and spirochete and tick biology. The signature-tagged transposon mutagenesis has been developed to investigate virulence determinants and pathogenesis of Borrelia burgdorferi. Mutants in genes important in virulence are identified by negative selection in which the mutants fail to colonize or disseminate in the animal host and tick vector. STM procedure combined with Luminex Flex ® Map™ technology and next-generation sequencing (e.g., Tn-seq) are the powerful high-throughput tools for the determination of Borrelia burgdorferi virulence determinants. The assessment of multiple tissue sites and two DNA resources at two different time points using Luminex Flex ® Map™ technology provides a robust data set. B. burgdorferi transposon mutant screening indicates that a high proportion of genes are the novel virulence determinants that are required for mouse and tick infection. In this protocol, an effective signature-tagged Himar1-based transposon suicide vector was developed and used to generate a sequence-defined library of nearly 4800 mutants in the infectious B. burgdorferi B31 clone. In STM, signature-tagged suicide vectors are constructed by inserting unique DNA sequences (tags) into the transposable elements. The signature-tagged transposon mutants are generated when transposon suicide vectors are transformed into an infectious B. burgdorferi clone, and the transposable element is transposed into the 5'-TA-3' sequence in the B. burgdorferi genome with the signature tag. The transposon library is created and consists of many sub-libraries, each sub-library has several hundreds of mutants with same tags. A group of mice or ticks are infected with a mixed

CMS: a web-based system for visualization and analysis of genome-wide methylation data of human cancers.

Science.gov (United States)

Gu, Fei; Doderer, Mark S; Huang, Yi-Wen; Roa, Juan C; Goodfellow, Paul J; Kizer, E Lynette; Huang, Tim H M; Chen, Yidong

2013-01-01

DNA methylation of promoter CpG islands is associated with gene suppression, and its unique genome-wide profiles have been linked to tumor progression. Coupled with high-throughput sequencing technologies, it can now efficiently determine genome-wide methylation profiles in cancer cells. Also, experimental and computational technologies make it possible to find the functional relationship between cancer-specific methylation patterns and their clinicopathological parameters. Cancer methylome system (CMS) is a web-based database application designed for the visualization, comparison and statistical analysis of human cancer-specific DNA methylation. Methylation intensities were obtained from MBDCap-sequencing, pre-processed and stored in the database. 191 patient samples (169 tumor and 22 normal specimen) and 41 breast cancer cell-lines are deposited in the database, comprising about 6.6 billion uniquely mapped sequence reads. This provides comprehensive and genome-wide epigenetic portraits of human breast cancer and endometrial cancer to date. Two views are proposed for users to better understand methylation structure at the genomic level or systemic methylation alteration at the gene level. In addition, a variety of annotation tracks are provided to cover genomic information. CMS includes important analytic functions for interpretation of methylation data, such as the detection of differentially methylated regions, statistical calculation of global methylation intensities, multiple gene sets of biologically significant categories, interactivity with UCSC via custom-track data. We also present examples of discoveries utilizing the framework. CMS provides visualization and analytic functions for cancer methylome datasets. A comprehensive collection of datasets, a variety of embedded analytic functions and extensive applications with biological and translational significance make this system powerful and unique in cancer methylation research. CMS is freely accessible

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas.

Science.gov (United States)

Mohapatra, Gayatry; Engler, David A; Starbuck, Kristen D; Kim, James C; Bernay, Derek C; Scangas, George A; Rousseau, Audrey; Batchelor, Tracy T; Betensky, Rebecca A; Louis, David N

2011-04-01

Array comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA). Because diffuse malignant gliomas are often sampled by small biopsies, formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis; FFPE tissues are also needed to study the intratumoral heterogeneity that characterizes these neoplasms. In this paper, we present a combination of evaluations and technical advances that provide strong support for the ready use of oligonucleotide aCGH on FFPE diffuse gliomas. We first compared aCGH using bacterial artificial chromosome (BAC) arrays in 45 paired frozen and FFPE gliomas, and demonstrate a high concordance rate between FFPE and frozen DNA in an individual clone-level analysis of sensitivity and specificity, assuring that under certain array conditions, frozen and FFPE DNA can perform nearly identically. However, because oligonucleotide arrays offer advantages to BAC arrays in genomic coverage and practical availability, we next developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. To demonstrate utility in FFPE tissues, we applied this approach to biphasic anaplastic oligoastrocytomas and demonstrate CNA differences between DNA obtained from the two components. Therefore, BAC and oligonucleotide aCGH can be sensitive and specific tools for detecting CNAs in FFPE DNA, and novel labeling techniques enable the routine use of oligonucleotide arrays for FFPE DNA. In combination, these advances should facilitate genome-wide analysis of rare, small and/or histologically heterogeneous gliomas from FFPE tissues.

A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

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Farkhondeh Saba

2017-01-01

Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

Directory of Open Access Journals (Sweden)

Farkhondeh Saba

2016-09-01

Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

Genomic signal processing for DNA sequence clustering.

Science.gov (United States)

Mendizabal-Ruiz, Gerardo; Román-Godínez, Israel; Torres-Ramos, Sulema; Salido-Ruiz, Ricardo A; Vélez-Pérez, Hugo; Morales, J Alejandro

2018-01-01

Genomic signal processing (GSP) methods which convert DNA data to numerical values have recently been proposed, which would offer the opportunity of employing existing digital signal processing methods for genomic data. One of the most used methods for exploring data is cluster analysis which refers to the unsupervised classification of patterns in data. In this paper, we propose a novel approach for performing cluster analysis of DNA sequences that is based on the use of GSP methods and the K-means algorithm. We also propose a visualization method that facilitates the easy inspection and analysis of the results and possible hidden behaviors. Our results support the feasibility of employing the proposed method to find and easily visualize interesting features of sets of DNA data.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

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Scott Cukras

Full Text Available Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

Science.gov (United States)

Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

2014-01-01

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

Genome-Wide Locations of Potential Epimutations Associated with Environmentally Induced Epigenetic Transgenerational Inheritance of Disease Using a Sequential Machine Learning Prediction Approach.

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M Muksitul Haque

Full Text Available Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation involves germline transmitted epimutations. The primary epimutations identified involve altered differential DNA methylation regions (DMRs. Different environmental toxicants have been shown to promote exposure (i.e., toxicant specific signatures of germline epimutations. Analysis of genomic features associated with these epimutations identified low-density CpG regions (<3 CpG / 100bp termed CpG deserts and a number of unique DNA sequence motifs. The rat genome was annotated for these and additional relevant features. The objective of the current study was to use a machine learning computational approach to predict all potential epimutations in the genome. A number of previously identified sperm epimutations were used as training sets. A novel machine learning approach using a sequential combination of Active Learning and Imbalance Class Learner analysis was developed. The transgenerational sperm epimutation analysis identified approximately 50K individual sites with a 1 kb mean size and 3,233 regions that had a minimum of three adjacent sites with a mean size of 3.5 kb. A select number of the most relevant genomic features were identified with the low density CpG deserts being a critical genomic feature of the features selected. A similar independent analysis with transgenerational somatic cell epimutation training sets identified a smaller number of 1,503 regions of genome-wide predicted sites and differences in genomic feature contributions. The predicted genome-wide germline (sperm epimutations were found to be distinct from the predicted somatic cell epimutations. Validation of the genome-wide germline predicted sites used two recently identified transgenerational sperm epimutation signature sets from the pesticides dichlorodiphenyltrichloroethane (DDT and methoxychlor (MXC exposure lineage F3 generation. Analysis of this positive validation

Genome-wide association study of multiplex schizophrenia pedigrees

DEFF Research Database (Denmark)

Levinson, Douglas F; Shi, Jianxin; Wang, Kai

2012-01-01

The authors used a genome-wide association study (GWAS) of multiply affected families to investigate the association of schizophrenia to common single-nucleotide polymorphisms (SNPs) and rare copy number variants (CNVs).......The authors used a genome-wide association study (GWAS) of multiply affected families to investigate the association of schizophrenia to common single-nucleotide polymorphisms (SNPs) and rare copy number variants (CNVs)....

Reciprocal Regulation between DNA-PKcs and Snail1 Conferring Genomic Instability

International Nuclear Information System (INIS)

Seo, Haeng Ran; Lee, Hae June; Jin, Yeung Bae; Bae, Sang Woo; Lee, Yun Sil; Kim, Nam Hee; Kim, Hyun Sil; Nam, Hyung Wook; Yook, Jong In

2010-01-01

Although the roles of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) involving non-homologous end joining (NHEJ) of DNA repair are well recognized, the biological mechanisms and regulators by which DNA-PKcs regulate genomic instability are not clearly defined. We show herein that DNA-PKcs activity resulting from DNA damage caused by ionizing radiation (IR) phosphorylates Snail1 at serine 100, which results in increased Snail1 expression and its function by inhibition of GSK-3-mediated phosphorylation. Furthermore, Snail1 phosphorylated at serine 100 can reciprocally inhibit kinase activity of DNA-PKcs, resulting in an inhibition to recruit DNA-PKcs or Ku70/80 to a DNA double-strand break site, and ultimately inhibition of DNA repair activity. The impairment of repair activity by a direct interaction between Snail1 and DNA-PKcs increases the resistance to DNA damaging agents, such as IR, and genomic instability. Our findings provide a novel cellular mechanism for induction of genomic instability by reciprocal regulation of DNA-PKcs and Snail1

Methylome-wide Sequencing Detects DNA Hypermethylation Distinguishing Indolent from Aggressive Prostate Cancer

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Jeffrey M. Bhasin

2015-12-01

Full Text Available A critical need in understanding the biology of prostate cancer is characterizing the molecular differences between indolent and aggressive cases. Because DNA methylation can capture the regulatory state of tumors, we analyzed differential methylation patterns genome-wide among benign prostatic tissue and low-grade and high-grade prostate cancer and found extensive, focal hypermethylation regions unique to high-grade disease. These hypermethylation regions occurred not only in the promoters of genes but also in gene bodies and at intergenic regions that are enriched for DNA-protein binding sites. Integration with existing RNA-sequencing (RNA-seq and survival data revealed regions where DNA methylation correlates with reduced gene expression associated with poor outcome. Regions specific to aggressive disease are proximal to genes with distinct functions from regions shared by indolent and aggressive disease. Our compendium of methylation changes reveals crucial molecular distinctions between indolent and aggressive prostate cancer.

Genome-wide association study of susceptibility loci for breast cancer in Sardinian population.

Science.gov (United States)

Palomba, Grazia; Loi, Angela; Porcu, Eleonora; Cossu, Antonio; Zara, Ilenia; Budroni, Mario; Dei, Mariano; Lai, Sandra; Mulas, Antonella; Olmeo, Nina; Ionta, Maria Teresa; Atzori, Francesco; Cuccuru, Gianmauro; Pitzalis, Maristella; Zoledziewska, Magdalena; Olla, Nazario; Lovicu, Mario; Pisano, Marina; Abecasis, Gonçalo R; Uda, Manuela; Tanda, Francesco; Michailidou, Kyriaki; Easton, Douglas F; Chanock, Stephen J; Hoover, Robert N; Hunter, David J; Schlessinger, David; Sanna, Serena; Crisponi, Laura; Palmieri, Giuseppe

2015-05-10

Despite progress in identifying genes associated with breast cancer, many more risk loci exist. Genome-wide association analyses in genetically-homogeneous populations, such as that of Sardinia (Italy), could represent an additional approach to detect low penetrance alleles. We performed a genome-wide association study comparing 1431 Sardinian patients with non-familial, BRCA1/2-mutation-negative breast cancer to 2171 healthy Sardinian blood donors. DNA was genotyped using GeneChip Human Mapping 500 K Arrays or Genome-Wide Human SNP Arrays 6.0. To increase genomic coverage, genotypes of additional SNPs were imputed using data from HapMap Phase II. After quality control filtering of genotype data, 1367 cases (9 men) and 1658 controls (1156 men) were analyzed on a total of 2,067,645 SNPs. Overall, 33 genomic regions (67 candidate SNPs) were associated with breast cancer risk at the p <  0(-6) level. Twenty of these regions contained defined genes, including one already associated with breast cancer risk: TOX3. With a lower threshold for preliminary significance to p < 10(-5), we identified 11 additional SNPs in FGFR2, a well-established breast cancer-associated gene. Ten candidate SNPs were selected, excluding those already associated with breast cancer, for technical validation as well as replication in 1668 samples from the same population. Only SNP rs345299, located in intron 1 of VAV3, remained suggestively associated (p-value, 1.16 x 10(-5)), but it did not associate with breast cancer risk in pooled data from two large, mixed-population cohorts. This study indicated the role of TOX3 and FGFR2 as breast cancer susceptibility genes in BRCA1/2-wild-type breast cancer patients from Sardinian population.

Genome-wide association study of susceptibility loci for breast cancer in Sardinian population

International Nuclear Information System (INIS)

Palomba, Grazia; Loi, Angela; Porcu, Eleonora; Cossu, Antonio; Zara, Ilenia

2015-01-01

Despite progress in identifying genes associated with breast cancer, many more risk loci exist. Genome-wide association analyses in genetically-homogeneous populations, such as that of Sardinia (Italy), could represent an additional approach to detect low penetrance alleles. We performed a genome-wide association study comparing 1431 Sardinian patients with non-familial, BRCA1/2-mutation-negative breast cancer to 2171 healthy Sardinian blood donors. DNA was genotyped using GeneChip Human Mapping 500 K Arrays or Genome-Wide Human SNP Arrays 6.0. To increase genomic coverage, genotypes of additional SNPs were imputed using data from HapMap Phase II. After quality control filtering of genotype data, 1367 cases (9 men) and 1658 controls (1156 men) were analyzed on a total of 2,067,645 SNPs. Overall, 33 genomic regions (67 candidate SNPs) were associated with breast cancer risk at the p < 10 −6 level. Twenty of these regions contained defined genes, including one already associated with breast cancer risk: TOX3. With a lower threshold for preliminary significance to p < 10 −5 , we identified 11 additional SNPs in FGFR2, a well-established breast cancer-associated gene. Ten candidate SNPs were selected, excluding those already associated with breast cancer, for technical validation as well as replication in 1668 samples from the same population. Only SNP rs345299, located in intron 1 of VAV3, remained suggestively associated (p-value, 1.16x10 −5 ), but it did not associate with breast cancer risk in pooled data from two large, mixed-population cohorts. This study indicated the role of TOX3 and FGFR2 as breast cancer susceptibility genes in BRCA1/2-wild-type breast cancer patients from Sardinian population. The online version of this article (doi:10.1186/s12885-015-1392-9) contains supplementary material, which is available to authorized users

Genome-wide characterization of microsatelittes and marker development in the carcinogenic liver fluke Clonorchis sinensis

Science.gov (United States)

Nguyen, Thao T.B.; Arimatsu, Yuji; Hong, Sung-Jong; Brindley, Paul J.; Blair, David; Laha, Thewarach; Sripa, Banchob

2015-01-01

Clonorchis sinensis is an important carcinogenic human liver fluke endemic in East and Southeast Asia. There are several conventional molecular markers have been used for identification and genetic diversity, however, no information about microsatellites of this liver fluke published so far. We here report microsatellite characterization and marker development for genetic diversity study in C. sinensis using genome-wide bioinformatics approach. Based on our search criteria, a total of 256,990 microsatellites (≥ 12 base pairs) were identified from genome database of C. sinensis with hexa-nucleotide motif being the most abundant (51%) followed by penta-nucleotide (18.3%) and tri-nucleotide (12.7%). The tetra-nucleotide, di-nucleotide and mononucleotide motifs accounted for 9.75 %, 7.63% and 0.14%, respectively. The total length of all microsatellites accounts for 0. 72 % of 547 Mb of the whole genome size and the frequency of microsatellites were found to be one microsatellite in every 2.13 kb of DNA. For the di-, tri, and tetra-nucleotide, the repeat numbers redundant are six (28%), four (45%) and three (76%), respectively. The ATC repeat is the most abundant microsatellites followed by AT, AAT and AC, respectively. Within 40 microsatellite loci developed, 24 microsatellite markers showed potential to differentiate between C. sinensis and O. viverrini. Seven out of 24 loci showed heterozygous with observed heterozygosity ranged from 0.467 to 1. Four-primer sets could amplify both C. sinensis and O. viverrini DNA with different sizes. This study provides basic information of C. sinensis microsatellites and the genome-wide markers developed may be a useful tool for genetic study of C. sinensis. PMID:25782682

Genome-wide characterization of microsatellites and marker development in the carcinogenic liver fluke Clonorchis sinensis.

Science.gov (United States)

Nguyen, Thao T B; Arimatsu, Yuji; Hong, Sung-Jong; Brindley, Paul J; Blair, David; Laha, Thewarach; Sripa, Banchob

2015-06-01

Clonorchis sinensis is an important carcinogenic human liver fluke endemic in East and Southeast Asia. There are several conventional molecular markers that have been used for identification and genetic diversity; however, no information about microsatellites of this liver fluke is published so far. We here report microsatellite characterization and marker development for a genetic diversity study in C. sinensis, using a genome-wide bioinformatics approach. Based on our search criteria, a total of 256,990 microsatellites (≥12 base pairs) were identified from a genome database of C. sinensis, with hexanucleotide motif being the most abundant (51%) followed by pentanucleotide (18.3%) and trinucleotide (12.7%). The tetranucleotide, dinucleotide, and mononucleotide motifs accounted for 9.75, 7.63, and 0.14%, respectively. The total length of all microsatellites accounts for 0. 72% of 547 Mb of the whole genome size, and the frequency of microsatellites was found to be one microsatellite in every 2.13 kb of DNA. For the di-, tri-, and tetranucleotide, the repeat numbers redundant are six (28%), four (45%), and three (76%), respectively. The ATC repeat is the most abundant microsatellites followed by AT, AAT, and AC, respectively. Within 40 microsatellite loci developed, 24 microsatellite markers showed potential to differentiate between C. sinensis and Opisthorchis viverrini. Seven out of 24 loci showed to be heterozygous with observed heterozygosity that ranged from 0.467 to 1. Four primer sets could amplify both C. sinensis and O. viverrini DNA with different sizes. This study provides basic information of C. sinensis microsatellites, and the genome-wide markers developed may be a useful tool for the genetic study of C. sinensis.

Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide-DNA Complexes.

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Yoshizumi, Takeshi; Oikawa, Kazusato; Chuah, Jo-Ann; Kodama, Yutaka; Numata, Keiji

2018-05-14

Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

A genome-wide association study by ImmunoChip reveals potential modifiers in myelodysplastic syndromes.

Science.gov (United States)

Danjou, Fabrice; Fozza, Claudio; Zoledziewska, Magdalena; Mulas, Antonella; Corda, Giovanna; Contini, Salvatore; Dore, Fausto; Galleu, Antonio; Di Tucci, Anna Angela; Caocci, Giovanni; Gaviano, Eleonora; Latte, Giancarlo; Gabbas, Attilio; Casula, Paolo; Delogu, Lucia Gemma; La Nasa, Giorgio; Angelucci, Emanuele; Cucca, Francesco; Longinotti, Maurizio

2016-11-01

Because different findings suggest that an immune dysregulation plays a role in the pathogenesis of myelodysplastic syndrome (MDS), we analyzed a large cohort of patients from a homogeneous Sardinian population using ImmunoChip, a genotyping array exploring 147,954 single-nucleotide polymorphisms (SNPs) localized in genomic regions displaying some degree of association with immune-mediated diseases or pathways. The population studied included 133 cases and 3,894 controls, and a total of 153,978 autosomal markers and 971 non-autosomal markers were genotyped. After association analysis, only one variant passed the genome-wide significance threshold: rs71325459 (p = 1.16 × 10 -12 ), which is situated on chromosome 20. The variant is in high linkage disequilibrium with rs35640778, an untested missense variant situated in the RTEL1 gene, an interesting candidate that encodes for an ATP-dependent DNA helicase implicated in telomere-length regulation, DNA repair, and maintenance of genomic stability. The second most associated signal is composed of five variants that fall slightly below the genome-wide significance threshold but point out another interesting gene candidate. These SNPs, with p values between 2.53 × 10 -6 and 3.34 × 10 -6 , are situated in the methylene tetrahydrofolate reductase (MTHFR) gene. The most associated of these variants, rs1537514, presents an increased frequency of the derived C allele in cases, with 11.4% versus 4.4% in controls. MTHFR is the rate-limiting enzyme in the methyl cycle and genetic variations in this gene have been strongly associated with the risk of neoplastic diseases. The current understanding of the MDS biology, which is based on the hypothesis of the sequential development of multiple subclonal molecular lesions, fits very well with the demonstration of a possible role for RTEL1 and MTHFR gene polymorphisms, both of which are related to a variable risk of genomic instability. Copyright © 2016 ISEH - International

Kidney Dysfunction in Adult Offspring Exposed In Utero to Type 1 Diabetes Is Associated with Alterations in Genome-Wide DNA Methylation.

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Jean-François Gautier

Full Text Available Fetal exposure to hyperglycemia impacts negatively kidney development and function.Our objective was to determine whether fetal exposure to moderate hyperglycemia is associated with epigenetic alterations in DNA methylation in peripheral blood cells and whether those alterations are related to impaired kidney function in adult offspring.Twenty nine adult, non-diabetic offspring of mothers with type 1 diabetes (T1D (case group were matched with 28 offspring of T1D fathers (control group for the study of their leukocyte genome-wide DNA methylation profile (27,578 CpG sites, Human Methylation 27 BeadChip, Illumina Infinium. In a subset of 19 cases and 18 controls, we assessed renal vascular development by measuring Glomerular Filtration Rate (GFR and Effective Renal Plasma Flow (ERPF at baseline and during vasodilatation produced by amino acid infusion.Globally, DNA was under-methylated in cases vs. controls. Among the 87 CpG sites differently methylated, 74 sites were less methylated and 13 sites more methylated in cases vs. controls. None of these CpG sites were located on a gene known to be directly involved in kidney development and/or function. However, the gene encoding DNA methyltransferase 1 (DNMT1--a key enzyme involved in gene expression during early development--was under-methylated in cases. The average methylation of the 74 under-methylated sites differently correlated with GFR in cases and controls.Alterations in methylation profile imprinted by the hyperglycemic milieu of T1D mothers during fetal development may impact kidney function in adult offspring. The involved pathways seem to be a nonspecific imprinting process rather than specific to kidney development or function.

Adiponectin Concentrations: A Genome-wide Association Study

OpenAIRE

Jee, Sun Ha; Sull, Jae Woong; Lee, Jong-Eun; Shin, Chol; Park, Jongkeun; Kimm, Heejin; Cho, Eun-Young; Shin, Eun-Soon; Yun, Ji Eun; Park, Ji Wan; Kim, Sang Yeun; Lee, Sun Ju; Jee, Eun Jung; Baik, Inkyung; Kao, Linda

2010-01-01

Adiponectin is associated with obesity and insulin resistance. To date, there has been no genome-wide association study (GWAS) of adiponectin levels in Asians. Here we present a GWAS of a cohort of Korean volunteers. A total of 4,001 subjects were genotyped by using a genome-wide marker panel in a two-stage design (979 subjects initially and 3,022 in a second stage). Another 2,304 subjects were used for follow-up replication studies with selected markers. In the discovery phase, the top SNP a...

Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae.

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Doudican, Nicole A; Song, Binwei; Shadel, Gerald S; Doetsch, Paul W

2005-06-01

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

Genome-Wide Mutational Signature of the Chemotherapeutic Agent Mitomycin C in Caenorhabditis elegans.

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Tam, Annie S; Chu, Jeffrey S C; Rose, Ann M

2015-11-12

Cancer therapy largely depends on chemotherapeutic agents that generate DNA lesions. However, our understanding of the nature of the resulting lesions as well as the mutational profiles of these chemotherapeutic agents is limited. Among these lesions, DNA interstrand crosslinks are among the more toxic types of DNA damage. Here, we have characterized the mutational spectrum of the commonly used DNA interstrand crosslinking agent mitomycin C (MMC). Using a combination of genetic mapping, whole genome sequencing, and genomic analysis, we have identified and confirmed several genomic lesions linked to MMC-induced DNA damage in Caenorhabditis elegans. Our data indicate that MMC predominantly causes deletions, with a 5'-CpG-3' sequence context prevalent in the deleted regions of DNA. Furthermore, we identified microhomology flanking the deletion junctions, indicative of DNA repair via nonhomologous end joining. Based on these results, we propose a general repair mechanism that is likely to be involved in the biological response to this highly toxic agent. In conclusion, the systematic study we have described provides insight into potential sequence specificity of MMC with DNA. Copyright © 2016 Tam et al.

Genome-Wide Mutational Signature of the Chemotherapeutic Agent Mitomycin C in Caenorhabditis elegans

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Annie S. Tam

2016-01-01

Full Text Available Cancer therapy largely depends on chemotherapeutic agents that generate DNA lesions. However, our understanding of the nature of the resulting lesions as well as the mutational profiles of these chemotherapeutic agents is limited. Among these lesions, DNA interstrand crosslinks are among the more toxic types of DNA damage. Here, we have characterized the mutational spectrum of the commonly used DNA interstrand crosslinking agent mitomycin C (MMC. Using a combination of genetic mapping, whole genome sequencing, and genomic analysis, we have identified and confirmed several genomic lesions linked to MMC-induced DNA damage in Caenorhabditis elegans. Our data indicate that MMC predominantly causes deletions, with a 5′-CpG-3′ sequence context prevalent in the deleted regions of DNA. Furthermore, we identified microhomology flanking the deletion junctions, indicative of DNA repair via nonhomologous end joining. Based on these results, we propose a general repair mechanism that is likely to be involved in the biological response to this highly toxic agent. In conclusion, the systematic study we have described provides insight into potential sequence specificity of MMC with DNA.

Genome-wide Studies of Mycolic Acid Bacteria: Computational Identification and Analysis of a Minimal Genome

KAUST Repository

Kamanu, Frederick Kinyua

2012-12-01

The mycolic acid bacteria are a distinct suprageneric group of asporogenous Grampositive, high GC-content bacteria, distinguished by the presence of mycolic acids in their cell envelope. They exhibit great diversity in their cell and morphology; although primarily non-pathogens, this group contains three major pathogens Mycobacterium leprae, Mycobacterium tuberculosis complex, and Corynebacterium diphtheria. Although the mycolic acid bacteria are a clearly defined group of bacteria, the taxonomic relationships between its constituent genera and species are less well defined. Two approaches were tested for their suitability in describing the taxonomy of the group. First, a Multilocus Sequence Typing (MLST) experiment was assessed and found to be superior to monophyletic (16S small ribosomal subunit) in delineating a total of 52 mycolic acid bacterial species. Phylogenetic inference was performed using the neighbor-joining method. To further refine phylogenetic analysis and to take advantage of the widespread availability of bacterial genome data, a computational framework that simulates DNA-DNA hybridisation was developed and validated using multiscale bootstrap resampling. The tool classifies microbial genomes based on whole genome DNA, and was deployed as a web-application using PHP and Javascript. It is accessible online at http://cbrc.kaust.edu.sa/dna_hybridization/ A third study was a computational and statistical methods in the identification and analysis of a putative minimal mycolic acid bacterial genome so as to better understand (1) the genomic requirements to encode a mycolic acid bacterial cell and (2) the role and type of genes and genetic elements that lead to the massive increase in genome size in environmental mycolic acid bacteria. Using a reciprocal comparison approach, a total of 690 orthologous gene clusters forming a putative minimal genome were identified across 24 mycolic acid bacterial species. In order to identify new potential drug

Genome-wide association study of response to cognitive–behavioural therapy in children with anxiety disorders

Science.gov (United States)

Coleman, Jonathan R. I.; Lester, Kathryn J.; Keers, Robert; Roberts, Susanna; Curtis, Charles; Arendt, Kristian; Bögels, Susan; Cooper, Peter; Creswell, Cathy; Dalgleish, Tim; Hartman, Catharina A.; Heiervang, Einar R.; Hötzel, Katrin; Hudson, Jennifer L.; In-Albon, Tina; Lavallee, Kristen; Lyneham, Heidi J.; Marin, Carla E.; Meiser-Stedman, Richard; Morris, Talia; Nauta, Maaike H.; Rapee, Ronald M.; Schneider, Silvia; Schneider, Sophie C.; Silverman, Wendy K.; Thastum, Mikael; Thirlwall, Kerstin; Waite, Polly; Wergeland, Gro Janne; Breen, Gerome; Eley, Thalia C.

2016-01-01

Background Anxiety disorders are common, and cognitive–behavioural therapy (CBT) is a first-line treatment. Candidate gene studies have suggested a genetic basis to treatment response, but findings have been inconsistent. Aims To perform the first genome-wide association study (GWAS) of psychological treatment response in children with anxiety disorders (n = 980). Method Presence and severity of anxiety was assessed using semi-structured interview at baseline, on completion of treatment (post-treatment), and 3 to 12 months after treatment completion (follow-up). DNA was genotyped using the Illumina Human Core Exome-12v1.0 array. Linear mixed models were used to test associations between genetic variants and response (change in symptom severity) immediately post-treatment and at 6-month follow-up. Results No variants passed a genome-wide significance threshold (P = 5 × 10−8) in either analysis. Four variants met criteria for suggestive significance (P<5 × 10−6) in association with response post-treatment, and three variants in the 6-month follow-up analysis. Conclusions This is the first genome-wide therapygenetic study. It suggests no common variants of very high effect underlie response to CBT. Future investigations should maximise power to detect single-variant and polygenic effects by using larger, more homogeneous cohorts. PMID:26989097

Next Generation DNA Sequencing and the Future of Genomic Medicine

OpenAIRE

Anderson, Matthew W.; Schrijver, Iris

2010-01-01

In the years since the first complete human genome sequence was reported, there has been a rapid development of technologies to facilitate high-throughput sequence analysis of DNA (termed “next-generation” sequencing). These novel approaches to DNA sequencing offer the promise of complete genomic analysis at a cost feasible for routine clinical diagnostics. However, the ability to more thoroughly interrogate genomic sequence raises a number of important issues with regard to result interpreta...

ATM signaling and genomic stability in response to DNA damage

International Nuclear Information System (INIS)

Lavin, Martin F.; Birrell, Geoff; Chen, Philip; Kozlov, Sergei; Scott, Shaun; Gueven, Nuri

2005-01-01

DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed

Genomic gigantism: DNA loss is slow in mountain grasshoppers.

Science.gov (United States)

Bensasson, D; Petrov, D A; Zhang, D X; Hartl, D L; Hewitt, G M

2001-02-01

Several studies have shown DNA loss to be inversely correlated with genome size in animals. These studies include a comparison between Drosophila and the cricket, Laupala, but there has been no assessment of DNA loss in insects with very large genomes. Podisma pedestris, the brown mountain grasshopper, has a genome over 100 times as large as that of Drosophila and 10 times as large as that of Laupala. We used 58 paralogous nuclear pseudogenes of mitochondrial origin to study the characteristics of insertion, deletion, and point substitution in P. pedestris and Italopodisma. In animals, these pseudogenes are "dead on arrival"; they are abundant in many different eukaryotes, and their mitochondrial origin simplifies the identification of point substitutions accumulated in nuclear pseudogene lineages. There appears to be a mononucleotide repeat within the 643-bp pseudogene sequence studied that acts as a strong hot spot for insertions or deletions (indels). Because the data for other insect species did not contain such an unusual region, hot spots were excluded from species comparisons. The rate of DNA loss relative to point substitution appears to be considerably and significantly lower in the grasshoppers studied than in Drosophila or Laupala. This suggests that the inverse correlation between genome size and the rate of DNA loss can be extended to comparisons between insects with large or gigantic genomes (i.e., Laupala and Podisma). The low rate of DNA loss implies that in grasshoppers, the accumulation of point mutations is a more potent force for obscuring ancient pseudogenes than their loss through indel accumulation, whereas the reverse is true for Drosophila. The main factor contributing to the difference in the rates of DNA loss estimated for grasshoppers, crickets, and Drosophila appears to be deletion size. Large deletions are relatively rare in Podisma and Italopodisma.

Genomic analysis of murine DNA-dependent protein kinase

International Nuclear Information System (INIS)

Fujimori, A.; Abe, M.

2003-01-01

Full text: The gene of catalytic subunit of DNA dependent protein kinase is responsible gene for SCID mice. The molecules play a critical role in non-homologous end joining including the V(D)J recombination. Contribution of the molecules to the difference of radiosensitivity and the susceptibility to cancer has been suggested. Here we show the entire nucleotide sequence of approximately 193 kbp and 84 kbp genomic regions encoding the entire DNA-PKcs gene in the mouse and chicken respectively. Retroposon was found in the intron 51 of mouse genomic DNA-PKcs gene but in human and chicken. Comparative analysis of these two species strongly suggested that only two genes, DNA-PKcs and MCM4, exist in the region of both species. Several conserved sequences and cis elements, however, were predicted. Recently, the orthologous region for the human DNA-PKcs locus was completed. The results of further comparative study will be discussed

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

Science.gov (United States)

Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

2006-02-01

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

The mitochondrial and plastid genomes of Volvox carteri: bloated molecules rich in repetitive DNA

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Lee Robert W

2009-03-01

Full Text Available Abstract Background The magnitude of noncoding DNA in organelle genomes can vary significantly; it is argued that much of this variation is attributable to the dissemination of selfish DNA. The results of a previous study indicate that the mitochondrial DNA (mtDNA of the green alga Volvox carteri abounds with palindromic repeats, which appear to be selfish elements. We became interested in the evolution and distribution of these repeats when, during a cursory exploration of the V. carteri nuclear DNA (nucDNA and plastid DNA (ptDNA sequences, we found palindromic repeats with similar structural features to those of the mtDNA. Upon this discovery, we decided to investigate the diversity and evolutionary implications of these palindromic elements by sequencing and characterizing large portions of mtDNA and ptDNA and then comparing these data to the V. carteri draft nuclear genome sequence. Results We sequenced 30 and 420 kilobases (kb of the mitochondrial and plastid genomes of V. carteri, respectively – resulting in partial assemblies of these genomes. The mitochondrial genome is the most bloated green-algal mtDNA observed to date: ~61% of the sequence is noncoding, most of which is comprised of short palindromic repeats spread throughout the intergenic and intronic regions. The plastid genome is the largest (>420 kb and most expanded (>80% noncoding ptDNA sequence yet discovered, with a myriad of palindromic repeats in the noncoding regions, which have a similar size and secondary structure to those of the mtDNA. We found that 15 kb (~0.01% of the nuclear genome are homologous to the palindromic elements of the mtDNA, and 50 kb (~0.05% are homologous to those of the ptDNA. Conclusion Selfish elements in the form of short palindromic repeats have propagated in the V. carteri mtDNA and ptDNA, resulting in the distension of these genomes. Copies of these same repeats are also found in a small fraction of the nucDNA, but appear to be inert in this

Genome-wide methylation analysis identifies a core set of hypermethylated genes in CIMP-H colorectal cancer.

Science.gov (United States)

McInnes, Tyler; Zou, Donghui; Rao, Dasari S; Munro, Francesca M; Phillips, Vicky L; McCall, John L; Black, Michael A; Reeve, Anthony E; Guilford, Parry J

2017-03-28

Aberrant DNA methylation profiles are a characteristic of all known cancer types, epitomized by the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). Hypermethylation has been observed at CpG islands throughout the genome, but it is unclear which factors determine whether an individual island becomes methylated in cancer. DNA methylation in CRC was analysed using the Illumina HumanMethylation450K array. Differentially methylated loci were identified using Significance Analysis of Microarrays (SAM) and the Wilcoxon Signed Rank (WSR) test. Unsupervised hierarchical clustering was used to identify methylation subtypes in CRC. In this study we characterized the DNA methylation profiles of 94 CRC tissues and their matched normal counterparts. Consistent with previous studies, unsupervized hierarchical clustering of genome-wide methylation data identified three subtypes within the tumour samples, designated CIMP-H, CIMP-L and CIMP-N, that showed high, low and very low methylation levels, respectively. Differential methylation between normal and tumour samples was analysed at the individual CpG level, and at the gene level. The distribution of hypermethylation in CIMP-N tumours showed high inter-tumour variability and appeared to be highly stochastic in nature, whereas CIMP-H tumours exhibited consistent hypermethylation at a subset of genes, in addition to a highly variable background of hypermethylated genes. EYA4, TFPI2 and TLX1 were hypermethylated in more than 90% of all tumours examined. One-hundred thirty-two genes were hypermethylated in 100% of CIMP-H tumours studied and these were highly enriched for functions relating to skeletal system development (Bonferroni adjusted p value =2.88E-15), segment specification (adjusted p value =9.62E-11), embryonic development (adjusted p value =1.52E-04), mesoderm development (adjusted p value =1.14E-20), and ectoderm development (adjusted p value =7.94E-16). Our genome-wide characterization of DNA

Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

Directory of Open Access Journals (Sweden)

Zdepski Anna

2011-05-01

Full Text Available Abstract Background High throughput sequencing (HTS technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR. We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

Cpf1-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cpf1.

Science.gov (United States)

Park, Jeongbin; Bae, Sangsu

2018-03-15

Following the type II CRISPR-Cas9 system, type V CRISPR-Cpf1 endonucleases have been found to be applicable for genome editing in various organisms in vivo. However, there are as yet no web-based tools capable of optimally selecting guide RNAs (gRNAs) among all possible genome-wide target sites. Here, we present Cpf1-Database, a genome-wide gRNA library design tool for LbCpf1 and AsCpf1, which have DNA recognition sequences of 5'-TTTN-3' at the 5' ends of target sites. Cpf1-Database provides a sophisticated but simple way to design gRNAs for AsCpf1 nucleases on the genome scale. One can easily access the data using a straightforward web interface, and using the powerful collections feature one can easily design gRNAs for thousands of genes in short time. Free access at http://www.rgenome.net/cpf1-database/. sangsubae@hanyang.ac.kr.

A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors.

Science.gov (United States)

Espinal, Allyson C; Wang, Dan; Yan, Li; Liu, Song; Tang, Li; Hu, Qiang; Morrison, Carl D; Ambrosone, Christine B; Higgins, Michael J; Sucheston-Campbell, Lara E

2017-02-28

DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER- tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER-) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.

Genome-wide study of correlations between genomic features and their relationship with the regulation of gene expression.

Science.gov (United States)

Kravatsky, Yuri V; Chechetkin, Vladimir R; Tchurikov, Nikolai A; Kravatskaya, Galina I

2015-02-01

The broad class of tasks in genetics and epigenetics can be reduced to the study of various features that are distributed over the genome (genome tracks). The rapid and efficient processing of the huge amount of data stored in the genome-scale databases cannot be achieved without the software packages based on the analytical criteria. However, strong inhomogeneity of genome tracks hampers the development of relevant statistics. We developed the criteria for the assessment of genome track inhomogeneity and correlations between two genome tracks. We also developed a software package, Genome Track Analyzer, based on this theory. The theory and software were tested on simulated data and were applied to the study of correlations between CpG islands and transcription start sites in the Homo sapiens genome, between profiles of protein-binding sites in chromosomes of Drosophila melanogaster, and between DNA double-strand breaks and histone marks in the H. sapiens genome. Significant correlations between transcription start sites on the forward and the reverse strands were observed in genomes of D. melanogaster, Caenorhabditis elegans, Mus musculus, H. sapiens, and Danio rerio. The observed correlations may be related to the regulation of gene expression in eukaryotes. Genome Track Analyzer is freely available at http://ancorr.eimb.ru/. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Detection of Non-Amplified Genomic DNA

CERN Document Server

Corradini, Roberto

2012-01-01

This book offers a state-of-the-art overview on non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. The importance of non-amplified DNA sequencing technologies will be also discussed. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specifi...

TALENs: customizable molecular DNA scissors for genome engineering of plants.

Science.gov (United States)

Chen, Kunling; Gao, Caixia

2013-06-20

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases (TALENs), consisting of an engineered specific (TALE) DNA binding domain and a Fok I cleavage domain, are newly developed versatile reagents for genome engineering in different organisms. Because of the simplicity of the DNA recognition code and their modular assembly, TALENs can act as customizable molecular DNA scissors inducing double-strand breaks (DSBs) at given genomic location. Thus, they provide a valuable approach to targeted genome modifications such as mutations, insertions, replacements or chromosome rearrangements. In this article, we review the development of TALENs, and summarize the principles and tools for TALEN-mediated gene targeting in plant cells, as well as current and potential strategies for use in plant research and crop improvement. Copyright © 2013. Published by Elsevier Ltd.

Recurrence time statistics: versatile tools for genomic DNA sequence analysis.

Science.gov (United States)

Cao, Yinhe; Tung, Wen-Wen; Gao, J B

2004-01-01

With the completion of the human and a few model organisms' genomes, and the genomes of many other organisms waiting to be sequenced, it has become increasingly important to develop faster computational tools which are capable of easily identifying the structures and extracting features from DNA sequences. One of the more important structures in a DNA sequence is repeat-related. Often they have to be masked before protein coding regions along a DNA sequence are to be identified or redundant expressed sequence tags (ESTs) are to be sequenced. Here we report a novel recurrence time based method for sequence analysis. The method can conveniently study all kinds of periodicity and exhaustively find all repeat-related features from a genomic DNA sequence. An efficient codon index is also derived from the recurrence time statistics, which has the salient features of being largely species-independent and working well on very short sequences. Efficient codon indices are key elements of successful gene finding algorithms, and are particularly useful for determining whether a suspected EST belongs to a coding or non-coding region. We illustrate the power of the method by studying the genomes of E. coli, the yeast S. cervisivae, the nematode worm C. elegans, and the human, Homo sapiens. Computationally, our method is very efficient. It allows us to carry out analysis of genomes on the whole genomic scale by a PC.

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

International Nuclear Information System (INIS)

Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung; Han, Myun Soo; Kim, Jong Jin; Kim, Wook; Lee, Jong Eun; Song, Joon Myong

2012-01-01

Highlights: ► Genomic DNA quantification were performed using a quantum dot-labeled Alu sequence. ► This probe provided PCR-free determination of human genomic DNA. ► Qdot-labeled Alu probe-hybridized genomic DNAs had a 2.5-femtogram detection limit. ► Qdot-labeled Alu sequence was used to assess DNA samples for human identification. - Abstract: Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

Mutagenic repair of double-stranded DNA breaks in vaccinia virus genomes requires cellular DNA ligase IV activity in the cytosol.

Science.gov (United States)

Luteijn, Rutger David; Drexler, Ingo; Smith, Geoffrey L; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

2018-04-20

Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.

Molecular analysis and genomic organization of major DNA satellites in banana (Musa spp.).

Science.gov (United States)

Čížková, Jana; Hřibová, Eva; Humplíková, Lenka; Christelová, Pavla; Suchánková, Pavla; Doležel, Jaroslav

2013-01-01

Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa.

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing.

Science.gov (United States)

Feehan, Joanna M; Scheibel, Katherine E; Bourras, Salim; Underwood, William; Keller, Beat; Somerville, Shauna C

2017-03-31

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 µg DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., > 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Genome-wide patterns of nucleotide polymorphism in domesticated rice

DEFF Research Database (Denmark)

Caicedo, Ana L; Williamson, Scott H; Hernandez, Ryan D

2007-01-01

Domesticated Asian rice (Oryza sativa) is one of the oldest domesticated crop species in the world, having fed more people than any other plant in human history. We report the patterns of DNA sequence variation in rice and its wild ancestor, O. rufipogon, across 111 randomly chosen gene fragments......, and use these to infer the evolutionary dynamics that led to the origins of rice. There is a genome-wide excess of high-frequency derived single nucleotide polymorphisms (SNPs) in O. sativa varieties, a pattern that has not been reported for other crop species. We developed several alternative models...... to explain contemporary patterns of polymorphisms in rice, including a (i) selectively neutral population bottleneck model, (ii) bottleneck plus migration model, (iii) multiple selective sweeps model, and (iv) bottleneck plus selective sweeps model. We find that a simple bottleneck model, which has been...

Genome-wide screening and identification of antigens for rickettsial vaccine development

Science.gov (United States)

The capacity to identify immunogens for vaccine development by genome-wide screening has been markedly enhanced by the availability of complete microbial genome sequences coupled to rapid proteomic and bioinformatic analysis. Critical to this genome-wide screening is in vivo testing in the context o...

Quality control and conduct of genome-wide association meta-analyses

DEFF Research Database (Denmark)

Winkler, Thomas W; Day, Felix R; Croteau-Chonka, Damien C

2014-01-01

Rigorous organization and quality control (QC) are necessary to facilitate successful genome-wide association meta-analyses (GWAMAs) of statistics aggregated across multiple genome-wide association studies. This protocol provides guidelines for (i) organizational aspects of GWAMAs, and for (ii) QC...

Genome-wide association study of pathological gambling.

Science.gov (United States)

Lang, M; Leménager, T; Streit, F; Fauth-Bühler, M; Frank, J; Juraeva, D; Witt, S H; Degenhardt, F; Hofmann, A; Heilmann-Heimbach, S; Kiefer, F; Brors, B; Grabe, H-J; John, U; Bischof, A; Bischof, G; Völker, U; Homuth, G; Beutel, M; Lind, P A; Medland, S E; Slutske, W S; Martin, N G; Völzke, H; Nöthen, M M; Meyer, C; Rumpf, H-J; Wurst, F M; Rietschel, M; Mann, K F

2016-08-01

Pathological gambling is a behavioural addiction with negative economic, social, and psychological consequences. Identification of contributing genes and pathways may improve understanding of aetiology and facilitate therapy and prevention. Here, we report the first genome-wide association study of pathological gambling. Our aims were to identify pathways involved in pathological gambling, and examine whether there is a genetic overlap between pathological gambling and alcohol dependence. Four hundred and forty-five individuals with a diagnosis of pathological gambling according to the Diagnostic and Statistical Manual of Mental Disorders were recruited in Germany, and 986 controls were drawn from a German general population sample. A genome-wide association study of pathological gambling comprising single marker, gene-based, and pathway analyses, was performed. Polygenic risk scores were generated using data from a German genome-wide association study of alcohol dependence. No genome-wide significant association with pathological gambling was found for single markers or genes. Pathways for Huntington's disease (P-value=6.63×10(-3)); 5'-adenosine monophosphate-activated protein kinase signalling (P-value=9.57×10(-3)); and apoptosis (P-value=1.75×10(-2)) were significant. Polygenic risk score analysis of the alcohol dependence dataset yielded a one-sided nominal significant P-value in subjects with pathological gambling, irrespective of comorbid alcohol dependence status. The present results accord with previous quantitative formal genetic studies which showed genetic overlap between non-substance- and substance-related addictions. Furthermore, pathway analysis suggests shared pathology between Huntington's disease and pathological gambling. This finding is consistent with previous imaging studies. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

Science.gov (United States)

Arnaiz, Olivier; Mathy, Nathalie; Baudry, Céline; Malinsky, Sophie; Aury, Jean-Marc; Denby Wilkes, Cyril; Garnier, Olivier; Labadie, Karine; Lauderdale, Benjamin E; Le Mouël, Anne; Marmignon, Antoine; Nowacki, Mariusz; Poulain, Julie; Prajer, Malgorzata; Wincker, Patrick; Meyer, Eric; Duharcourt, Sandra; Duret, Laurent; Bétermier, Mireille; Sperling, Linda

2012-01-01

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

Directory of Open Access Journals (Sweden)

Olivier Arnaiz

Full Text Available Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a

Network Biomarkers of Bladder Cancer Based on a Genome-Wide Genetic and Epigenetic Network Derived from Next-Generation Sequencing Data.

Science.gov (United States)

Li, Cheng-Wei; Chen, Bor-Sen

2016-01-01

Epigenetic and microRNA (miRNA) regulation are associated with carcinogenesis and the development of cancer. By using the available omics data, including those from next-generation sequencing (NGS), genome-wide methylation profiling, candidate integrated genetic and epigenetic network (IGEN) analysis, and drug response genome-wide microarray analysis, we constructed an IGEN system based on three coupling regression models that characterize protein-protein interaction networks (PPINs), gene regulatory networks (GRNs), miRNA regulatory networks (MRNs), and epigenetic regulatory networks (ERNs). By applying system identification method and principal genome-wide network projection (PGNP) to IGEN analysis, we identified the core network biomarkers to investigate bladder carcinogenic mechanisms and design multiple drug combinations for treating bladder cancer with minimal side-effects. The progression of DNA repair and cell proliferation in stage 1 bladder cancer ultimately results not only in the derepression of miR-200a and miR-200b but also in the regulation of the TNF pathway to metastasis-related genes or proteins, cell proliferation, and DNA repair in stage 4 bladder cancer. We designed a multiple drug combination comprising gefitinib, estradiol, yohimbine, and fulvestrant for treating stage 1 bladder cancer with minimal side-effects, and another multiple drug combination comprising gefitinib, estradiol, chlorpromazine, and LY294002 for treating stage 4 bladder cancer with minimal side-effects.

Genome-wide analysis of Tol2 transposon reintegration in zebrafish.

Science.gov (United States)

Kondrychyn, Igor; Garcia-Lecea, Marta; Emelyanov, Alexander; Parinov, Sergey; Korzh, Vladimir

2009-09-08

Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. We performed a large-scale enhancer trap (ET) screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

Science.gov (United States)

Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

2016-02-01

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

Genome-wide methylation patterns in Salmonella enterica Subsp. enterica Serovars.

Directory of Open Access Journals (Sweden)

Cary Pirone-Davies

Full Text Available The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N6-methyladenine (m6A methylated motifs, one N4-methylcytosine (m4C motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems, indicating the high diversity of methylation patterns present in Salmonella.

Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver.

Directory of Open Access Journals (Sweden)

Guillaume Rey

2011-02-01

Full Text Available The mammalian circadian clock uses interlocked negative feedback loops in which the heterodimeric basic helix-loop-helix transcription factor BMAL1/CLOCK is a master regulator. While there is prominent control of liver functions by the circadian clock, the detailed links between circadian regulators and downstream targets are poorly known. Using chromatin immunoprecipitation combined with deep sequencing we obtained a time-resolved and genome-wide map of BMAL1 binding in mouse liver, which allowed us to identify over 2,000 binding sites, with peak binding narrowly centered around Zeitgeber time 6. Annotation of BMAL1 targets confirms carbohydrate and lipid metabolism as the major output of the circadian clock in mouse liver. Moreover, transcription regulators are largely overrepresented, several of which also exhibit circadian activity. Genes of the core circadian oscillator stand out as strongly bound, often at promoter and distal sites. Genomic sequence analysis of the sites identified E-boxes and tandem E1-E2 consensus elements. Electromobility shift assays showed that E1-E2 sites are bound by a dimer of BMAL1/CLOCK heterodimers with a spacing-dependent cooperative interaction, a finding that was further validated in transactivation assays. BMAL1 target genes showed cyclic mRNA expression profiles with a phase distribution centered at Zeitgeber time 10. Importantly, sites with E1-E2 elements showed tighter phases both in binding and mRNA accumulation. Finally, analyzing the temporal profiles of BMAL1 binding, precursor mRNA and mature mRNA levels showed how transcriptional and post-transcriptional regulation contribute differentially to circadian expression phase. Together, our analysis of a dynamic protein-DNA interactome uncovered how genes of the core circadian oscillator crosstalk and drive phase-specific circadian output programs in a complex tissue.

Genome-wide comparison of medieval and modern Mycobacterium leprae

DEFF Research Database (Denmark)

Schuenemann, Verena J; Singh, Pushpendra; Mendum, Thomas A

2013-01-01

Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly...... origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human...

Whole-genome methylation caller designed for methyl- DNA ...

African Journals Online (AJOL)

etchie

2013-02-20

Feb 20, 2013 ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, Hidden ... its response to environmental cues. .... have a great potential to become the most cost-effective ... hg18 reference genome (set to 0 if not present in retrieved reads). ..... DNA methylation patterns and epigenetic memory.

Genome-wide association study of clinical dimensions of schizophrenia

DEFF Research Database (Denmark)

Fanous, Ayman H; Zhou, Baiyu; Aggen, Steven H

2012-01-01

Multiple sources of evidence suggest that genetic factors influence variation in clinical features of schizophrenia. The authors present the first genome-wide association study (GWAS) of dimensional symptom scores among individuals with schizophrenia.......Multiple sources of evidence suggest that genetic factors influence variation in clinical features of schizophrenia. The authors present the first genome-wide association study (GWAS) of dimensional symptom scores among individuals with schizophrenia....

Systematic identification of fragile sites via genome-wide location analysis of γ-H2AX

Science.gov (United States)

Szilard, Rachel K.; Jacques, Pierre-Étienne; Laramée, Louise; Cheng, Benjamin; Galicia, Sarah; Bataille, Alain R.; Yeung, ManTek; Mendez, Megan; Bergeron, Maxime; Robert, François; Durocher, Daniel

2011-01-01

Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication fork stalling results in histone H2A phosphorylation to yield γ-H2A (yeast γ-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)- dependent manner. Here, we describe the genome-wide location analysis of γ-H2A as a strategy to identify loci prone to engage the Mec1 and Tel1 pathways. Remarkably, γ-H2A enrichment overlaps with loci prone to replication fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for γ-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of γ-H2A at these loci. Finally, our work indicates that high resolution mapping of γ-H2AX is a fruitful route to map fragile sites in eukaryotic genomes. PMID:20139982

GWIS: Genome-Wide Inferred Statistics for Functions of Multiple Phenotypes

NARCIS (Netherlands)

Nieuwboer, H.A.; Pool, R.; Dolan, C.V.; Boomsma, D.I.; Nivard, M.G.

2016-01-01

Here we present a method of genome-wide inferred study (GWIS) that provides an approximation of genome-wide association study (GWAS) summary statistics for a variable that is a function of phenotypes for which GWAS summary statistics, phenotypic means, and covariances are available. A GWIS can be

Simplified extraction of good quality genomic DNA from a variety of ...

African Journals Online (AJOL)

Depending on the nature and complexity of plant material, proper method needs to be employed for extraction of genomic DNA, along with its performance evaluation by different molecular techniques. Here, we optimized and employed a simple genomic DNA isolation protocol suitable for a variety of plant materials ...

Genome-wide map of Apn1 binding sites under oxidative stress in Saccharomyces cerevisiae.

Science.gov (United States)

Morris, Lydia P; Conley, Andrew B; Degtyareva, Natalya; Jordan, I King; Doetsch, Paul W

2017-11-01

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large-scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress-induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large-scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large-scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Gene interactions in the DNA damage-response pathway identified by genome-wide RNA-interference analysis of synthetic lethality

NARCIS (Netherlands)

van Haaften, Gijs; Vastenhouw, Nadine L; Nollen, Ellen A A; Plasterk, Ronald H A; Tijsterman, Marcel

2004-01-01

Here, we describe a systematic search for synthetic gene interactions in a multicellular organism, the nematode Caenorhabditis elegans. We established a high-throughput method to determine synthetic gene interactions by genome-wide RNA interference and identified genes that are required to protect

Next-Generation Sequencing Approaches in Genome-Wide Discovery of Single Nucleotide Polymorphism Markers Associated with Pungency and Disease Resistance in Pepper.

Science.gov (United States)

Manivannan, Abinaya; Kim, Jin-Hee; Yang, Eun-Young; Ahn, Yul-Kyun; Lee, Eun-Su; Choi, Sena; Kim, Do-Sun

2018-01-01

Pepper is an economically important horticultural plant that has been widely used for its pungency and spicy taste in worldwide cuisines. Therefore, the domestication of pepper has been carried out since antiquity. Owing to meet the growing demand for pepper with high quality, organoleptic property, nutraceutical contents, and disease tolerance, genomics assisted breeding techniques can be incorporated to develop novel pepper varieties with desired traits. The application of next-generation sequencing (NGS) approaches has reformed the plant breeding technology especially in the area of molecular marker assisted breeding. The availability of genomic information aids in the deeper understanding of several molecular mechanisms behind the vital physiological processes. In addition, the NGS methods facilitate the genome-wide discovery of DNA based markers linked to key genes involved in important biological phenomenon. Among the molecular markers, single nucleotide polymorphism (SNP) indulges various benefits in comparison with other existing DNA based markers. The present review concentrates on the impact of NGS approaches in the discovery of useful SNP markers associated with pungency and disease resistance in pepper. The information provided in the current endeavor can be utilized for the betterment of pepper breeding in future.

Next-Generation Sequencing Approaches in Genome-Wide Discovery of Single Nucleotide Polymorphism Markers Associated with Pungency and Disease Resistance in Pepper

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Abinaya Manivannan

2018-01-01

Full Text Available Pepper is an economically important horticultural plant that has been widely used for its pungency and spicy taste in worldwide cuisines. Therefore, the domestication of pepper has been carried out since antiquity. Owing to meet the growing demand for pepper with high quality, organoleptic property, nutraceutical contents, and disease tolerance, genomics assisted breeding techniques can be incorporated to develop novel pepper varieties with desired traits. The application of next-generation sequencing (NGS approaches has reformed the plant breeding technology especially in the area of molecular marker assisted breeding. The availability of genomic information aids in the deeper understanding of several molecular mechanisms behind the vital physiological processes. In addition, the NGS methods facilitate the genome-wide discovery of DNA based markers linked to key genes involved in important biological phenomenon. Among the molecular markers, single nucleotide polymorphism (SNP indulges various benefits in comparison with other existing DNA based markers. The present review concentrates on the impact of NGS approaches in the discovery of useful SNP markers associated with pungency and disease resistance in pepper. The information provided in the current endeavor can be utilized for the betterment of pepper breeding in future.

Genome-Wide Detection and Analysis of Multifunctional Genes

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Pritykin, Yuri; Ghersi, Dario; Singh, Mona

2015-01-01

Many genes can play a role in multiple biological processes or molecular functions. Identifying multifunctional genes at the genome-wide level and studying their properties can shed light upon the complexity of molecular events that underpin cellular functioning, thereby leading to a better understanding of the functional landscape of the cell. However, to date, genome-wide analysis of multifunctional genes (and the proteins they encode) has been limited. Here we introduce a computational approach that uses known functional annotations to extract genes playing a role in at least two distinct biological processes. We leverage functional genomics data sets for three organisms—H. sapiens, D. melanogaster, and S. cerevisiae—and show that, as compared to other annotated genes, genes involved in multiple biological processes possess distinct physicochemical properties, are more broadly expressed, tend to be more central in protein interaction networks, tend to be more evolutionarily conserved, and are more likely to be essential. We also find that multifunctional genes are significantly more likely to be involved in human disorders. These same features also hold when multifunctionality is defined with respect to molecular functions instead of biological processes. Our analysis uncovers key features about multifunctional genes, and is a step towards a better genome-wide understanding of gene multifunctionality. PMID:26436655

Adolescent binge-pattern alcohol exposure alters genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve male offspring.

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Asimes, AnnaDorothea; Torcaso, Audrey; Pinceti, Elena; Kim, Chun K; Zeleznik-Le, Nancy J; Pak, Toni R

2017-05-01

Teenage binge drinking is a major health concern in the United States, with 21% of teenagers reporting binge-pattern drinking behavior in the previous 30 days. Recently, our lab showed that alcohol-naïve offspring of rats exposed to alcohol during adolescence exhibited altered gene expression profiles in the hypothalamus, a brain region involved in stress regulation. We employed Enhanced Reduced Representation Bisulfite Sequencing as an unbiased approach to test the hypothesis that parental exposure to binge-pattern alcohol during adolescence alters DNA methylation profiles in their alcohol-naïve offspring. Wistar rats were administered a repeated binge-ethanol exposure paradigm during early (postnatal day (PND) 37-44) and late (PND 67-74) adolescent development. Animals were mated 24 h after the last ethanol dose and subsequent offspring were produced. Analysis of male PND7 offspring revealed that offspring of alcohol-exposed parents exhibited differential DNA methylation patterns in the hypothalamus. The differentially methylated cytosines (DMCs) were distinct between offspring depending on which parent was exposed to ethanol. Moreover, novel DMCs were observed when both parents were exposed to ethanol and many DMCs from single parent ethanol exposure were not recapitulated with dual parent exposure. We also measured mRNA expression of several differentially methylated genes and some, but not all, showed correlative changes in expression. Importantly, methylation was not a direct predictor of expression levels, underscoring the complexity of transcriptional regulation. Overall, we demonstrate that adolescent binge ethanol exposure causes altered genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve offspring. Copyright © 2016 Elsevier Inc. All rights reserved.

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas

Science.gov (United States)

Mohapatra, Gayatry; Engler, David A.; Starbuck, Kristen D.; Kim, James C.; Bernay, Derek C.; Scangas, George A.; Rousseau, Audrey; Batchelor, Tracy T.; Betensky, Rebecca A.; Louis, David N.

2010-01-01

Molecular genetic analysis of cancer is rapidly evolving as a result of improvement in genomic technologies and the growing applicability of such analyses to clinical oncology. Array based comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA), particularly in solid tumors, and has been applied to the study of malignant gliomas. In the clinical setting, however, gliomas are often sampled by small biopsies and thus formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis, especially for rare types of gliomas. Moreover, the biological basis for the marked intratumoral heterogeneity in gliomas is most readily addressed in FFPE material. Therefore, for gliomas, the ability to use DNA from FFPE tissue is essential for both clinical and research applications. In this study, we have constructed a custom bacterial artificial chromosome (BAC) array and show excellent sensitivity and specificity for detecting CNAs in a panel of paired frozen and FFPE glioma samples. Our study demonstrates a high concordance rate between CNAs detected in FFPE compared to frozen DNA. We have also developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. This labeling technique was applied to a panel of biphasic anaplastic oligoastrocytomas (AOA) to identify genetic changes unique to each component. Together, results from these studies suggest that BAC and oligonucleotide aCGH are sensitive tools for detecting CNAs in FFPE DNA, and can enable genome-wide analysis of rare, small and/or histologically heterogeneous gliomas. PMID:21080181

A protocol for large scale genomic DNA isolation for cacao genetics ...

African Journals Online (AJOL)

Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the preservation of tissue samples for future use in cacao genome studies. The present study proposes a method for the ...

Regenerant arabidopsis lineages display a distinct genome-wide spectrum of mutations conferring variant phenotypes

KAUST Repository

Jiang, Caifu

2011-07-28

Multicellular organisms can be regenerated from totipotent differentiated somatic cell or nuclear founders [1-3]. Organisms regenerated from clonally related isogenic founders might a priori have been expected to be phenotypically invariant. However, clonal regenerant animals display variant phenotypes caused by defective epigenetic reprogramming of gene expression [2], and clonal regenerant plants exhibit poorly understood heritable phenotypic ("somaclonal") variation [4-7]. Here we show that somaclonal variation in regenerant Arabidopsis lineages is associated with genome-wide elevation in DNA sequence mutation rate. We also show that regenerant mutations comprise a distinctive molecular spectrum of base substitutions, insertions, and deletions that probably results from decreased DNA repair fidelity. Finally, we show that while regenerant base substitutions are a likely major genetic cause of the somaclonal variation of regenerant Arabidopsis lineages, transposon movement is unlikely to contribute substantially to that variation. We conclude that the phenotypic variation of regenerant plants, unlike that of regenerant animals, is substantially due to DNA sequence mutation. 2011 Elsevier Ltd. All rights reserved.

Regenerant arabidopsis lineages display a distinct genome-wide spectrum of mutations conferring variant phenotypes

KAUST Repository

Jiang, Caifu; Mithani, Aziz; Gan, Xiangchao; Belfield, Eric J.; Klingler, John  P.; Zhu, Jian-Kang; Ragoussis, Jiannis; Mott, Richard; Harberd, Nicholas  P.

2011-01-01

Multicellular organisms can be regenerated from totipotent differentiated somatic cell or nuclear founders [1-3]. Organisms regenerated from clonally related isogenic founders might a priori have been expected to be phenotypically invariant. However, clonal regenerant animals display variant phenotypes caused by defective epigenetic reprogramming of gene expression [2], and clonal regenerant plants exhibit poorly understood heritable phenotypic ("somaclonal") variation [4-7]. Here we show that somaclonal variation in regenerant Arabidopsis lineages is associated with genome-wide elevation in DNA sequence mutation rate. We also show that regenerant mutations comprise a distinctive molecular spectrum of base substitutions, insertions, and deletions that probably results from decreased DNA repair fidelity. Finally, we show that while regenerant base substitutions are a likely major genetic cause of the somaclonal variation of regenerant Arabidopsis lineages, transposon movement is unlikely to contribute substantially to that variation. We conclude that the phenotypic variation of regenerant plants, unlike that of regenerant animals, is substantially due to DNA sequence mutation. 2011 Elsevier Ltd. All rights reserved.

Genomic DNA extraction from sapwood of Pinus roxburghii for ...

African Journals Online (AJOL)

A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, where collection of needle tissues is extremely difficult has been standardized. The extracted DNA was comparable to that obtained from the needle tissue in terms of yield and purity. The yield of extracted DNA ranged ...

Isolating silkworm genomic DNA without liquid nitrogen suitable for ...

African Journals Online (AJOL)

Genomic DNA was isolated from posterior silk gland of silkworms, Antheraea assama. Absolute alcohol was used as tissue fixing solution instead of grinding in liquid nitrogen, which yielded high molecular weight DNA (>40 kb). Samples yielded similar amount of DNA when fixed in absolute alcohol (400 μmg/g of silk gland ...

Analysis of the giant genomes of Fritillaria (Liliaceae) indicates that a lack of DNA removal characterizes extreme expansions in genome size.

Science.gov (United States)

Kelly, Laura J; Renny-Byfield, Simon; Pellicer, Jaume; Macas, Jiří; Novák, Petr; Neumann, Pavel; Lysak, Martin A; Day, Peter D; Berger, Madeleine; Fay, Michael F; Nichols, Richard A; Leitch, Andrew R; Leitch, Ilia J

2015-10-01

Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smallest and largest recorded values. In the absence of polyploidy, changes in the amount of repetitive DNA (transposable elements and tandem repeats) are primarily responsible for genome size differences between species. However, there is ongoing debate regarding the relative importance of amplification of repetitive DNA versus its deletion in governing genome size. Using data from 454 sequencing, we analysed the most repetitive fraction of some of the largest known genomes for diploid plant species, from members of Fritillaria. We revealed that genomic expansion has not resulted from the recent massive amplification of just a handful of repeat families, as shown in species with smaller genomes. Instead, the bulk of these immense genomes is composed of highly heterogeneous, relatively low-abundance repeat-derived DNA, supporting a scenario where amplified repeats continually accumulate due to infrequent DNA removal. Our results indicate that a lack of deletion and low turnover of repetitive DNA are major contributors to the evolution of extremely large genomes and show that their size cannot simply be accounted for by the activity of a small number of high-abundance repeat families. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Genome-wide analysis of Tol2 transposon reintegration in zebrafish

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Parinov Sergey

2009-09-01

Full Text Available Abstract Background Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. Results We performed a large-scale enhancer trap (ET screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Conclusion Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

Functional interrogation of non-coding DNA through CRISPR genome editing.

Science.gov (United States)

Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H

2017-05-15

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome-Wide Association Study (GWAS) and Genome-Wide Environment Interaction Study (GWEIS) of Depressive Symptoms in African American and Hispanic/Latina Women

Science.gov (United States)

Dunn, Erin C.; Wiste, Anna; Radmanesh, Farid; Almli, Lynn M.; Gogarten, Stephanie M.; Sofer, Tamar; Faul, Jessica D.; Kardia, Sharon L.R.; Smith, Jennifer A.; Weir, David R.; Zhao, Wei; Soare, Thomas W.; Mirza, Saira S.; Hek, Karin; Tiemeier, Henning W.; Goveas, Joseph S.; Sarto, Gloria E.; Snively, Beverly M.; Cornelis, Marilyn; Koenen, Karestan C.; Kraft, Peter; Purcell, Shaun; Ressler, Kerry J.; Rosand, Jonathan; Wassertheil-Smoller, Sylvia; Smoller, Jordan W.

2016-01-01

Background Genome-wide association studies (GWAS) have been unable to identify variants linked to depression. We hypothesized that examining depressive symptoms and considering gene-environment interaction (G×E) might improve efficiency for gene discovery. We therefore conducted a GWAS and genome-wide environment interaction study (GWEIS) of depressive symptoms. Methods Using data from the SHARe cohort of the Women’s Health Initiative, comprising African Americans (n=7179) and Hispanics/Latinas (n=3138), we examined genetic main effects and G×E with stressful life events and social support. We also conducted a heritability analysis using genome-wide complex trait analysis (GCTA). Replication was attempted in four independent cohorts. Results No SNPs achieved genome-wide significance for main effects in either discovery sample. The top signals in African Americans were rs73531535 (located 20kb from GPR139, p=5.75×10−8) and rs75407252 (intronic to CACNA2D3, p=6.99×10−7). In Hispanics/Latinas, the top signals were rs2532087 (located 27kb from CD38, p=2.44×10−7) and rs4542757 (intronic to DCC, p=7.31×10−7). In the GWEIS with stressful life events, one interaction signal was genome-wide significant in African Americans (rs4652467; p=4.10×10−10; located 14kb from CEP350). This interaction was not observed in a smaller replication cohort. Although heritability estimates for depressive symptoms and stressful life events were each less than 10%, they were strongly genetically correlated (rG=0.95), suggesting that common variation underlying depressive symptoms and stressful life event exposure, though modest on their own, were highly overlapping in this sample. Conclusions Our results underscore the need for larger samples, more GWEIS, and greater investigation into genetic and environmental determinants of depressive symptoms in minorities. PMID:27038408

Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

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De Marzo Angelo M

2011-06-01

Full Text Available Abstract Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.

Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip.

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Jang, Jun Hyeong; Kim, Sun-Joong; Yoon, Bo Hyun; Ryu, Jee-Hoon; Gu, Man Bock; Chang, Hyo-Ihl

2011-06-01

This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.

Genome-Wide Analysis of Heteroduplex DNA in Mismatch Repair–Deficient Yeast Cells Reveals Novel Properties of Meiotic Recombination Pathways

Science.gov (United States)

Martini, Emmanuelle; Borde, Valérie; Legendre, Matthieu; Audic, Stéphane; Regnault, Béatrice; Soubigou, Guillaume; Dujon, Bernard; Llorente, Bertrand

2011-01-01

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR–proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans–hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates. PMID

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

Directory of Open Access Journals (Sweden)

Emmanuelle Martini

2011-09-01

Full Text Available Meiotic DNA double-strand breaks (DSBs initiate crossover (CO recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs. Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs. First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

Analysis of the genome-wide variations among multiple strains of the plant pathogenic bacterium Xylella fastidiosa

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Walker M Andrew

2006-09-01

Full Text Available Abstract Background The Gram-negative, xylem-limited phytopathogenic bacterium Xylella fastidiosa is responsible for causing economically important diseases in grapevine, citrus and many other plant species. Despite its economic impact, relatively little is known about the genomic variations among strains isolated from different hosts and their influence on the population genetics of this pathogen. With the availability of genome sequence information for four strains, it is now possible to perform genome-wide analyses to identify and categorize such DNA variations and to understand their influence on strain functional divergence. Results There are 1,579 genes and 194 non-coding homologous sequences present in the genomes of all four strains, representing a 76. 2% conservation of the sequenced genome. About 60% of the X. fastidiosa unique sequences exist as tandem gene clusters of 6 or more genes. Multiple alignments identified 12,754 SNPs and 14,449 INDELs in the 1528 common genes and 20,779 SNPs and 10,075 INDELs in the 194 non-coding sequences. The average SNP frequency was 1.08 × 10-2 per base pair of DNA and the average INDEL frequency was 2.06 × 10-2 per base pair of DNA. On an average, 60.33% of the SNPs were synonymous type while 39.67% were non-synonymous type. The mutation frequency, primarily in the form of external INDELs was the main type of sequence variation. The relative similarity between the strains was discussed according to the INDEL and SNP differences. The number of genes unique to each strain were 60 (9a5c, 54 (Dixon, 83 (Ann1 and 9 (Temecula-1. A sub-set of the strain specific genes showed significant differences in terms of their codon usage and GC composition from the native genes suggesting their xenologous origin. Tandem repeat analysis of the genomic sequences of the four strains identified associations of repeat sequences with hypothetical and phage related functions. Conclusion INDELs and strain specific genes

Identification of networks of co-occurring, tumor-related DNA copy number changes using a genome-wide scoring approach.

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Christiaan Klijn

2010-01-01

Full Text Available Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors following the accumulation of genetic mutations that enable them to evade the growth control checkpoints that would normally suppress their growth or result in apoptosis. It is therefore important to identify those combinations of mutations that collaborate in cancer development and progression. DNA copy number alterations (CNAs are one of the ways in which cancer genes are deregulated in tumor cells. We hypothesized that synergistic interactions between cancer genes might be identified by looking for regions of co-occurring gain and/or loss. To this end we developed a scoring framework to separate truly co-occurring aberrations from passenger mutations and dominant single signals present in the data. The resulting regions of high co-occurrence can be investigated for between-region functional interactions. Analysis of high-resolution DNA copy number data from a panel of 95 hematological tumor cell lines correctly identified co-occurring recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell malignancies, respectively, showing that we can recover truly co-occurring genomic alterations. In addition, our analysis revealed networks of co-occurring genomic losses and gains that are enriched for cancer genes. These networks are also highly enriched for functional relationships between genes. We further examine sub-networks of these networks, core networks, which contain many known cancer genes. The core network for co-occurring DNA losses we find seems to be independent of the canonical cancer genes within the network. Our findings suggest that large-scale, low-intensity copy number alterations may be an important feature of cancer development or maintenance by affecting gene dosage of a large interconnected network of functionally related genes.

Sequencing of chloroplast genome using whole cellular DNA and Solexa sequencing technology

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Jian eWu

2012-11-01

Full Text Available Sequencing of the chloroplast genome using traditional sequencing methods has been difficult because of its size (>120 kb and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the chloroplast genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassica rapa accessions with one lane per accession. In total, 246 Mb, 362Mb, 361 Mb sequence data were generated for the three accessions Chiifu-401-42, Z16 and FT, respectively. Microreads were assembled by reference-guided assembly using the cpDNA sequences of B. rapa, Arabidopsis thaliana, and Nicotiana tabacum. We achieved coverage of more than 99.96% of the cp genome in the three tested accessions using the B. rapa sequence as the reference. When A. thaliana or N. tabacum sequences were used as references, 99.7–99.8% or 95.5–99.7% of the B. rapa chloroplast genome was covered, respectively. These results demonstrated that sequencing of whole cellular DNA isolated from young leaves using the Illumina Genome Analyzer is an efficient method for high-throughput sequencing of chloroplast genome.

A Genome-Wide Breast Cancer Scan in African Americans

Science.gov (United States)

2010-06-01

SNPs from the African American breast cancer scan to COGs , a European collaborative study which is has designed a SNP array with that will be genotyped...Award Number: W81XWH-08-1-0383 TITLE: A Genome-wide Breast Cancer Scan in African Americans PRINCIPAL INVESTIGATOR: Christopher A...SUBTITLE A Genome-wide Breast Cancer Scan in African Americans 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-08-1-0383 5c. PROGRAM

The genome-wide landscape of DNA methylation and hydroxymethylation in response to sleep deprivation impacts on synaptic plasticity genes.

Science.gov (United States)

Massart, R; Freyburger, M; Suderman, M; Paquet, J; El Helou, J; Belanger-Nelson, E; Rachalski, A; Koumar, O C; Carrier, J; Szyf, M; Mongrain, V

2014-01-21

Sleep is critical for normal brain function and mental health. However, the molecular mechanisms mediating the impact of sleep loss on both cognition and the sleep electroencephalogram remain mostly unknown. Acute sleep loss impacts brain gene expression broadly. These data contributed to current hypotheses regarding the role for sleep in metabolism, synaptic plasticity and neuroprotection. These changes in gene expression likely underlie increased sleep intensity following sleep deprivation (SD). Here we tested the hypothesis that epigenetic mechanisms coordinate the gene expression response driven by SD. We found that SD altered the cortical genome-wide distribution of two major epigenetic marks: DNA methylation and hydroxymethylation. DNA methylation differences were enriched in gene pathways involved in neuritogenesis and synaptic plasticity, whereas large changes (>4000 sites) in hydroxymethylation where observed in genes linked to cytoskeleton, signaling and neurotransmission, which closely matches SD-dependent changes in the transcriptome. Moreover, this epigenetic remodeling applied to elements previously linked to sleep need (for example, Arc and Egr1) and synaptic partners of Neuroligin-1 (Nlgn1; for example, Dlg4, Nrxn1 and Nlgn3), which we recently identified as a regulator of sleep intensity following SD. We show here that Nlgn1 mutant mice display an enhanced slow-wave slope during non-rapid eye movement sleep following SD but this mutation does not affect SD-dependent changes in gene expression, suggesting that the Nlgn pathway acts downstream to mechanisms triggering gene expression changes in SD. These data reveal that acute SD reprograms the epigenetic landscape, providing a unique molecular route by which sleep can impact brain function and health.

The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

Energy Technology Data Exchange (ETDEWEB)

Smith, David R.; Lee, Robert W.; Cushman, John C.; Magnuson, Jon K.; Tran, Duc; Polle, Juergen E.

2010-05-07

Abstract Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results: The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable

The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

Directory of Open Access Journals (Sweden)

Tran Duc

2010-05-01

Full Text Available Abstract Background Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the

Genome-wide DNA methylation sequencing reveals miR-663a is a novel epimutation candidate in CIMP-high endometrial cancer

OpenAIRE

Yanokura, Megumi; Banno, Kouji; Adachi, Masataka; Aoki, Daisuke; Abe, Kuniya

2017-01-01

Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three ...

High-throughput sequencing of three Lemnoideae (duckweeds chloroplast genomes from total DNA.

Directory of Open Access Journals (Sweden)

Wenqin Wang

Full Text Available BACKGROUND: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. METHODS: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. CONCLUSIONS: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

Genome-wide association studies and resting heart rate

DEFF Research Database (Denmark)

Oskari Kilpeläinen, Tuomas

2016-01-01

Genome-wide association studies (GWASs) have revolutionized the search for genetic variants regulating resting heart rate. In the last 10 years, GWASs have led to the identification of at least 21 novel heart rate loci. These discoveries have provided valuable insights into the mechanisms...... and pathways that regulate heart rate and link heart rate to cardiovascular morbidity and mortality. GWASs capture majority of genetic variation in a population sample by utilizing high-throughput genotyping chips measuring genotypes for up to several millions of SNPs across the genome in thousands...... of individuals. This allows the identification of the strongest heart rate associated signals at genome-wide level. While GWASs provide robust statistical evidence of the association of a given genetic locus with heart rate, they are only the starting point for detailed follow-up studies to locate the causal...

Genome-wide Anaplasma phagocytophilum AnkA-DNA interactions are enriched in intergenic regions and gene promoters and correlate with infection-induced differential gene expression.

Directory of Open Access Journals (Sweden)

J Stephen Dumler

2016-09-01

Full Text Available Anaplasma phagocytophilum, an obligate intracellular prokaryote, infects neutrophils and alters cardinal functions via reprogrammed transcription. Large contiguous regions of neutrophil chromosomes are differentially expressed during infection. Secreted A. phagocytophilum effector AnkA transits into the neutrophil or granulocyte nucleus to complex with DNA in heterochromatin across all chromosomes. AnkA binds to gene promoters to dampen cis-transcription and also has features of matrix attachment region (MAR-binding proteins that regulate three-dimensional chromatin architecture and coordinate transcriptional programs encoded in topologically-associated chromatin domains. We hypothesize that identification of additional AnkA binding sites will better delineate how A. phagocytophilum infection results in reprogramming of the neutrophil genome. Using AnkA-binding ChIP-seq, we showed that AnkA binds broadly throughout all chromosomes in a reproducible pattern, especially at: i intergenic regions predicted to be matrix attachment regions (MARs; ii within predicted lamina-associated domains; and iii at promoters ≤3,000 bp upstream of transcriptional start sites. These findings provide genome-wide support for AnkA as a regulator of cis-gene transcription. Moreover, the dominant mark of AnkA in distal intergenic regions known to be AT-enriched, coupled with frequent enrichment in the nuclear lamina, provides strong support for its role as a MAR-binding protein and genome re-organizer. AnkA must be considered a prime candidate to promote neutrophil reprogramming and subsequent functional changes that belie improved microbial fitness and pathogenicity.

Functional role of a highly repetitive DNA sequence in anchorage of the mouse genome.

Science.gov (United States)

Neuer-Nitsche, B; Lu, X N; Werner, D

1988-09-12

The major portion of the eukaryotic genome consists of various categories of repetitive DNA sequences which have been studied with respect to their base compositions, organizations, copy numbers, transcription and species specificities; their biological roles, however, are still unclear. A novel quality of a highly repetitive mouse DNA sequence is described which points to a functional role: All copies (approximately 50,000 per haploid genome) of this DNA sequence reside on genomic Alu I DNA fragments each associated with nuclear polypeptides that are not released from DNA by proteinase K, SDS and phenol extraction. By this quality the repetitive DNA sequence is classified as a member of the sub-set of DNA sequences involved in tight DNA-polypeptide complexes which have been previously shown to be components of the subnuclear structure termed 'nuclear matrix'. From these results it has to be concluded that the repetitive DNA sequence characterized in this report represents or comprises a signal for a large number of site specific attachment points of the mouse genome in the nuclear matrix.

Genome wide characterization of simple sequence repeats in watermelon genome and their application in comparative mapping and genetic diversity analysis.

Science.gov (United States)

Zhu, Huayu; Song, Pengyao; Koo, Dal-Hoe; Guo, Luqin; Li, Yanman; Sun, Shouru; Weng, Yiqun; Yang, Luming

2016-08-05

Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been difficult and costly. The whole genome sequencing with next-generation sequencing (NGS) technologies provides large amounts of sequence data to develop numerous microsatellite markers at whole genome scale. SSR markers have great advantage in cross-species comparisons and allow investigation of karyotype and genome evolution through highly efficient computation approaches such as in silico PCR. Here we described genome wide development and characterization of SSR markers in the watermelon (Citrullus lanatus) genome, which were then use in comparative analysis with two other important crop species in the Cucurbitaceae family: cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). We further applied these markers in evaluating the genetic diversity and population structure in watermelon germplasm collections. A total of 39,523 microsatellite loci were identified from the watermelon draft genome with an overall density of 111 SSRs/Mbp, and 32,869 SSR primers were designed with suitable flanking sequences. The dinucleotide SSRs were the most common type representing 34.09 % of the total SSR loci and the AT-rich motifs were the most abundant in all nucleotide repeat types. In silico PCR analysis identified 832 and 925 SSR markers with each having a single amplicon in the cucumber and melon draft genome, respectively. Comparative analysis with these cross-species SSR markers revealed complicated mosaic patterns of syntenic blocks among the genomes of three species. In addition, genetic diversity analysis of 134 watermelon accessions with 32 highly informative SSR loci placed these lines into two groups with all accessions of C.lanatus var. citorides and three accessions of C. colocynthis clustered in one group and all accessions of C. lanatus var. lanatus and the remaining accessions of C. colocynthis

Complete chloroplast genome and 45S nrDNA sequences of the medicinal plant species Glycyrrhiza glabra and Glycyrrhiza uralensis.

Science.gov (United States)

Kang, Sang-Ho; Lee, Jeong-Hoon; Lee, Hyun Oh; Ahn, Byoung Ohg; Won, So Youn; Sohn, Seong-Han; Kim, Jung Sun

2017-10-06

Glycyrrhiza uralensis and G. glabra, members of the Fabaceae, are medicinally important species that are native to Asia and Europe. Extracts from these plants are widely used as natural sweeteners because of their much greater sweetness than sucrose. In this study, the three complete chloroplast genomes and five 45S nuclear ribosomal (nr)DNA sequences of these two licorice species and an interspecific hybrid are presented. The chloroplast genomes of G. glabra, G. uralensis and G. glabra × G. uralensis were 127,895 bp, 127,716 bp and 127,939 bp, respectively. The three chloroplast genomes harbored 110 annotated genes, including 76 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The 45S nrDNA sequences were either 5,947 or 5,948 bp in length. Glycyrrhiza glabra and G. glabra × G. uralensis showed two types of nrDNA, while G. uralensis contained a single type. The complete 45S nrDNA sequence unit contains 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 26S rRNA. We identified simple sequence repeat and tandem repeat sequences. We also developed four reliable markers for analysis of Glycyrrhiza diversity authentication.

Adiponectin Concentrations: A Genome-wide Association Study

Science.gov (United States)

Jee, Sun Ha; Sull, Jae Woong; Lee, Jong-Eun; Shin, Chol; Park, Jongkeun; Kimm, Heejin; Cho, Eun-Young; Shin, Eun-Soon; Yun, Ji Eun; Park, Ji Wan; Kim, Sang Yeun; Lee, Sun Ju; Jee, Eun Jung; Baik, Inkyung; Kao, Linda; Yoon, Sungjoo Kim; Jang, Yangsoo; Beaty, Terri H.

2010-01-01

Adiponectin is associated with obesity and insulin resistance. To date, there has been no genome-wide association study (GWAS) of adiponectin levels in Asians. Here we present a GWAS of a cohort of Korean volunteers. A total of 4,001 subjects were genotyped by using a genome-wide marker panel in a two-stage design (979 subjects initially and 3,022 in a second stage). Another 2,304 subjects were used for follow-up replication studies with selected markers. In the discovery phase, the top SNP associated with mean log adiponectin was rs3865188 in CDH13 on chromosome 16 (p = 1.69 × 10−15 in the initial sample, p = 6.58 × 10−39 in the second genome-wide sample, and p = 2.12 × 10−32 in the replication sample). The meta-analysis p value for rs3865188 in all 6,305 individuals was 2.82 × 10−83. The association of rs3865188 with high-molecular-weight adiponectin (p = 7.36 × 10−58) was even stronger in the third sample. A reporter assay that evaluated the effects of a CDH13 promoter SNP in complete linkage disequilibrium with rs3865188 revealed that the major allele increased expression 2.2-fold. This study clearly shows that genetic variants in CDH13 influence adiponectin levels in Korean adults. PMID:20887962

Novel approach for deriving genome wide SNP analysis data from archived blood spots

Science.gov (United States)

2012-01-01

Background The ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman™TM technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman™TM cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored. Findings DNA was extracted from FTA Whatman™TM cards (following adaptations of the manufacturer’s instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way. Conclusions DNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies. PMID:22974252

Genome-wide association study of pigmentary traits (skin and iris color in individuals of East Asian ancestry

Directory of Open Access Journals (Sweden)

Lida Rawofi

2017-11-01

Full Text Available Background Currently, there is limited knowledge about the genetics underlying pigmentary traits in East Asian populations. Here, we report the results of the first genome-wide association study of pigmentary traits (skin and iris color in individuals of East Asian ancestry. Methods We obtained quantitative skin pigmentation measures (M-index in the inner upper arm of the participants using a portable reflectometer (N = 305. Quantitative measures of iris color (expressed as L*, a* and b* CIELab coordinates were extracted from high-resolution iris pictures (N = 342. We also measured the color differences between the pupillary and ciliary regions of the iris (e.g., iris heterochromia. DNA samples were genotyped with Illumina’s Infinium Multi-Ethnic Global Array (MEGA and imputed using the 1000 Genomes Phase 3 samples as reference haplotypes. Results For skin pigmentation, we did not observe any genome-wide significant signal. We followed-up in three independent Chinese samples the lead SNPs of five regions showing multiple common markers (minor allele frequency ≥ 5% with good imputation scores and suggestive evidence of association (p-values < 10−5. One of these markers, rs2373391, which is located in an intron of the ZNF804B gene on chromosome 7, was replicated in one of the Chinese samples (p = 0.003. For iris color, we observed genome-wide signals in the OCA2 region on chromosome 15. This signal is driven by the non-synonymous rs1800414 variant, which explains 11.9%, 10.4% and 6% of the variation observed in the b*, a* and L* coordinates in our sample, respectively. However, the OCA2 region was not associated with iris heterochromia. Discussion Additional genome-wide association studies in East Asian samples will be necessary to further disentangle the genetic architecture of pigmentary traits in East Asian populations.

A genome-wide expression profile of salt-responsive genes in the apple rootstock Malus zumi.

Science.gov (United States)

Li, Qingtian; Liu, Jia; Tan, Dunxian; Allan, Andrew C; Jiang, Yuzhuang; Xu, Xuefeng; Han, Zhenhai; Kong, Jin

2013-10-18

In some areas of cultivation, a lack of salt tolerance severely affects plant productivity. Apple, Malus x domestica Borkh., is sensitive to salt, and, as a perennial woody plant the mechanism of salt stress adaption will be different from that of annual herbal model plants, such as Arabidopsis. Malus zumi is a salt tolerant apple rootstock, which survives high salinity (up to 0.6% NaCl). To examine the mechanism underlying this tolerance, a genome-wide expression analysis was performed, using a cDNA library constructed from salt-treated seedlings of Malus zumi. A total of 15,000 cDNA clones were selected for microarray analysis. In total a group of 576 cDNAs, of which expression changed more than four-fold, were sequenced and 18 genes were selected to verify their expression pattern under salt stress by semi-quantitative RT-PCR. Our genome-wide expression analysis resulted in the isolation of 50 novel Malus genes and the elucidation of a new apple-specific mechanism of salt tolerance, including the stabilization of photosynthesis under stress, involvement of phenolic compounds, and sorbitol in ROS scavenging and osmoprotection. The promoter regions of 111 genes were analyzed by PlantCARE, suggesting an intensive cross-talking of abiotic stress in Malus zumi. An interaction network of salt responsive genes was constructed and molecular regulatory pathways of apple were deduced. Our research will contribute to gene function analysis and further the understanding of salt-tolerance mechanisms in fruit trees.

Genome-wide differential gene expression in children exposed to air pollution in the Czech Republic

DEFF Research Database (Denmark)

van Leeuwen, D M; van Herwijnen, M H M; Pedersen, Marie

2006-01-01

The Teplice area in the Czech Republic is a mining district where elevated levels of air pollution including airborne carcinogens, have been demonstrated, especially during winter time. This environmental exposure can impact human health; in particular children may be more vulnerable. To study....... This suggests an effect of air pollution on the primary structural unit of the condensed DNA. In addition, several other pathways were modulated. Based on the results of this study, we suggest that transcriptomic analysis represents a promising biomarker for environmental carcinogenesis....... the impact of air pollution in children at the transcriptional level, peripheral blood cells were subjected to whole genome response analysis, in order to identify significantly modulated biological pathways and processes as a result of exposure. Using genome-wide oligonucleotide microarrays, we investigated...

Genome-wide association study identifies five new schizophrenia loci.

LENUS (Irish Health Repository)

Ripke, Stephan

2011-10-01

We examined the role of common genetic variation in schizophrenia in a genome-wide association study of substantial size: a stage 1 discovery sample of 21,856 individuals of European ancestry and a stage 2 replication sample of 29,839 independent subjects. The combined stage 1 and 2 analysis yielded genome-wide significant associations with schizophrenia for seven loci, five of which are new (1p21.3, 2q32.3, 8p23.2, 8q21.3 and 10q24.32-q24.33) and two of which have been previously implicated (6p21.32-p22.1 and 18q21.2). The strongest new finding (P = 1.6 × 10(-11)) was with rs1625579 within an intron of a putative primary transcript for MIR137 (microRNA 137), a known regulator of neuronal development. Four other schizophrenia loci achieving genome-wide significance contain predicted targets of MIR137, suggesting MIR137-mediated dysregulation as a previously unknown etiologic mechanism in schizophrenia. In a joint analysis with a bipolar disorder sample (16,374 affected individuals and 14,044 controls), three loci reached genome-wide significance: CACNA1C (rs4765905, P = 7.0 × 10(-9)), ANK3 (rs10994359, P = 2.5 × 10(-8)) and the ITIH3-ITIH4 region (rs2239547, P = 7.8 × 10(-9)).

Evaluation of FTA ® paper for storage of oral meta-genomic DNA.

Science.gov (United States)

Foitzik, Magdalena; Stumpp, Sascha N; Grischke, Jasmin; Eberhard, Jörg; Stiesch, Meike

2014-10-01

The purpose of the present study was to evaluate the short-term storage of meta-genomic DNA from native oral biofilms on FTA(®) paper. Thirteen volunteers of both sexes received an acrylic splint for intraoral biofilm formation over a period of 48 hours. The biofilms were collected, resuspended in phosphate-buffered saline, and either stored on FTA(®) paper or directly processed by standard laboratory DNA extraction. The nucleic acid extraction efficiencies were evaluated by 16S rDNA targeted SSCP fingerprinting. The acquired banding pattern of FTA-derived meta-genomic DNA was compared to a standard DNA preparation protocol. Sensitivity and positive predictive values were calculated. The volunteers showed inter-individual differences in their bacterial species composition. A total of 200 bands were found for both methods and 85% of the banding patterns were equal, representing a sensitivity of 0.941 and a false-negative predictive value of 0.059. Meta-genomic DNA sampling, extraction, and adhesion using FTA(®) paper is a reliable method for storage of microbial DNA for a short period of time.

[Whole Genome Sequencing of Human mtDNA Based on Ion Torrent PGM™ Platform].

Science.gov (United States)

Cao, Y; Zou, K N; Huang, J P; Ma, K; Ping, Y

2017-08-01

To analyze and detect the whole genome sequence of human mitochondrial DNA （mtDNA） by Ion Torrent PGM™ platform and to study the differences of mtDNA sequence in different tissues. Samples were collected from 6 unrelated individuals by forensic postmortem examination, including chest blood, hair, costicartilage, nail, skeletal muscle and oral epithelium. Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer. Libraries were constructed with Ion Shear™ Plus Reagents kit and Ion Plus Fragment Library kit. Whole genome sequencing of mtDNA was performed using Ion Torrent PGM™ platform. Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HVⅠ region. The whole genome sequence of mtDNA from all samples were amplified successfully. Six unrelated individuals belonged to 6 different haplotypes. Different tissues in one individual had heteroplasmy difference. The heteroplasmy positions and the mutation positions on HVⅠ region were verified by Sanger sequencing. After a consistency check by the Kappa method, it was found that the results of mtDNA sequence had a high consistency in different tissues. The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues, which have good consistency. The results provide guidance for the further applications of mtDNA in forensic science. Copyright© by the Editorial Department of Journal of Forensic Medicine

Study on the relationship between DNA-PKcs and genomic instability and hyper-radiosensitivity

International Nuclear Information System (INIS)

Yang Kang; Zhu Jiayun; Ding Nan; Li Junhong; Hu Wentao; Su Fengtao; He Jinpeng; Li Sha

2010-01-01

To investigate the relationship between DNA-PKcs and genome instability and hyper-radiosensitivity, human glioma cell lines M059K and M059J, as a model expressing wild-type DNA-PKcs and a model defective in DNA-PKcs activity, were exposed to low doses of X-rays. Cells survival fractions were assessed by colony-forming assay and Cytochalasin-B micronucleus assay was employed to detect the genomic instability happening in each single irradiated colony. It has been found that as the post-incubation time increased, M059K cells expressing wild-type DNA-PKcs exhibited low-dose hyper-radiosensitivity and showed a similar genomic instability after 0.2 Gy and 0.6 Gy irradiations, but the M059J cells lacking in DNA-PKcs didn't present low-dose hyper-radiosensitivity and showed a higher genomic instability of 0.6 Gy than that of 0.2 Gy. The results indicate that DNA-PKcs may act as one of the key factors that lead to low-dose hyper-radiosensitivity. (authors)

Genome-wide DNA polymorphism in the indica rice varieties RGD-7S and Taifeng B as revealed by whole genome re-sequencing.

Science.gov (United States)

Fu, Chong-Yun; Liu, Wu-Ge; Liu, Di-Lin; Li, Ji-Hua; Zhu, Man-Shan; Liao, Yi-Long; Liu, Zhen-Rong; Zeng, Xue-Qin; Wang, Feng

2016-03-01

Next-generation sequencing technologies provide opportunities to further understand genetic variation, even within closely related cultivars. We performed whole genome resequencing of two elite indica rice varieties, RGD-7S and Taifeng B, whose F1 progeny showed hybrid weakness and hybrid vigor when grown in the early- and late-cropping seasons, respectively. Approximately 150 million 100-bp pair-end reads were generated, which covered ∼86% of the rice (Oryza sativa L. japonica 'Nipponbare') reference genome. A total of 2,758,740 polymorphic sites including 2,408,845 SNPs and 349,895 InDels were detected in RGD-7S and Taifeng B, respectively. Applying stringent parameters, we identified 961,791 SNPs and 46,640 InDels between RGD-7S and Taifeng B (RGD-7S/Taifeng B). The density of DNA polymorphisms was 256.8 SNPs and 12.5 InDels per 100 kb for RGD-7S/Taifeng B. Copy number variations (CNVs) were also investigated. In RGD-7S, 1989 of 2727 CNVs were overlapped in 218 genes, and 1231 of 2010 CNVs were annotated in 175 genes in Taifeng B. In addition, we verified a subset of InDels in the interval of hybrid weakness genes, Hw3 and Hw4, and obtained some polymorphic InDel markers, which will provide a sound foundation for cloning hybrid weakness genes. Analysis of genomic variations will also contribute to understanding the genetic basis of hybrid weakness and heterosis.

Isolation of Retroelement from Plant Genomic DNA

OpenAIRE

sprotocols

2014-01-01

Author: Pat Heslop-Harrison ### Abstract: Retroelements and their derivatives are an ubiquitous and abundant component of plant genomes. From the 1990s, PCR based techniques have been developed to isolate the elements from genomic DNA of different plants, and the methods and primers used are presented here. Major classes of retroelements include the Ty1-copia, the Ty3-gypsy and the LINE (non-LTR) groups. Mixed PCR products representing the full heterogeneous pool of retrotransposo...

Comparison of protocols for genomic DNA extraction from 'velame ...

African Journals Online (AJOL)

usuario

2013-07-24

Jul 24, 2013 ... involving C. linearifolius, we compared the efficiency of six protocols for genomic DNA extraction previously ... phytic, with diverse aspect and floristics, average rainfall between ..... The variation observed for DNA concentrations estimated with .... performed with protocol 1 (data not shown), or still, bands.

FGWAS: Functional genome wide association analysis.

Science.gov (United States)

Huang, Chao; Thompson, Paul; Wang, Yalin; Yu, Yang; Zhang, Jingwen; Kong, Dehan; Colen, Rivka R; Knickmeyer, Rebecca C; Zhu, Hongtu

2017-10-01

Functional phenotypes (e.g., subcortical surface representation), which commonly arise in imaging genetic studies, have been used to detect putative genes for complexly inherited neuropsychiatric and neurodegenerative disorders. However, existing statistical methods largely ignore the functional features (e.g., functional smoothness and correlation). The aim of this paper is to develop a functional genome-wide association analysis (FGWAS) framework to efficiently carry out whole-genome analyses of functional phenotypes. FGWAS consists of three components: a multivariate varying coefficient model, a global sure independence screening procedure, and a test procedure. Compared with the standard multivariate regression model, the multivariate varying coefficient model explicitly models the functional features of functional phenotypes through the integration of smooth coefficient functions and functional principal component analysis. Statistically, compared with existing methods for genome-wide association studies (GWAS), FGWAS can substantially boost the detection power for discovering important genetic variants influencing brain structure and function. Simulation studies show that FGWAS outperforms existing GWAS methods for searching sparse signals in an extremely large search space, while controlling for the family-wise error rate. We have successfully applied FGWAS to large-scale analysis of data from the Alzheimer's Disease Neuroimaging Initiative for 708 subjects, 30,000 vertices on the left and right hippocampal surfaces, and 501,584 SNPs. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome-wide SNP and haplotype analyses reveal a rich history underlying dog domestication

Science.gov (United States)

vonHoldt, Bridgett M.; Pollinger, John P.; Lohmueller, Kirk E.; Han, Eunjung; Parker, Heidi G.; Quignon, Pascale; Degenhardt, Jeremiah D.; Boyko, Adam R.; Earl, Dent A.; Auton, Adam; Reynolds, Andy; Bryc, Kasia; Brisbin, Abra; Knowles, James C.; Mosher, Dana S.; Spady, Tyrone C.; Elkahloun, Abdel; Geffen, Eli; Pilot, Malgorzata; Jedrzejewski, Wlodzimierz; Greco, Claudia; Randi, Ettore; Bannasch, Danika; Wilton, Alan; Shearman, Jeremy; Musiani, Marco; Cargill, Michelle; Jones, Paul G.; Qian, Zuwei; Huang, Wei; Ding, Zhao-Li; Zhang, Ya-ping; Bustamante, Carlos D.; Ostrander, Elaine A.; Novembre, John; Wayne, Robert K.

2010-01-01

Advances in genome technology have facilitated a new understanding of the historical and genetic processes crucial to rapid phenotypic evolution under domestication1,2. To understand the process of dog diversification better, we conducted an extensive genome-wide survey of more than 48,000 single nucleotide polymorphisms in dogs and their wild progenitor, the grey wolf. Here we show that dog breeds share a higher proportion of multi-locus haplotypes unique to grey wolves from the Middle East, indicating that they are a dominant source of genetic diversity for dogs rather than wolves from east Asia, as suggested by mitochondrial DNA sequence data3. Furthermore, we find a surprising correspondence between genetic and phenotypic/functional breed groupings but there are exceptions that suggest phenotypic diversification depended in part on the repeated crossing of individuals with novel phenotypes. Our results show that Middle Eastern wolves were a critical source of genome diversity, although interbreeding with local wolf populations clearly occurred elsewhere in the early history of specific lineages. More recently, the evolution of modern dog breeds seems to have been an iterative process that drew on a limited genetic toolkit to create remarkable phenotypic diversity. PMID:20237475

Identification of Genetic Susceptibility Loci for Colorectal Tumors in a Genome-Wide Meta-analysis.

Science.gov (United States)

Peters, Ulrike; Jiao, Shuo; Schumacher, Fredrick R; Hutter, Carolyn M; Aragaki, Aaron K; Baron, John A; Berndt, Sonja I; Bézieau, Stéphane; Brenner, Hermann; Butterbach, Katja; Caan, Bette J; Campbell, Peter T; Carlson, Christopher S; Casey, Graham; Chan, Andrew T; Chang-Claude, Jenny; Chanock, Stephen J; Chen, Lin S; Coetzee, Gerhard A; Coetzee, Simon G; Conti, David V; Curtis, Keith R; Duggan, David; Edwards, Todd; Fuchs, Charles S; Gallinger, Steven; Giovannucci, Edward L; Gogarten, Stephanie M; Gruber, Stephen B; Haile, Robert W; Harrison, Tabitha A; Hayes, Richard B; Henderson, Brian E; Hoffmeister, Michael; Hopper, John L; Hudson, Thomas J; Hunter, David J; Jackson, Rebecca D; Jee, Sun Ha; Jenkins, Mark A; Jia, Wei-Hua; Kolonel, Laurence N; Kooperberg, Charles; Küry, Sébastien; Lacroix, Andrea Z; Laurie, Cathy C; Laurie, Cecelia A; Le Marchand, Loic; Lemire, Mathieu; Levine, David; Lindor, Noralane M; Liu, Yan; Ma, Jing; Makar, Karen W; Matsuo, Keitaro; Newcomb, Polly A; Potter, John D; Prentice, Ross L; Qu, Conghui; Rohan, Thomas; Rosse, Stephanie A; Schoen, Robert E; Seminara, Daniela; Shrubsole, Martha; Shu, Xiao-Ou; Slattery, Martha L; Taverna, Darin; Thibodeau, Stephen N; Ulrich, Cornelia M; White, Emily; Xiang, Yongbing; Zanke, Brent W; Zeng, Yi-Xin; Zhang, Ben; Zheng, Wei; Hsu, Li

2013-04-01

Heritable factors contribute to the development of colorectal cancer. Identifying the genetic loci associated with colorectal tumor formation could elucidate the mechanisms of pathogenesis. We conducted a genome-wide association study that included 14 studies, 12,696 cases of colorectal tumors (11,870 cancer, 826 adenoma), and 15,113 controls of European descent. The 10 most statistically significant, previously unreported findings were followed up in 6 studies; these included 3056 colorectal tumor cases (2098 cancer, 958 adenoma) and 6658 controls of European and Asian descent. Based on the combined analysis, we identified a locus that reached the conventional genome-wide significance level at less than 5.0 × 10(-8): an intergenic region on chromosome 2q32.3, close to nucleic acid binding protein 1 (most significant single nucleotide polymorphism: rs11903757; odds ratio [OR], 1.15 per risk allele; P = 3.7 × 10(-8)). We also found evidence for 3 additional loci with P values less than 5.0 × 10(-7): a locus within the laminin gamma 1 gene on chromosome 1q25.3 (rs10911251; OR, 1.10 per risk allele; P = 9.5 × 10(-8)), a locus within the cyclin D2 gene on chromosome 12p13.32 (rs3217810 per risk allele; OR, 0.84; P = 5.9 × 10(-8)), and a locus in the T-box 3 gene on chromosome 12q24.21 (rs59336; OR, 0.91 per risk allele; P = 3.7 × 10(-7)). In a large genome-wide association study, we associated polymorphisms close to nucleic acid binding protein 1 (which encodes a DNA-binding protein involved in DNA repair) with colorectal tumor risk. We also provided evidence for an association between colorectal tumor risk and polymorphisms in laminin gamma 1 (this is the second gene in the laminin family to be associated with colorectal cancers), cyclin D2 (which encodes for cyclin D2), and T-box 3 (which encodes a T-box transcription factor and is a target of Wnt signaling to β-catenin). The roles of these genes and their products in cancer pathogenesis warrant further

DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis.

Science.gov (United States)

Enriquez-Rios, Vanessa; Dumitrache, Lavinia C; Downing, Susanna M; Li, Yang; Brown, Eric J; Russell, Helen R; McKinnon, Peter J

2017-01-25

The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G 2 /M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system. The DNA damage response (DDR) is essential for prevention of a broad spectrum of different human neurologic diseases. However, a detailed understanding of the DDR at a physiological level is lacking. In contrast to many in

Studies on the effects of persistent RNA priming on DNA replication and genomic stability

OpenAIRE

Stuckey, Ruth

2014-01-01

[EN]: DNA replication and transcription take place on the same DNA template, and the correct interplay between these processes ensures faithful genome duplication. DNA replication must be highly coordinated with other cell cycle events, such as segregation of fully replicated DNA in order to maintain genomic integrity. Transcription generates RNA:DNA hybrids, transient intermediate structures that are degraded by the ribonuclease H (RNaseH) class of enzymes. RNA:DNA hybrids can form R-loops, ...

Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner

DEFF Research Database (Denmark)

Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan

2009-01-01

MOTIVATION: The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary...... determinant of alignment accuracy, while heuristics that prevent consideration of certain alignments are a primary determinant of runtime and memory usage. Both accuracy and speed are important considerations in choosing an alignment algorithm, but scoring systems have received much less attention than...

Genome-Wide Mapping of 5mC and 5hmC Identified Differentially Modified Genomic Regions in Late-Onset Severe Preeclampsia: A Pilot Study.

Directory of Open Access Journals (Sweden)

Lisha Zhu

Full Text Available Preeclampsia (PE is a leading cause of perinatal morbidity and mortality. However, as a common form of PE, the etiology of late-onset PE is elusive. We analyzed 5-methylcytosine (5mC and 5-hydroxymethylcytosine (5hmC levels in the placentas of late-onset severe PE patients (n = 4 and normal controls (n = 4 using a (hydroxymethylated DNA immunoprecipitation approach combined with deep sequencing ([h]MeDIP-seq, and the results were verified by (hMeDIP-qPCR. The most significant differentially methylated regions (DMRs were verified by MassARRAY EppiTYPER in an enlarged sample size (n = 20. Bioinformatics analysis identified 714 peaks of 5mC that were associated with 403 genes and 119 peaks of 5hmC that were associated with 61 genes, thus showing significant differences between the PE patients and the controls (>2-fold, p<0.05. Further, only one gene, PTPRN2, had both 5mC and 5hmC changes in patients. The ErbB signaling pathway was enriched in those 403 genes that had significantly different 5mC level between the groups. This genome-wide mapping of 5mC and 5hmC in late-onset severe PE and normal controls demonstrates that both 5mC and 5hmC play epigenetic roles in the regulation of the disease, but work independently. We reveal the genome-wide mapping of DNA methylation and DNA hydroxymethylation in late-onset PE placentas for the first time, and the identified ErbB signaling pathway and the gene PTPRN2 may be relevant to the epigenetic pathogenesis of late-onset PE.

Meta-analysis of genome-wide association from genomic prediction models

Science.gov (United States)

A limitation of many genome-wide association studies (GWA) in animal breeding is that there are many loci with small effect sizes; thus, larger sample sizes (N) are required to guarantee suitable power of detection. To increase sample size, results from different GWA can be combined in a meta-analys...

Epigenome-wide association study of DNA methylation in narcolepsy: an integrated genetic and epigenetic approach.

Science.gov (United States)

Shimada, Mihoko; Miyagawa, Taku; Toyoda, Hiromi; Tokunaga, Katsushi; Honda, Makoto

2018-04-01

Narcolepsy with cataplexy, which is a hypersomnia characterized by excessive daytime sleepiness and cataplexy, is a multifactorial disease caused by both genetic and environmental factors. Several genetic factors including HLA-DQB1*06:02 have been identified; however, the disease etiology is still unclear. Epigenetic modifications, such as DNA methylation, have been suggested to play an important role in the pathogenesis of complex diseases. Here, we examined DNA methylation profiles of blood samples from narcolepsy and healthy control individuals and performed an epigenome-wide association study (EWAS) to investigate methylation loci associated with narcolepsy. Moreover, data from the EWAS and a previously performed narcolepsy genome-wide association study were integrated to search for methylation loci with causal links to the disease. We found that (1) genes annotated to the top-ranked differentially methylated positions (DMPs) in narcolepsy were associated with pathways of hormone secretion and monocarboxylic acid metabolism. (2) Top-ranked narcolepsy-associated DMPs were significantly more abundant in non-CpG island regions and more than 95 per cent of such sites were hypomethylated in narcolepsy patients. (3) The integrative analysis identified the CCR3 region where both a single methylation site and multiple single-nucleotide polymorphisms were found to be associated with the disease as a candidate region responsible for narcolepsy. The findings of this study suggest the importance of future replication studies, using methylation technologies with wider genome coverage and/or larger number of samples, to confirm and expand on these results.

An automated annotation tool for genomic DNA sequences using

Indian Academy of Sciences (India)

Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated ...

Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

International Nuclear Information System (INIS)

Okamura, K; Hisada, T; Takata, K; Hiraishi, A

2013-01-01

Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

The Mapping of Predicted Triplex DNA:RNA in the Drosophila Genome Reveals a Prominent Location in Development- and Morphogenesis-Related Genes

Directory of Open Access Journals (Sweden)

Claude Pasquier

2017-07-01

Full Text Available Double-stranded DNA is able to form triple-helical structures by accommodating a third nucleotide strand. A nucleic acid triplex occurs according to Hoogsteen rules that predict the stability and affinity of the third strand bound to the Watson–Crick duplex. The “triplex-forming oligonucleotide” (TFO can be a short sequence of RNA that binds to the major groove of the targeted duplex only when this duplex presents a sequence of purine or pyrimidine bases in one of the DNA strands. Many nuclear proteins are known to bind triplex DNA or DNA:RNA, but their biological functions are unexplored. We identified sequences that are capable of engaging as the “triplex-forming oligonucleotide” in both the pre-lncRNA and pre-mRNA collections of Drosophila melanogaster. These motifs were matched against the Drosophila genome in order to identify putative sequences of triplex formation in intergenic regions, promoters, and introns/exons. Most of the identified TFOs appear to be located in the intronic region of the analyzed genes. Computational prediction of the most targeted genes by TFOs originating from pre-lncRNAs and pre-mRNAs revealed that they are restrictively associated with development- and morphogenesis-related gene networks. The refined analysis by Gene Ontology enrichment demonstrates that some individual TFOs present genome-wide scale matches that are located in numerous genes and regulatory sequences. The triplex DNA:RNA computational mapping at the genome-wide scale suggests broad interference in the regulatory process of the gene networks orchestrated by TFO RNAs acting in association simultaneously at multiple sites.

Revisiting the classification of curtoviruses based on genome-wide pairwise identity

KAUST Repository

Varsani, Arvind; Martin, Darren Patrick; Navas-Castillo, Jesú s; Moriones, Enrique; Herná ndez-Zepeda, Cecilia; Idris, Ali; Murilo Zerbini, F.; Brown, Judith K.

2014-01-01

Members of the genus Curtovirus (family Geminiviridae) are important pathogens of many wild and cultivated plant species. Until recently, relatively few full curtovirus genomes have been characterised. However, with the 19 full genome sequences now available in public databases, we revisit the proposed curtovirus species and strain classification criteria. Using pairwise identities coupled with phylogenetic evidence, revised species and strain demarcation guidelines have been instituted. Specifically, we have established 77% genome-wide pairwise identity as a species demarcation threshold and 94% genome-wide pairwise identity as a strain demarcation threshold. Hence, whereas curtovirus sequences with >77% genome-wide pairwise identity would be classified as belonging to the same species, those sharing >94% identity would be classified as belonging to the same strain. We provide step-by-step guidelines to facilitate the classification of newly discovered curtovirus full genome sequences and a set of defined criteria for naming new species and strains. The revision yields three curtovirus species: Beet curly top virus (BCTV), Spinach severe surly top virus (SpSCTV) and Horseradish curly top virus (HrCTV). © 2014 Springer-Verlag Wien.

Revisiting the classification of curtoviruses based on genome-wide pairwise identity

KAUST Repository

Varsani, Arvind

2014-01-25

Members of the genus Curtovirus (family Geminiviridae) are important pathogens of many wild and cultivated plant species. Until recently, relatively few full curtovirus genomes have been characterised. However, with the 19 full genome sequences now available in public databases, we revisit the proposed curtovirus species and strain classification criteria. Using pairwise identities coupled with phylogenetic evidence, revised species and strain demarcation guidelines have been instituted. Specifically, we have established 77% genome-wide pairwise identity as a species demarcation threshold and 94% genome-wide pairwise identity as a strain demarcation threshold. Hence, whereas curtovirus sequences with >77% genome-wide pairwise identity would be classified as belonging to the same species, those sharing >94% identity would be classified as belonging to the same strain. We provide step-by-step guidelines to facilitate the classification of newly discovered curtovirus full genome sequences and a set of defined criteria for naming new species and strains. The revision yields three curtovirus species: Beet curly top virus (BCTV), Spinach severe surly top virus (SpSCTV) and Horseradish curly top virus (HrCTV). © 2014 Springer-Verlag Wien.

Resurrection of DNA function in vivo from an extinct genome.

Directory of Open Access Journals (Sweden)

Andrew J Pask

2008-05-01

Full Text Available There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine, obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

Science.gov (United States)

Chan, Yi-Wah; Mohr, Remus; Millard, Andrew D; Holmes, Antony B; Larkum, Anthony W; Whitworth, Anna L; Mann, Nicholas H; Scanlan, David J; Hess, Wolfgang R; Clokie, Martha R J

2011-08-01

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

a potential source of spurious associations in genome-wide ...

Indian Academy of Sciences (India)

2010-04-01

Apr 1, 2010 ... Genome-wide association studies (GWAS) examine the entire human genome with the goal of identifying genetic variants. (usually single nucleotide polymorphisms (SNPs)) that are associated with phenotypic traits such as disease status and drug response. The discordance of significantly associated ...

Genome-wide association study of smoking initiation and current smoking

DEFF Research Database (Denmark)

Vink, Jacqueline M; Smit, August B; de Geus, Eco J C

2009-01-01

For the identification of genes associated with smoking initiation and current smoking, genome-wide association analyses were carried out in 3497 subjects. Significant genes that replicated in three independent samples (n = 405, 5810, and 1648) were visualized into a biologically meaningful network......) and cell-adhesion molecules (e.g., CDH23). We conclude that a network-based genome-wide association approach can identify genes influencing smoking behavior....

Suicidal function of DNA methylation in age-related genome disintegration.

Science.gov (United States)

Mazin, Alexander L

2009-10-01

This article is dedicated to the 60th anniversary of 5-methylcytosine discovery in DNA. Cytosine methylation can affect genetic and epigenetic processes, works as a part of the genome-defense system and has mutagenic activity; however, the biological functions of this enzymatic modification are not well understood. This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome. The proposed model considers DNA methylation as the generator of 5mC>T transitions that induce 40-70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes. The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.

A genome-wide association study of aging.

Science.gov (United States)

Walter, Stefan; Atzmon, Gil; Demerath, Ellen W; Garcia, Melissa E; Kaplan, Robert C; Kumari, Meena; Lunetta, Kathryn L; Milaneschi, Yuri; Tanaka, Toshiko; Tranah, Gregory J; Völker, Uwe; Yu, Lei; Arnold, Alice; Benjamin, Emelia J; Biffar, Reiner; Buchman, Aron S; Boerwinkle, Eric; Couper, David; De Jager, Philip L; Evans, Denis A; Harris, Tamara B; Hoffmann, Wolfgang; Hofman, Albert; Karasik, David; Kiel, Douglas P; Kocher, Thomas; Kuningas, Maris; Launer, Lenore J; Lohman, Kurt K; Lutsey, Pamela L; Mackenbach, Johan; Marciante, Kristin; Psaty, Bruce M; Reiman, Eric M; Rotter, Jerome I; Seshadri, Sudha; Shardell, Michelle D; Smith, Albert V; van Duijn, Cornelia; Walston, Jeremy; Zillikens, M Carola; Bandinelli, Stefania; Baumeister, Sebastian E; Bennett, David A; Ferrucci, Luigi; Gudnason, Vilmundur; Kivimaki, Mika; Liu, Yongmei; Murabito, Joanne M; Newman, Anne B; Tiemeier, Henning; Franceschini, Nora

2011-11-01

Human longevity and healthy aging show moderate heritability (20%-50%). We conducted a meta-analysis of genome-wide association studies from 9 studies from the Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium for 2 outcomes: (1) all-cause mortality, and (2) survival free of major disease or death. No single nucleotide polymorphism (SNP) was a genome-wide significant predictor of either outcome (p < 5 × 10(-8)). We found 14 independent SNPs that predicted risk of death, and 8 SNPs that predicted event-free survival (p < 10(-5)). These SNPs are in or near genes that are highly expressed in the brain (HECW2, HIP1, BIN2, GRIA1), genes involved in neural development and function (KCNQ4, LMO4, GRIA1, NETO1) and autophagy (ATG4C), and genes that are associated with risk of various diseases including cancer and Alzheimer's disease. In addition to considerable overlap between the traits, pathway and network analysis corroborated these findings. These findings indicate that variation in genes involved in neurological processes may be an important factor in regulating aging free of major disease and achieving longevity. Copyright © 2011 Elsevier Inc. All rights reserved.

Gigwa-Genotype investigator for genome-wide analyses.

Science.gov (United States)

Sempéré, Guilhem; Philippe, Florian; Dereeper, Alexis; Ruiz, Manuel; Sarah, Gautier; Larmande, Pierre

2016-06-06

Exploring the structure of genomes and analyzing their evolution is essential to understanding the ecological adaptation of organisms. However, with the large amounts of data being produced by next-generation sequencing, computational challenges arise in terms of storage, search, sharing, analysis and visualization. This is particularly true with regards to studies of genomic variation, which are currently lacking scalable and user-friendly data exploration solutions. Here we present Gigwa, a web-based tool that provides an easy and intuitive way to explore large amounts of genotyping data by filtering it not only on the basis of variant features, including functional annotations, but also on genotype patterns. The data storage relies on MongoDB, which offers good scalability properties. Gigwa can handle multiple databases and may be deployed in either single- or multi-user mode. In addition, it provides a wide range of popular export formats. The Gigwa application is suitable for managing large amounts of genomic variation data. Its user-friendly web interface makes such processing widely accessible. It can either be simply deployed on a workstation or be used to provide a shared data portal for a given community of researchers.

Susceptibility to Childhood Pneumonia: A Genome-Wide Analysis.

Science.gov (United States)

Hayden, Lystra P; Cho, Michael H; McDonald, Merry-Lynn N; Crapo, James D; Beaty, Terri H; Silverman, Edwin K; Hersh, Craig P

2017-01-01

Previous studies have indicated that in adult smokers, a history of childhood pneumonia is associated with reduced lung function and chronic obstructive pulmonary disease. There have been few previous investigations using genome-wide association studies to investigate genetic predisposition to pneumonia. This study aims to identify the genetic variants associated with the development of pneumonia during childhood and over the course of the lifetime. Study subjects included current and former smokers with and without chronic obstructive pulmonary disease participating in the COPDGene Study. Pneumonia was defined by subject self-report, with childhood pneumonia categorized as having the first episode at pneumonia (843 cases, 9,091 control subjects) and lifetime pneumonia (3,766 cases, 5,659 control subjects) were performed separately in non-Hispanic whites and African Americans. Non-Hispanic white and African American populations were combined in the meta-analysis. Top genetic variants from childhood pneumonia were assessed in network analysis. No single-nucleotide polymorphisms reached genome-wide significance, although we identified potential regions of interest. In the childhood pneumonia analysis, this included variants in NGR1 (P = 6.3 × 10 -8 ), PAK6 (P = 3.3 × 10 -7 ), and near MATN1 (P = 2.8 × 10 -7 ). In the lifetime pneumonia analysis, this included variants in LOC339862 (P = 8.7 × 10 -7 ), RAPGEF2 (P = 8.4 × 10 -7 ), PHACTR1 (P = 6.1 × 10 -7 ), near PRR27 (P = 4.3 × 10 -7 ), and near MCPH1 (P = 2.7 × 10 -7 ). Network analysis of the genes associated with childhood pneumonia included top networks related to development, blood vessel morphogenesis, muscle contraction, WNT signaling, DNA damage, apoptosis, inflammation, and immune response (P ≤ 0.05). We have identified genes potentially associated with the risk of pneumonia. Further research will be required to confirm these

Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets.

Science.gov (United States)

Kline, Margaret C; Romsos, Erica L; Duewer, David L

2016-02-16

Polymerase chain reaction (PCR) multiplexed assays perform best when the input quantity of template DNA is controlled to within about a factor of √2. To help ensure that PCR assays yield consistent results over time and place, results from methods used to determine DNA quantity need to be metrologically traceable to a common reference. Many DNA quantitation systems can be accurately calibrated with solutions of DNA in aqueous buffer. Since they do not require external calibration, end-point limiting dilution technologies, collectively termed "digital PCR (dPCR)", have been proposed as suitable for value assigning such DNA calibrants. The performance characteristics of several commercially available dPCR systems have recently been documented using plasmid, viral, or fragmented genomic DNA; dPCR performance with more complex materials, such as human genomic DNA, has been less studied. With the goal of providing a human genomic reference material traceably certified for mass concentration, we are investigating the measurement characteristics of several dPCR systems. We here report results of measurements from multiple PCR assays, on four human genomic DNAs treated with four endonuclease restriction enzymes using both chamber and droplet dPCR platforms. We conclude that dPCR does not estimate the absolute number of PCR targets in a given volume but rather the number of accessible and amplifiable targets. While enzymatic restriction of human genomic DNA increases accessibility for some assays, in well-optimized PCR assays it can reduce the number of amplifiable targets and increase assay variability relative to uncut sample.

Effect of nickel chloride on Arabidopsis genomic DNA and methylation of 18S rDNA

Directory of Open Access Journals (Sweden)

Zhongai Li

2015-01-01

Conclusions: NiCl2 application caused variation of DNA methylation of the Arabidopsis genomic and offspring's. NiCl2 also resulted in nucleolar injury and deformity of root tip cells. The methylation rate of 18S rDNA also changed by adding NiCl2.

cDNA structure, genomic organization and expression patterns of ...

African Journals Online (AJOL)

Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin cDNA cloned from the liver was ...

RAD-seq derived genome-wide nuclear markers resolve the phylogeny of tunas

KAUST Repository

Díaz-Arce, Natalia

2016-06-07

Although species from the genus Thunnus include some of the most commercially important and most severely overexploited fishes, the phylogeny of this genus is still unresolved, hampering evolutionary and traceability studies that could help improve conservation and management strategies for these species. Previous attempts based on mitochondrial and nuclear markers were unsuccessful in inferring a congruent and reliable phylogeny, probably due to mitochondrial introgression events and lack of enough phylogenetically informative markers. Here we infer the first genome-wide nuclear marker-based phylogeny of tunas using restriction site associated DNA sequencing (RAD-seq) data. Our results, derived from phylogenomic inferences obtained from 128 nucleotide matrices constructed using alternative data assembly procedures, support a single Thunnus evolutionary history that challenges previous assumptions based on morphological and molecular data.

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

Science.gov (United States)

Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

2015-11-20

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

Reconstructing Roma history from genome-wide data.

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Priya Moorjani

Full Text Available The Roma people, living throughout Europe and West Asia, are a diverse population linked by the Romani language and culture. Previous linguistic and genetic studies have suggested that the Roma migrated into Europe from South Asia about 1,000-1,500 years ago. Genetic inferences about Roma history have mostly focused on the Y chromosome and mitochondrial DNA. To explore what additional information can be learned from genome-wide data, we analyzed data from six Roma groups that we genotyped at hundreds of thousands of single nucleotide polymorphisms (SNPs. We estimate that the Roma harbor about 80% West Eurasian ancestry-derived from a combination of European and South Asian sources-and that the date of admixture of South Asian and European ancestry was about 850 years before present. We provide evidence for Eastern Europe being a major source of European ancestry, and North-west India being a major source of the South Asian ancestry in the Roma. By computing allele sharing as a measure of linkage disequilibrium, we estimate that the migration of Roma out of the Indian subcontinent was accompanied by a severe founder event, which appears to have been followed by a major demographic expansion after the arrival in Europe.

Genome-Wide Locations of Potential Epimutations Associated with Environmentally Induced Epigenetic Transgenerational Inheritance of Disease Using a Sequential Machine Learning Prediction Approach.

Science.gov (United States)

Haque, M Muksitul; Holder, Lawrence B; Skinner, Michael K

2015-01-01

Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation involves germline transmitted epimutations. The primary epimutations identified involve altered differential DNA methylation regions (DMRs). Different environmental toxicants have been shown to promote exposure (i.e., toxicant) specific signatures of germline epimutations. Analysis of genomic features associated with these epimutations identified low-density CpG regions (machine learning computational approach to predict all potential epimutations in the genome. A number of previously identified sperm epimutations were used as training sets. A novel machine learning approach using a sequential combination of Active Learning and Imbalance Class Learner analysis was developed. The transgenerational sperm epimutation analysis identified approximately 50K individual sites with a 1 kb mean size and 3,233 regions that had a minimum of three adjacent sites with a mean size of 3.5 kb. A select number of the most relevant genomic features were identified with the low density CpG deserts being a critical genomic feature of the features selected. A similar independent analysis with transgenerational somatic cell epimutation training sets identified a smaller number of 1,503 regions of genome-wide predicted sites and differences in genomic feature contributions. The predicted genome-wide germline (sperm) epimutations were found to be distinct from the predicted somatic cell epimutations. Validation of the genome-wide germline predicted sites used two recently identified transgenerational sperm epimutation signature sets from the pesticides dichlorodiphenyltrichloroethane (DDT) and methoxychlor (MXC) exposure lineage F3 generation. Analysis of this positive validation data set showed a 100% prediction accuracy for all the DDT-MXC sperm epimutations. Observations further elucidate the genomic features associated with transgenerational germline epimutations and identify a genome-wide

Genome-wide association study of Tourette's syndrome

NARCIS (Netherlands)

Scharf, J. M.; Yu, D.; Mathews, C. A.; Neale, B. M.; Stewart, S. E.; Fagerness, J. A.; Evans, P.; Gamazon, E.; Edlund, C. K.; Service, S. K.; Tikhomirov, A.; Osiecki, L.; Illmann, C.; Pluzhnikov, A.; Konkashbaev, A.; Davis, L. K.; Han, B.; Crane, J.; Moorjani, P.; Crenshaw, A. T.; Parkin, M. A.; Reus, V. I.; Lowe, T. L.; Rangel-Lugo, M.; Chouinard, S.; Dion, Y.; Girard, S.; Cath, D. C.; Smit, J. H.; King, R. A.; Fernandez, T. V.; Leckman, J. F.; Kidd, K. K.; Kidd, J. R.; Pakstis, A. J.; State, M. W.; Herrera, L. D.; Romero, R.; Fournier, E.; Sandor, P.; Barr, C. L.; Phan, N.; Gross-Tsur, V.; Benarroch, F.; Pollak, Y.; Budman, C. L.; Bruun, R. D.; Erenberg, G.; Naarden, A. L.; Hoekstra, P. J.

2013-01-01

Tourette's syndrome (TS) is a developmental disorder that has one of the highest familial recurrence rates among neuropsychiatric diseases with complex inheritance. However, the identification of definitive TS susceptibility genes remains elusive. Here, we report the first genome-wide association

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

KAUST Repository

Belfield, E.J.; Gan, X.; Mithani, A.; Brown, C.; Jiang, C.; Franklin, K.; Alvey, E.; Wibowo, A.; Jung, M.; Bailey, K.; Kalwani, S.; Ragoussis, J.; Mott, R.; Harberd, N.P.

2012-01-01

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

KAUST Repository

Belfield, E.J.

2012-04-12

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

Whole-genome methylation caller designed for methyl- DNA ...

African Journals Online (AJOL)

etchie

2013-02-20

Feb 20, 2013 ... Our method uses a single-CpG-resolution, whole-genome methylation ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, ...... methylation is prevalent in embryonic stem cells andmaybe mediated.

Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

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Yang Moon-Sik

2004-09-01

Full Text Available Abstract Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1. After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

De novo DNA methylation during monkey pre-implantation embryogenesis.

Science.gov (United States)

Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-04-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis.

TEGS-CN: A Statistical Method for Pathway Analysis of Genome-wide Copy Number Profile.

Science.gov (United States)

Huang, Yen-Tsung; Hsu, Thomas; Christiani, David C

2014-01-01

The effects of copy number alterations make up a significant part of the tumor genome profile, but pathway analyses of these alterations are still not well established. We proposed a novel method to analyze multiple copy numbers of genes within a pathway, termed Test for the Effect of a Gene Set with Copy Number data (TEGS-CN). TEGS-CN was adapted from TEGS, a method that we previously developed for gene expression data using a variance component score test. With additional development, we extend the method to analyze DNA copy number data, accounting for different sizes and thus various numbers of copy number probes in genes. The test statistic follows a mixture of X (2) distributions that can be obtained using permutation with scaled X (2) approximation. We conducted simulation studies to evaluate the size and the power of TEGS-CN and to compare its performance with TEGS. We analyzed a genome-wide copy number data from 264 patients of non-small-cell lung cancer. With the Molecular Signatures Database (MSigDB) pathway database, the genome-wide copy number data can be classified into 1814 biological pathways or gene sets. We investigated associations of the copy number profile of the 1814 gene sets with pack-years of cigarette smoking. Our analysis revealed five pathways with significant P values after Bonferroni adjustment (number data, and causal mechanisms of the five pathways require further study.

Continued colonization of the human genome by mitochondrial DNA.

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Miria Ricchetti

2004-09-01

Full Text Available Integration of mitochondrial DNA fragments into nuclear chromosomes (giving rise to nuclear DNA sequences of mitochondrial origin, or NUMTs is an ongoing process that shapes nuclear genomes. In yeast this process depends on double-strand-break repair. Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus. From sequence analysis of the genome of Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4-6 million years. Thus, we measured the fixation rate of NUMTs in the human genome. Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics. We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs. Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns. Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor. Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences. This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults. We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.

Genome dynamics of short oligonucleotides: the example of bacterial DNA uptake enhancing sequences.

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Mohammed Bakkali

Full Text Available Among the many bacteria naturally competent for transformation by DNA uptake-a phenomenon with significant clinical and financial implications- Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences. The genomic overrepresentation of these DNA uptake enhancing sequences (DUES causes preferential uptake of conspecific DNA, but the function(s behind this overrepresentation and its evolution are still a matter for discovery. Here I analyze DUES genome dynamics and evolution and test the validity of the results to other selectively constrained oligonucleotides. I use statistical methods and computer simulations to examine DUESs accumulation in Haemophilus influenzae and Neisseria gonorrhoeae genomes. I analyze DUESs sequence and nucleotide frequencies, as well as those of all their mismatched forms, and prove the dependence of DUESs genomic overrepresentation on their preferential uptake by quantifying and correlating both characteristics. I then argue that mutation, uptake bias, and weak selection against DUESs in less constrained parts of the genome combined are sufficient enough to cause DUESs accumulation in susceptible parts of the genome with no need for other DUES function. The distribution of overrepresentation values across sequences with different mismatch loads compared to the DUES suggests a gradual yet not linear molecular drive of DNA sequences depending on their similarity to the DUES. Other genomically overrepresented sequences, both pro- and eukaryotic, show similar distribution of frequencies suggesting that the molecular drive reported above applies to other frequent oligonucleotides. Rare oligonucleotides, however, seem to be gradually drawn to genomic underrepresentation, thus, suggesting a molecular drag. To my knowledge this work provides the first clear evidence of the gradual evolution of selectively constrained oligonucleotides, including repeated, palindromic and protein

Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

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Antoine Marmignon

2014-08-01

Full Text Available During somatic differentiation, physiological DNA double-strand breaks (DSB can drive programmed genome rearrangements (PGR, during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES. IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium

Genome-wide transcriptional reorganization associated with senescence-to-immortality switch during human hepatocellular carcinogenesis.

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Gokhan Yildiz

Full Text Available Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal" by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15

Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

Science.gov (United States)

Christen, Matthias; Del Medico, Luca; Christen, Heinz; Christen, Beat

2017-01-01

Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

Directory of Open Access Journals (Sweden)

Matthias Christen

Full Text Available Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

Altered DNA methylation associated with a translocation linked to major mental illness

OpenAIRE

McCartney, Daniel L; Walker, Rosie M; Morris, Stewart W; Anderson, Susan M; Duff, Barbara J; Marioni, Riccardo E; Millar, J Kirsty; McCarthy, Shane E; Ryan, Niamh M; Lawrie, Stephen M; Watson, Andrew R; Blackwood, Douglas H R; Thomson, Pippa A; McIntosh, Andrew M; McCombie, W Richard

2018-01-01

Recent work has highlighted a possible role for altered epigenetic modifications, including differential DNA methylation, in susceptibility to psychiatric illness. Here, we investigate blood-based DNA methylation in a large family where a balanced translocation between chromosomes 1 and 11 shows genome-wide significant linkage to psychiatric illness. Genome-wide DNA methylation was profiled in whole-blood-derived DNA from 41 individuals using the Infinium HumanMethylation450 BeadChip (Illumin...

Genome-wide linkage analysis for human longevity

DEFF Research Database (Denmark)

Beekman, Marian; Blanché, Hélène; Perola, Markus

2013-01-01

Clear evidence exists for heritability of human longevity, and much interest is focused on identifying genes associated with longer lives. To identify such longevity alleles, we performed the largest genome-wide linkage scan thus far reported. Linkage analyses included 2118 nonagenarian Caucasian...

Phylogeographic and genome-wide investigations of Vietnam ethnic groups reveal signatures of complex historical demographic movements.

Science.gov (United States)

Pischedda, S; Barral-Arca, R; Gómez-Carballa, A; Pardo-Seco, J; Catelli, M L; Álvarez-Iglesias, V; Cárdenas, J M; Nguyen, N D; Ha, H H; Le, A T; Martinón-Torres, F; Vullo, C; Salas, A

2017-10-03

The territory of present-day Vietnam was the cradle of one of the world's earliest civilizations, and one of the first world regions to develop agriculture. We analyzed the mitochondrial DNA (mtDNA) complete control region of six ethnic groups and the mitogenomes from Vietnamese in The 1000 Genomes Project (1000G). Genome-wide data from 1000G (~55k SNPs) were also investigated to explore different demographic scenarios. All Vietnamese carry South East Asian (SEA) haplotypes, which show a moderate geographic and ethnic stratification, with the Mong constituting the most distinctive group. Two new mtDNA clades (M7b1a1f1 and F1f1) point to historical gene flow between the Vietnamese and other neighboring countries. Bayesian-based inferences indicate a time-deep and continuous population growth of Vietnamese, although with some exceptions. The dramatic population decrease experienced by the Cham 700 years ago (ya) fits well with the Nam tiến ("southern expansion") southwards from their original heartland in the Red River Delta. Autosomal SNPs consistently point to important historical gene flow within mainland SEA, and add support to a main admixture event occurring between Chinese and a southern Asian ancestral composite (mainly represented by the Malay). This admixture event occurred ~800 ya, again coinciding with the Nam tiến.

Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

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Ben Beheshti

2003-01-01

Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

The Genomic Pattern of tDNA Operon Expression in E. coli.

Directory of Open Access Journals (Sweden)

2005-06-01

Full Text Available In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates.

Genome-wide comparative analysis of four Indian Drosophila species.

Science.gov (United States)

Mohanty, Sujata; Khanna, Radhika

2017-12-01

Comparative analysis of multiple genomes of closely or distantly related Drosophila species undoubtedly creates excitement among evolutionary biologists in exploring the genomic changes with an ecology and evolutionary perspective. We present herewith the de novo assembled whole genome sequences of four Drosophila species, D. bipectinata, D. takahashii, D. biarmipes and D. nasuta of Indian origin using Next Generation Sequencing technology on an Illumina platform along with their detailed assembly statistics. The comparative genomics analysis, e.g. gene predictions and annotations, functional and orthogroup analysis of coding sequences and genome wide SNP distribution were performed. The whole genome of Zaprionus indianus of Indian origin published earlier by us and the genome sequences of previously sequenced 12 Drosophila species available in the NCBI database were included in the analysis. The present work is a part of our ongoing genomics project of Indian Drosophila species.

Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner.

Science.gov (United States)

Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan; Brent, Michael R

2009-07-01

The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary determinant of alignment accuracy, while heuristics that prevent consideration of certain alignments are a primary determinant of runtime and memory usage. Both accuracy and speed are important considerations in choosing an alignment algorithm, but scoring systems have received much less attention than heuristics. We present Pairagon, a pair hidden Markov model based cDNA-to-genome alignment program, as the most accurate aligner for sequences with high- and low-identity levels. We conducted a series of experiments testing alignment accuracy with varying sequence identity. We first created 'perfect' simulated cDNA sequences by splicing the sequences of exons in the reference genome sequences of fly and human. The complete reference genome sequences were then mutated to various degrees using a realistic mutation simulator and the perfect cDNAs were aligned to them using Pairagon and 12 other aligners. To validate these results with natural sequences, we performed cross-species alignment using orthologous transcripts from human, mouse and rat. We found that aligner accuracy is heavily dependent on sequence identity. For sequences with 100% identity, Pairagon achieved accuracy levels of >99.6%, with one quarter of the errors of any other aligner. Furthermore, for human/mouse alignments, which are only 85% identical, Pairagon achieved 87% accuracy, higher than any other aligner. Pairagon source and executables are freely available at http://mblab.wustl.edu/software/pairagon/

Epigenetic control of mobile DNA as an interface between experience and genome change

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James A. Shapiro

2014-04-01

Full Text Available Mobile DNA in the genome is subject to RNA-targeted epigenetic control. This control regulates the activity of transposons, retrotransposons and genomic proviruses. Many different life history experiences alter the activities of mobile DNA and the expression of genetic loci regulated by nearby insertions. The same experiences induce alterations in epigenetic formatting and lead to trans-generational modifications of genome expression and stability. These observations lead to the hypothesis that epigenetic formatting directed by non-coding RNA provides a molecular interface between life history events and genome alteration.

Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

International Nuclear Information System (INIS)

Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

2005-01-01

Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [ 14 C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [ 14 C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 (micro)g of DNA. This sensitivity may allow the method to be used for clinical applications

Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

2005-12-29

Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.

Genome-wide association studies (GWAS) of adiposity

DEFF Research Database (Denmark)

Oskari Kilpeläinen, Tuomas; Ingelsson, Erik

2016-01-01

Adiposity is strongly heritable and one of the leading risk factors for type 2 diabetes, cardiovascular disease, cancer, and premature death. In the past 8 years, genome-wide association studies (GWAS) have greatly increased our understanding of the genes and biological pathways that regulate...

BuD, a helix–loop–helix DNA-binding domain for genome modification

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Stella, Stefano [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark); Molina, Rafael; López-Méndez, Blanca [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Campos-Olivas, Ramon [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Duchateau, Phillippe [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Montoya, Guillermo, E-mail: guillermo.montoya@cpr.ku.dk [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark)

2014-07-01

Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

Meta-analysis of Genome-Wide Association Studies for Extraversion

DEFF Research Database (Denmark)

van den Berg, Stéphanie M; de Moor, Marleen H M; Verweij, K. J. H.

2016-01-01

small sample sizes of those studies. Here, we report on a large meta-analysis of GWA studies for extraversion in 63,030 subjects in 29 cohorts. Extraversion item data from multiple personality inventories were harmonized across inventories and cohorts. No genome-wide significant associations were found...... at the single nucleotide polymorphism (SNP) level but there was one significant hit at the gene level for a long non-coding RNA site (LOC101928162). Genome-wide complex trait analysis in two large cohorts showed that the additive variance explained by common SNPs was not significantly different from zero...

Simultaneous genome-wide inference of physical, genetic, regulatory, and functional pathway components.

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Christopher Y Park

2010-11-01

Full Text Available Biomolecular pathways are built from diverse types of pairwise interactions, ranging from physical protein-protein interactions and modifications to indirect regulatory relationships. One goal of systems biology is to bridge three aspects of this complexity: the growing body of high-throughput data assaying these interactions; the specific interactions in which individual genes participate; and the genome-wide patterns of interactions in a system of interest. Here, we describe methodology for simultaneously predicting specific types of biomolecular interactions using high-throughput genomic data. This results in a comprehensive compendium of whole-genome networks for yeast, derived from ∼3,500 experimental conditions and describing 30 interaction types, which range from general (e.g. physical or regulatory to specific (e.g. phosphorylation or transcriptional regulation. We used these networks to investigate molecular pathways in carbon metabolism and cellular transport, proposing a novel connection between glycogen breakdown and glucose utilization supported by recent publications. Additionally, 14 specific predicted interactions in DNA topological change and protein biosynthesis were experimentally validated. We analyzed the systems-level network features within all interactomes, verifying the presence of small-world properties and enrichment for recurring network motifs. This compendium of physical, synthetic, regulatory, and functional interaction networks has been made publicly available through an interactive web interface for investigators to utilize in future research at http://function.princeton.edu/bioweaver/.

Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

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Yi Yin

2013-10-01

Full Text Available In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs. Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH. In this study, LOH events induced by ultraviolet (UV light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

The Glyphosate-Based Herbicide Roundup Does not Elevate Genome-Wide Mutagenesis of Escherichia coli.

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Tincher, Clayton; Long, Hongan; Behringer, Megan; Walker, Noah; Lynch, Michael

2017-10-05

Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS) method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH) Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria. Copyright © 2017 Tincher et al.

Novel genomes and genome constitutions identified by GISH and 5S rDNA and knotted1 genomic sequences in the genus Setaria.

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Zhao, Meicheng; Zhi, Hui; Doust, Andrew N; Li, Wei; Wang, Yongfang; Li, Haiquan; Jia, Guanqing; Wang, Yongqiang; Zhang, Ning; Diao, Xianmin

2013-04-11

The Setaria genus is increasingly of interest to researchers, as its two species, S. viridis and S. italica, are being developed as models for understanding C4 photosynthesis and plant functional genomics. The genome constitution of Setaria species has been studied in the diploid species S. viridis, S. adhaerans and S. grisebachii, where three genomes A, B and C were identified respectively. Two allotetraploid species, S. verticillata and S. faberi, were found to have AABB genomes, and one autotetraploid species, S. queenslandica, with an AAAA genome, has also been identified. The genomes and genome constitutions of most other species remain unknown, even though it was thought there are approximately 125 species in the genus distributed world-wide. GISH was performed to detect the genome constitutions of Eurasia species of S. glauca, S. plicata, and S. arenaria, with the known A, B and C genomes as probes. No or very poor hybridization signal was detected indicating that their genomes are different from those already described. GISH was also performed reciprocally between S. glauca, S. plicata, and S. arenaria genomes, but no hybridization signals between each other were found. The two sets of chromosomes of S. lachnea both hybridized strong signals with only the known C genome of S. grisebachii. Chromosomes of Qing 9, an accession formerly considered as S. viridis, hybridized strong signal only to B genome of S. adherans. Phylogenetic trees constructed with 5S rDNA and knotted1 markers, clearly classify the samples in this study into six clusters, matching the GISH results, and suggesting that the F genome of S. arenaria is basal in the genus. Three novel genomes in the Setaria genus were identified and designated as genome D (S. glauca), E (S. plicata) and F (S. arenaria) respectively. The genome constitution of tetraploid S. lachnea is putatively CCC'C'. Qing 9 is a B genome species indigenous to China and is hypothesized to be a newly identified species. The

Genome-Wide SNP Discovery, Genotyping and Their Preliminary Applications for Population Genetic Inference in Spotted Sea Bass (Lateolabrax maculatus.

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Juan Wang

Full Text Available Next-generation sequencing and the collection of genome-wide single-nucleotide polymorphisms (SNPs allow identifying fine-scale population genetic structure and genomic regions under selection. The spotted sea bass (Lateolabrax maculatus is a non-model species of ecological and commercial importance and widely distributed in northwestern Pacific. A total of 22 648 SNPs was discovered across the genome of L. maculatus by paired-end sequencing of restriction-site associated DNA (RAD-PE for 30 individuals from two populations. The nucleotide diversity (π for each population was 0.0028±0.0001 in Dandong and 0.0018±0.0001 in Beihai, respectively. Shallow but significant genetic differentiation was detected between the two populations analyzed by using both the whole data set (FST = 0.0550, P < 0.001 and the putatively neutral SNPs (FST = 0.0347, P < 0.001. However, the two populations were highly differentiated based on the putatively adaptive SNPs (FST = 0.6929, P < 0.001. Moreover, a total of 356 SNPs representing 298 unique loci were detected as outliers putatively under divergent selection by FST-based outlier tests as implemented in BAYESCAN and LOSITAN. Functional annotation of the contigs containing putatively adaptive SNPs yielded hits for 22 of 55 (40% significant BLASTX matches. Candidate genes for local selection constituted a wide array of functions, including binding, catalytic and metabolic activities, etc. The analyses with the SNPs developed in the present study highlighted the importance of genome-wide genetic variation for inference of population structure and local adaptation in L. maculatus.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

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Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-06-02

Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Multiple displacement amplification of whole genomic DNA from urediospores of Puccinia striiformis f. sp. tritici.

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Zhang, R; Ma, Z H; Wu, B M

2015-05-01

Biotrophic fungi, such as Puccinia striiformis f. sp. tritici, because they cannot be cultured on nutrient media, to obtain adequate quantity of DNA for molecular genetic analysis, are usually propagated on living hosts, wheat plants in case of P. striiformis f. sp. tritici. The propagation process is time-, space- and labor-consuming and has been a bottleneck to molecular genetic analysis of this pathogen. In this study we evaluated multiple displacement amplification (MDA) of pathogen genomic DNA from urediospores as an alternative approach to traditional propagation of urediospores followed by DNA extraction. The quantities of pathogen genomic DNA in the products were further determined via real-time PCR with a pair of primers specific for the β-tubulin gene of P. striiformis f. sp. tritici. The amplified fragment length polymorphism (AFLP) fingerprints were also compared between the DNA products. The results demonstrated that adequate genomic DNA at fragment size larger than 23 Kb could be amplified from 20 to 30 urediospores via MDA method. The real-time PCR results suggested that although fresh urediospores collected from diseased leaves were the best, spores picked from diseased leaves stored for a prolonged period could also be used for amplification. AFLP fingerprints exhibited no significant differences between amplified DNA and DNA extracted with CTAB method, suggesting amplified DNA can represent the pathogen's genomic DNA very well. Therefore, MDA could be used to obtain genomic DNA from small precious samples (dozens of spores) for molecular genetic analysis of wheat stripe rust pathogen, and other fungi that are difficult to propagate.

Vitamin D receptor signaling and its therapeutic implications: Genome-wide and structural view.

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Carlberg, Carsten; Molnár, Ferdinand

2015-05-01

Vitamin D3 is one of the few natural compounds that has, via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. For efficiently applying the therapeutic and disease-preventing potential of 1,25(OH)2D3 and its synthetic analogs, the key steps in vitamin D signaling need to be understood. These are the different types of molecular interactions with the VDR, such as (i) the complex formation of VDR with genomic DNA, (ii) the interaction of VDR with its partner transcription factors, (iii) the binding of 1,25(OH)2D3 or its synthetic analogs within the ligand-binding pocket of the VDR, and (iv) the resulting conformational change on the surface of the VDR leading to a change of the protein-protein interaction profile of the receptor with other proteins. This review will present the latest genome-wide insight into vitamin D signaling, and will discuss its therapeutic implications.

Concentrating and labeling genomic DNA in a nanofluidic array

DEFF Research Database (Denmark)

Marie, Rodolphe; Pedersen, Jonas Nyvold; Mir, Kalim U.

2018-01-01

, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due...... to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min-1. The final...

Instability of plastid DNA in the nuclear genome.

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Anna E Sheppard

2009-01-01

Full Text Available Functional gene transfer from the plastid (chloroplast and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.

Measuring the Levels of Ribonucleotides Embedded in Genomic DNA.

Science.gov (United States)

Meroni, Alice; Nava, Giulia M; Sertic, Sarah; Plevani, Paolo; Muzi-Falconi, Marco; Lazzaro, Federico

2018-01-01

Ribonucleotides (rNTPs) are incorporated into genomic DNA at a relatively high frequency during replication. They have beneficial effects but, if not removed from the chromosomes, increase genomic instability. Here, we describe a fast method to easily estimate the amounts of embedded ribonucleotides into the genome. The protocol described is performed in Saccharomyces cerevisiae and allows us to quantify altered levels of rNMPs due to different mutations in the replicative polymerase ε. However, this protocol can be easily applied to cells derived from any organism.

Genome-wide distribution and organization of microsatellites in plants: an insight into marker development in Brachypodium.

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Humira Sonah

Full Text Available Plant genomes are complex and contain large amounts of repetitive DNA including microsatellites that are distributed across entire genomes. Whole genome sequences of several monocot and dicot plants that are available in the public domain provide an opportunity to study the origin, distribution and evolution of microsatellites, and also facilitate the development of new molecular markers. In the present investigation, a genome-wide analysis of microsatellite distribution in monocots (Brachypodium, sorghum and rice and dicots (Arabidopsis, Medicago and Populus was performed. A total of 797,863 simple sequence repeats (SSRs were identified in the whole genome sequences of six plant species. Characterization of these SSRs revealed that mono-nucleotide repeats were the most abundant repeats, and that the frequency of repeats decreased with increase in motif length both in monocots and dicots. However, the frequency of SSRs was higher in dicots than in monocots both for nuclear and chloroplast genomes. Interestingly, GC-rich repeats were the dominant repeats only in monocots, with the majority of them being present in the coding region. These coding GC-rich repeats were found to be involved in different biological processes, predominantly binding activities. In addition, a set of 22,879 SSR markers that were validated by e-PCR were developed and mapped on different chromosomes in Brachypodium for the first time, with a frequency of 101 SSR markers per Mb. Experimental validation of 55 markers showed successful amplification of 80% SSR markers in 16 Brachypodium accessions. An online database 'BraMi' (Brachypodium microsatellite markers of these genome-wide SSR markers was developed and made available in the public domain. The observed differential patterns of SSR marker distribution would be useful for studying microsatellite evolution in a monocot-dicot system. SSR markers developed in this study would be helpful for genomic studies in Brachypodium

Genome-Wide Association Study and Linkage Analysis of the Healthy Aging Index

DEFF Research Database (Denmark)

Minster, Ryan L; Sanders, Jason L; Singh, Jatinder

2015-01-01

BACKGROUND: The Healthy Aging Index (HAI) is a tool for measuring the extent of health and disease across multiple systems. METHODS: We conducted a genome-wide association study and a genome-wide linkage analysis to map quantitative trait loci associated with the HAI and a modified HAI weighted...

Genome-wide divergence, haplotype distribution and population demographic histories for Gossypium hirsutum and Gossypium barbadense as revealed by genome-anchored SNPs

Science.gov (United States)

Use of 10,129 singleton SNPs of known genomic location in tetraploid cotton provided unique opportunities to characterize genome-wide diversity among 440 Gossypium hirsutum and 219 G. barbadense cultivars and landrace accessions of widespread origin. Using the SNPs distributed genome-wide, we exami...

Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.

Science.gov (United States)

Rudd, K E; Miller, W; Ostell, J; Benson, D A

1990-01-25

We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.

Genome-wide association study identifies a maternal copy-number deletion in PSG11 enriched among preeclampsia patients

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Zhao Linlu

2012-06-01

Full Text Available Abstract Background Specific genetic contributions for preeclampsia (PE are currently unknown. This genome-wide association study (GWAS aims to identify maternal single nucleotide polymorphisms (SNPs and copy-number variants (CNVs involved in the etiology of PE. Methods A genome-wide scan was performed on 177 PE cases (diagnosed according to National Heart, Lung and Blood Institute guidelines and 116 normotensive controls. White female study subjects from Iowa were genotyped on Affymetrix SNP 6.0 microarrays. CNV calls made using a combination of four detection algorithms (Birdseye, Canary, PennCNV, and QuantiSNP were merged using CNVision and screened with stringent prioritization criteria. Due to limited DNA quantities and the deleterious nature of copy-number deletions, it was decided a priori that only deletions would be selected for assay on the entire case-control dataset using quantitative real-time PCR. Results The top four SNP candidates had an allelic or genotypic p-value between 10-5 and 10-6, however, none surpassed the Bonferroni-corrected significance threshold. Three recurrent rare deletions meeting prioritization criteria detected in multiple cases were selected for targeted genotyping. A locus of particular interest was found showing an enrichment of case deletions in 19q13.31 (5/169 cases and 1/114 controls, which encompasses the PSG11 gene contiguous to a highly plastic genomic region. All algorithm calls for these regions were assay confirmed. Conclusions CNVs may confer risk for PE and represent interesting regions that warrant further investigation. Top SNP candidates identified from the GWAS, although not genome-wide significant, may be useful to inform future studies in PE genetics.

In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

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Janet L Smith

2015-05-01

Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

Genome-wide association study identifies genetic loci associated with iron deficiency.

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Christine E McLaren

2011-03-01

Full Text Available The existence of multiple inherited disorders of iron metabolism in man, rodents and other vertebrates suggests genetic contributions to iron deficiency. To identify new genomic locations associated with iron deficiency, a genome-wide association study (GWAS was performed using DNA collected from white men aged≥25 y and women≥50 y in the Hemochromatosis and Iron Overload Screening (HEIRS Study with serum ferritin (SF≤12 µg/L (cases and iron replete controls (SF>100 µg/L in men, SF>50 µg/L in women. Regression analysis was used to examine the association between case-control status (336 cases, 343 controls and quantitative serum iron measures and 331,060 single nucleotide polymorphism (SNP genotypes, with replication analyses performed in a sample of 71 cases and 161 controls from a population of white male and female veterans screened at a US Veterans Affairs (VA medical center. Five SNPs identified in the GWAS met genome-wide statistical significance for association with at least one iron measure, rs2698530 on chr. 2p14; rs3811647 on chr. 3q22, a known SNP in the transferrin (TF gene region; rs1800562 on chr. 6p22, the C282Y mutation in the HFE gene; rs7787204 on chr. 7p21; and rs987710 on chr. 22q11 (GWAS observed P<1.51×10(-7 for all. An association between total iron binding capacity and SNP rs3811647 in the TF gene (GWAS observed P=7.0×10(-9, corrected P=0.012 was replicated within the VA samples (observed P=0.012. Associations with the C282Y mutation in the HFE gene also were replicated. The joint analysis of the HEIRS and VA samples revealed strong associations between rs2698530 on chr. 2p14 and iron status outcomes. These results confirm a previously-described TF polymorphism and implicate one potential new locus as a target for gene identification.

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 V; Olesen, Morten S

2011-01-01

BACKGROUND: 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...

Genome-wide association study of Tourette Syndrome

Science.gov (United States)

Scharf, Jeremiah M.; Yu, Dongmei; Mathews, Carol A.; Neale, Benjamin M.; Stewart, S. Evelyn; Fagerness, Jesen A; Evans, Patrick; Gamazon, Eric; Edlund, Christopher K.; Service, Susan; Tikhomirov, Anna; Osiecki, Lisa; Illmann, Cornelia; Pluzhnikov, Anna; Konkashbaev, Anuar; Davis, Lea K; Han, Buhm; Crane, Jacquelyn; Moorjani, Priya; Crenshaw, Andrew T.; Parkin, Melissa A.; Reus, Victor I.; Lowe, Thomas L.; Rangel-Lugo, Martha; Chouinard, Sylvain; Dion, Yves; Girard, Simon; Cath, Danielle C; Smit, Jan H; King, Robert A.; Fernandez, Thomas; Leckman, James F.; Kidd, Kenneth K.; Kidd, Judith R.; Pakstis, Andrew J.; State, Matthew; Herrera, Luis Diego; Romero, Roxana; Fournier, Eduardo; Sandor, Paul; Barr, Cathy L; Phan, Nam; Gross-Tsur, Varda; Benarroch, Fortu; Pollak, Yehuda; Budman, Cathy L.; Bruun, Ruth D.; Erenberg, Gerald; Naarden, Allan L; Lee, Paul C; Weiss, Nicholas; Kremeyer, Barbara; Berrío, Gabriel Bedoya; Campbell, Desmond; Silgado, Julio C. Cardona; Ochoa, William Cornejo; Restrepo, Sandra C. Mesa; Muller, Heike; Duarte, Ana V. Valencia; Lyon, Gholson J; Leppert, Mark; Morgan, Jubel; Weiss, Robert; Grados, Marco A.; Anderson, Kelley; Davarya, Sarah; Singer, Harvey; Walkup, John; Jankovic, Joseph; Tischfield, Jay A.; Heiman, Gary A.; Gilbert, Donald L.; Hoekstra, Pieter J.; Robertson, Mary M.; Kurlan, Roger; Liu, Chunyu; Gibbs, J. Raphael; Singleton, Andrew; Hardy, John; Strengman, Eric; Ophoff, Roel; Wagner, Michael; Moessner, Rainald; Mirel, Daniel B.; Posthuma, Danielle; Sabatti, Chiara; Eskin, Eleazar; Conti, David V.; Knowles, James A.; Ruiz-Linares, Andres; Rouleau, Guy A.; Purcell, Shaun; Heutink, Peter; Oostra, Ben A.; McMahon, William; Freimer, Nelson; Cox, Nancy J.; Pauls, David L.

2012-01-01

Tourette Syndrome (TS) is a developmental disorder that has one of the highest familial recurrence rates among neuropsychiatric diseases with complex inheritance. However, the identification of definitive TS susceptibility genes remains elusive. Here, we report the first genome-wide association study (GWAS) of TS in 1285 cases and 4964 ancestry-matched controls of European ancestry, including two European-derived population isolates, Ashkenazi Jews from North America and Israel, and French Canadians from Quebec, Canada. In a primary meta-analysis of GWAS data from these European ancestry samples, no markers achieved a genome-wide threshold of significance (p<5 × 10−8); the top signal was found in rs7868992 on chromosome 9q32 within COL27A1 (p=1.85 × 10−6). A secondary analysis including an additional 211 cases and 285 controls from two closely-related Latin-American population isolates from the Central Valley of Costa Rica and Antioquia, Colombia also identified rs7868992 as the top signal (p=3.6 × 10−7 for the combined sample of 1496 cases and 5249 controls following imputation with 1000 Genomes data). This study lays the groundwork for the eventual identification of common TS susceptibility variants in larger cohorts and helps to provide a more complete understanding of the full genetic architecture of this disorder. PMID:22889924

Whole genome DNA copy number changes identified by high density oligonucleotide arrays

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Huang Jing

2004-05-01

Full Text Available Abstract Changes in DNA copy number are one of the hallmarks of the genetic instability common to most human cancers. Previous micro-array-based methods have been used to identify chromosomal gains and losses; however, they are unable to genotype alleles at the level of single nucleotide polymorphisms (SNPs. Here we describe a novel algorithm that uses a recently developed high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA, to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10,000 SNPs by allele-specific hybridisation to perfect match (PM and mismatch (MM probes synthesised on a single array. The copy number algorithm jointly uses PM intensity and discrimination ratios between paired PM and MM intensity values to identify and estimate genetic copy number changes. Values from an experimental sample are compared with SNP-specific distributions derived from a reference set containing over 100 normal individuals to gain statistical power. Genomic regions with statistically significant copy number changes can be identified using both single point analysis and contiguous point analysis of SNP intensities. We identified multiple regions of amplification and deletion using a panel of human breast cancer cell lines. We verified these results using an independent method based on quantitative polymerase chain reaction and found that our approach is both sensitive and specific and can tolerate samples which contain a mixture of both tumour and normal DNA. In addition, by using known allele frequencies from the reference set, statistically significant genomic intervals can be identified containing contiguous stretches of homozygous markers, potentially allowing the detection of regions undergoing loss of heterozygosity (LOH without the need for a matched normal control sample. The coupling of LOH analysis, via SNP genotyping, with copy number

Genome-wide identification of the regulatory targets of a transcription factor using biochemical characterization and computational genomic analysis

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Jolly Emmitt R

2005-11-01

Full Text Available Abstract Background A major challenge in computational genomics is the development of methodologies that allow accurate genome-wide prediction of the regulatory targets of a transcription factor. We present a method for target identification that combines experimental characterization of binding requirements with computational genomic analysis. Results Our method identified potential target genes of the transcription factor Ndt80, a key transcriptional regulator involved in yeast sporulation, using the combined information of binding affinity, positional distribution, and conservation of the binding sites across multiple species. We have also developed a mathematical approach to compute the false positive rate and the total number of targets in the genome based on the multiple selection criteria. Conclusion We have shown that combining biochemical characterization and computational genomic analysis leads to accurate identification of the genome-wide targets of a transcription factor. The method can be extended to other transcription factors and can complement other genomic approaches to transcriptional regulation.

Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

DEFF Research Database (Denmark)

Knudsen, Berith Elkær; Bergmark, Lasse; Munk, Patrick

2016-01-01

that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10......Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required...... for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial...

Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole

KAUST Repository

Campos, Mônica C.

2017-10-25

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 5–8 million people in Latin America. Although the nitroheterocyclic compound benznidazole has been the front-line drug for several decades, treatment failures are common. Benznidazole is a pro-drug and is bio-activated within the parasite by the mitochondrial nitroreductase TcNTR-1, leading to the generation of reactive metabolites that have trypanocidal activity. To better assess drug action and resistance, we sequenced the genomes of T. cruzi Y strain (35.5 Mb) and three benznidazole-resistant clones derived from a single drug-selected population. This revealed the genome-wide accumulation of mutations in the resistant parasites, in addition to variations in DNA copy-number. We observed mutations in DNA repair genes, linked with increased susceptibility to DNA alkylating and inter-strand cross-linking agents. Stop-codon-generating mutations in TcNTR-1 were associated with cross-resistance to other nitroheterocyclic drugs. Unexpectedly, the clones were also highly resistant to the ergosterol biosynthesis inhibitor posaconazole, a drug proposed for use against T. cruzi infections, in combination with benznidazole. Our findings therefore identify the highly mutagenic activity of benznidazole metabolites in T. cruzi, demonstrate that this can result in multi-drug resistance, and indicate that vigilance will be required if benznidazole is used in combination therapy.

G-Quadruplexes Involving Both Strands of Genomic DNA Are Highly Abundant and Colocalize with Functional Sites in the Human Genome.

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Andrzej S Kudlicki

Full Text Available The G-quadruplex is a non-canonical DNA structure biologically significant in DNA replication, transcription and telomere stability. To date, only G4s with all guanines originating from the same strand of DNA have been considered in the context of the human nuclear genome. Here, I discuss interstrand topological configurations of G-quadruplex DNA, consisting of guanines from both strands of genomic DNA; an algorithm is presented for predicting such structures. I have identified over 550,000 non-overlapping interstrand G-quadruplex forming sequences in the human genome--significantly more than intrastrand configurations. Functional analysis of interstrand G-quadruplex sites shows strong association with transcription initiation, the results are consistent with the XPB and XPD transcriptional helicases binding only to G-quadruplex DNA with interstrand topology. Interstrand quadruplexes are also enriched in origin of replication sites. Several topology classes of interstrand quadruplex-forming sequences are possible, and different topologies are enriched in different types of structural elements. The list of interstrand quadruplex forming sequences, and the computer program used for their prediction are available at the web address http://moment.utmb.edu/allquads.

Accurate DNA assembly and genome engineering with optimized uracil excision cloning

DEFF Research Database (Denmark)

Cavaleiro, Mafalda; Kim, Se Hyeuk; Seppala, Susanna

2015-01-01

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that pro......Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway...... that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly...... facilitating the advanced engineering of robust cell factories....

Studies on the Interaction between Zinc-Hydroxybenzoite Complex and Genomic DNA

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Hacali Necefoglu

2006-04-01

Full Text Available Zinc-Hydroxybenzoite ([Zn (H206] (p-HO-C6H4COO22H20 complex which wassynthesized and characterized by instrumental methods and the DNA samples which hadbeen isolated from cattle were allowed to interact at 37 oC for different time periods. Theinteraction of genomic DNA with this complex has been followed by agarose gelelectrophoresis at 50 V for 2 h. When DNA samples were allowed to interact with this metalcomplex, it was found that band intensities changed with the concentrations of the complex.In the result of interaction between this complex and genomic DNA samples, it wasdetermined that the intensities of bands were changed at the different concentrations of thecomplex. The brightness of the bands was increased and mobility of the bands wasdecreased, indicating the occurrence of increased covalent binding of the metal complexwith DNA. In this study it was concluded that the damage effect of ascorbate was reducedby Zinc-Hydroxybenzoite.

Links between DNA methylation and nucleosome occupancy in the human genome.

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Collings, Clayton K; Anderson, John N

2017-01-01

DNA methylation is an epigenetic modification that is enriched in heterochromatin but depleted at active promoters and enhancers. However, the debate on whether or not DNA methylation is a reliable indicator of high nucleosome occupancy has not been settled. For example, the methylation levels of DNA flanking CTCF sites are higher in linker DNA than in nucleosomal DNA, while other studies have shown that the nucleosome core is the preferred site of methylation. In this study, we make progress toward understanding these conflicting phenomena by implementing a bioinformatics approach that combines MNase-seq and NOMe-seq data and by comprehensively profiling DNA methylation and nucleosome occupancy throughout the human genome. The results demonstrated that increasing methylated CpG density is correlated with nucleosome occupancy in the total genome and within nearly all subgenomic regions. Features with elevated methylated CpG density such as exons, SINE-Alu sequences, H3K36-trimethylated peaks, and methylated CpG islands are among the highest nucleosome occupied elements in the genome, while some of the lowest occupancies are displayed by unmethylated CpG islands and unmethylated transcription factor binding sites. Additionally, outside of CpG islands, the density of CpGs within nucleosomes was shown to be important for the nucleosomal location of DNA methylation with low CpG frequencies favoring linker methylation and high CpG frequencies favoring core particle methylation. Prominent exceptions to the correlations between methylated CpG density and nucleosome occupancy include CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, distinguish the major silencing mechanisms of the human epigenome. Thus, the relationship between DNA methylation and nucleosome occupancy is influenced by the density of methylated CpG dinucleotides and by other epigenomic components in chromatin.

Genome-wide association study of the four-constitution medicine.

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Yin, Chang Shik; Park, Hi Joon; Chung, Joo-Ho; Lee, Hye-Jung; Lee, Byung-Cheol

2009-12-01

Four-constitution medicine (FCM), also known as Sasang constitutional medicine, and the heritage of the long history of individualized acupuncture medicine tradition, is one of the holistic and traditional systems of constitution to appraise and categorize individual differences into four major types. This study first reports a genome-wide association study on FCM, to explore the genetic basis of FCM and facilitate the integration of FCM with conventional individual differences research. Healthy individuals of the Korean population were classified into the four constitutional types (FCTs). A total of 353,202 single nucleotide polymorphisms (SNPs) were typed using whole genome amplified samples, and six-way comparison of FCM types provided lists of significantly differential SNPs. In one-to-one FCT comparisons, 15,944 SNPs were significantly differential, and 5 SNPs were commonly significant in all of the three comparisons. In one-to-two FCT comparisons, 22,616 SNPs were significantly differential, and 20 SNPs were commonly significant in all of the three comparison groups. This study presents the association between genome-wide SNP profiles and the categorization of the FCM, and it could further provide a starting point of genome-based identification and research of the constitutions of FCM.

A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome.

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Yu, Shoukai; Lemos, Bernardo

2016-12-31

Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genome-wide DNA methylation profiles reveal novel candidate genes associated with meat quality at different age stages in hens.

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Zhang, Meng; Yan, Feng-Bin; Li, Fang; Jiang, Ke-Ren; Li, Dong-Hua; Han, Rui-Li; Li, Zhuan-Jan; Jiang, Rui-Rui; Liu, Xiao-Jun; Kang, Xiang-Tao; Sun, Gui-Rong

2017-04-05

Poultry meat quality is associated with breed, age, tissue and other factors. Many previous studies have focused on distinct breeds; however, little is known regarding the epigenetic regulatory mechanisms in different age stages, such as DNA methylation. Here, we compared the global DNA methylation profiles between juvenile (20 weeks old) and later laying-period (55 weeks old) hens and identified candidate genes related to the development and meat quality of breast muscle using whole-genome bisulfite sequencing. The results showed that the later laying-period hens, which had a higher intramuscular fat (IMF) deposition capacity and water holding capacity (WHC) and less tenderness, exhibited higher global DNA methylation levels than the juvenile hens. A total of 2,714 differentially methylated regions were identified in the present study, which corresponded to 378 differentially methylated genes, mainly affecting muscle development, lipid metabolism, and the ageing process. Hypermethylation of the promoters of the genes ABCA1, COL6A1 and GSTT1L and the resulting transcriptional down-regulation in the later laying-period hens may be the reason for the significant difference in the meat quality between the juvenile and later laying-period hens. These findings contribute to a better understanding of epigenetic regulation in the skeletal muscle development and meat quality of chicken.

Googling DNA sequences on the World Wide Web.

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Hajibabaei, Mehrdad; Singer, Gregory A C

2009-11-10

New web-based technologies provide an excellent opportunity for sharing and accessing information and using web as a platform for interaction and collaboration. Although several specialized tools are available for analyzing DNA sequence information, conventional web-based tools have not been utilized for bioinformatics applications. We have developed a novel algorithm and implemented it for searching species-specific genomic sequences, DNA barcodes, by using popular web-based methods such as Google. We developed an alignment independent character based algorithm based on dividing a sequence library (DNA barcodes) and query sequence to words. The actual search is conducted by conventional search tools such as freely available Google Desktop Search. We implemented our algorithm in two exemplar packages. We developed pre and post-processing software to provide customized input and output services, respectively. Our analysis of all publicly available DNA barcode sequences shows a high accuracy as well as rapid results. Our method makes use of conventional web-based technologies for specialized genetic data. It provides a robust and efficient solution for sequence search on the web. The integration of our search method for large-scale sequence libraries such as DNA barcodes provides an excellent web-based tool for accessing this information and linking it to other available categories of information on the web.

Chapter 10: Mining genome-wide genetic markers.

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Xiang Zhang

Full Text Available Genome-wide association study (GWAS aims to discover genetic factors underlying phenotypic traits. The large number of genetic factors poses both computational and statistical challenges. Various computational approaches have been developed for large scale GWAS. In this chapter, we will discuss several widely used computational approaches in GWAS. The following topics will be covered: (1 An introduction to the background of GWAS. (2 The existing computational approaches that are widely used in GWAS. This will cover single-locus, epistasis detection, and machine learning methods that have been recently developed in biology, statistic, and computer science communities. This part will be the main focus of this chapter. (3 The limitations of current approaches and future directions.

Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter.

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Duewer, David L; Kline, Margaret C; Romsos, Erica L; Toman, Blaza

2018-05-01

The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA. Graphical abstract The roots of metrological traceability for human nuclear DNA mass concentration results. Values for the factors in blue must be established experimentally. Values for the factors in red have been established from authoritative source materials. HHGE stands for "haploid human genome equivalent"; there are two HHGE per diploid human genome.

Genome-wide association analyses of expression phenotypes.

Science.gov (United States)

Chen, Gary K; Zheng, Tian; Witte, John S; Goode, Ellen L; Gao, Lei; Hu, Pingzhao; Suh, Young Ju; Suktitipat, Bhoom; Szymczak, Silke; Woo, Jung Hoon; Zhang, Wei

2007-01-01

A number of issues arise when analyzing the large amount of data from high-throughput genotype and expression microarray experiments, including design and interpretation of genome-wide association studies of expression phenotypes. These issues were considered by contributions submitted to Group 1 of the Genetic Analysis Workshop 15 (GAW15), which focused on the association of quantitative expression data. These contributions evaluated diverse hypotheses, including those relevant to cancer and obesity research, and used various analytic techniques, many of which were derived from information theory. Several observations from these reports stand out. First, one needs to consider the genetic model of the trait of interest and carefully select which single nucleotide polymorphisms and individuals are included early in the design stage of a study. Second, by targeting specific pathways when analyzing genome-wide data, one can generate more interpretable results than agnostic approaches. Finally, for datasets with small sample sizes but a large number of features like the Genetic Analysis Workshop 15 dataset, machine learning approaches may be more practical than traditional parametric approaches. (c) 2007 Wiley-Liss, Inc.

Genome-wide analysis of tandem repeats in plants and green algae

Science.gov (United States)

Zhixin Zhao; Cheng Guo; Sreeskandarajan Sutharzan; Pei Li; Craig Echt; Jie Zhang; Chun Liang

2014-01-01

Tandem repeats (TRs) extensively exist in the genomes of prokaryotes and eukaryotes. Based on the sequenced genomes and gene annotations of 31 plant and algal species in Phytozome version 8.0 (http://www.phytozome.net/), we examined TRs in a genome-wide scale, characterized their distributions and motif features, and explored their putative biological functions. Among...

Investigation of common, low-frequency and rare genome-wide variation in anorexia nervosa

Science.gov (United States)

Huckins, L M; Hatzikotoulas, K; Southam, L; Thornton, L M; Steinberg, J; Aguilera-McKay, F; Treasure, J; Schmidt, U; Gunasinghe, C; Romero, A; Curtis, C; Rhodes, D; Moens, J; Kalsi, G; Dempster, D; Leung, R; Keohane, A; Burghardt, R; Ehrlich, S; Hebebrand, J; Hinney, A; Ludolph, A; Walton, E; Deloukas, P; Hofman, A; Palotie, A; Palta, P; van Rooij, F J A; Stirrups, K; Adan, R; Boni, C; Cone, R; Dedoussis, G; van Furth, E; Gonidakis, F; Gorwood, P; Hudson, J; Kaprio, J; Kas, M; Keski-Rahonen, A; Kiezebrink, K; Knudsen, G-P; Slof-Op 't Landt, M C T; Maj, M; Monteleone, A M; Monteleone, P; Raevuori, A H; Reichborn-Kjennerud, T; Tozzi, F; Tsitsika, A; van Elburg, A; Adan, R A H; Alfredsson, L; Ando, T; Andreassen, O A; Aschauer, H; Baker, J H; Barrett, J C; Bencko, V; Bergen, A W; Berrettini, W H; Birgegard, A; Boni, C; Boraska Perica, V; Brandt, H; Breen, G; Bulik, C M; Carlberg, L; Cassina, M; Cichon, S; Clementi, M; Cohen-Woods, S; Coleman, J; Cone, R D; Courtet, P; Crawford, S; Crow, S; Crowley, J; Danner, U N; Davis, O S P; de Zwaan, M; Dedoussis, G; Degortes, D; DeSocio, J E; Dick, D M; Dikeos, D; Dina, C; Ding, B; Dmitrzak-Weglarz, M; Docampo, E; Duncan, L; Egberts, K; Ehrlich, S; Escaramís, G; Esko, T; Espeseth, T; Estivill, X; Favaro, A; Fernández-Aranda, F; Fichter, M M; Finan, C; Fischer, K; Floyd, J A B; Foretova, L; Forzan, M; Franklin, C S; Gallinger, S; Gambaro, G; Gaspar, H A; Giegling, I; Gonidakis, F; Gorwood, P; Gratacos, M; Guillaume, S; Guo, Y; Hakonarson, H; Halmi, K A; Hatzikotoulas, K; Hauser, J; Hebebrand, J; Helder, S; Herms, S; Herpertz-Dahlmann, B; Herzog, W; Hilliard, C E; Hinney, A; Hübel, C; Huckins, L M; Hudson, J I; Huemer, J; Inoko, H; Janout, V; Jiménez-Murcia, S; Johnson, C; Julià, A; Juréus, A; Kalsi, G; Kaminska, D; Kaplan, A S; Kaprio, J; Karhunen, L; Karwautz, A; Kas, M J H; Kaye, W; Kennedy, J L; Keski-Rahkonen, A; Kiezebrink, K; Klareskog, L; Klump, K L; Knudsen, G P S; Koeleman, B P C; Koubek, D; La Via, M C; Landén, M; Le Hellard, S; Levitan, R D; Li, D; Lichtenstein, P; Lilenfeld, L; Lissowska, J; Lundervold, A; Magistretti, P; Maj, M; Mannik, K; Marsal, S; Martin, N; Mattingsdal, M; McDevitt, S; McGuffin, P; Merl, E; Metspalu, A; Meulenbelt, I; Micali, N; Mitchell, J; Mitchell, K; Monteleone, P; Monteleone, A M; Mortensen, P; Munn-Chernoff, M A; Navratilova, M; Nilsson, I; Norring, C; Ntalla, I; Ophoff, R A; O'Toole, J K; Palotie, A; Pante, J; Papezova, H; Pinto, D; Rabionet, R; Raevuori, A; Rajewski, A; Ramoz, N; Rayner, N W; Reichborn-Kjennerud, T; Ripatti, S; Roberts, M; Rotondo, A; Rujescu, D; Rybakowski, F; Santonastaso, P; Scherag, A; Scherer, S W; Schmidt, U; Schork, N J; Schosser, A; Slachtova, L; Sladek, R; Slagboom, P E; Slof-Op 't Landt, M C T; Slopien, A; Soranzo, N; Southam, L; Steen, V M; Strengman, E; Strober, M; Sullivan, P F; Szatkiewicz, J P; Szeszenia-Dabrowska, N; Tachmazidou, I; Tenconi, E; Thornton, L M; Tortorella, A; Tozzi, F; Treasure, J; Tsitsika, A; Tziouvas, K; van Elburg, A A; van Furth, E F; Wagner, G; Walton, E; Watson, H; Wichmann, H-E; Widen, E; Woodside, D B; Yanovski, J; Yao, S; Yilmaz, Z; Zeggini, E; Zerwas, S; Zipfel, S; Collier, D A; Sullivan, P F; Breen, G; Bulik, C M; Zeggini, E

2018-01-01

Anorexia nervosa (AN) is a complex neuropsychiatric disorder presenting with dangerously low body weight, and a deep and persistent fear of gaining weight. To date, only one genome-wide significant locus associated with AN has been identified. We performed an exome-chip based genome-wide association studies (GWAS) in 2158 cases from nine populations of European origin and 15 485 ancestrally matched controls. Unlike previous studies, this GWAS also probed association in low-frequency and rare variants. Sixteen independent variants were taken forward for in silico and de novo replication (11 common and 5 rare). No findings reached genome-wide significance. Two notable common variants were identified: rs10791286, an intronic variant in OPCML (P=9.89 × 10−6), and rs7700147, an intergenic variant (P=2.93 × 10−5). No low-frequency variant associations were identified at genome-wide significance, although the study was well-powered to detect low-frequency variants with large effect sizes, suggesting that there may be no AN loci in this genomic search space with large effect sizes. PMID:29155802

Role of oxidative DNA damage in genome instability and cancer

International Nuclear Information System (INIS)

Bignami, M.; Kunkel, T.

2009-01-01

Inactivation of mismatch repair (MMR) is associated with a dramatic genomic instability that is observed experimentally as a mutator phenotype and micro satellite instability (MSI). It has been implicit that the massive genetic instability in MMR defective cells simply reflects the accumulation of spontaneous DNA polymerase errors during DNA replication. We recently identified oxidation damage, a common threat to DNA integrity to which purines are very susceptible, as an important cofactor in this genetic instability

A genome-wide methylation study on obesity: differential variability and differential methylation.

Science.gov (United States)

Xu, Xiaojing; Su, Shaoyong; Barnes, Vernon A; De Miguel, Carmen; Pollock, Jennifer; Ownby, Dennis; Shi, Hidong; Zhu, Haidong; Snieder, Harold; Wang, Xiaoling

2013-05-01

Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common complex disease. We analyzed genome-wide methylation profiles of over 470,000 CpGs in peripheral blood samples from 48 obese and 48 lean African-American youth aged 14-20 y old. A substantial number of differentially variable CpG sites (DVCs), using statistics based on variances, as well as a substantial number of differentially methylated CpG sites (DMCs), using statistics based on means, were identified. Similar to the findings in cancers, DVCs generally exhibited an outlier structure and were more variable in cases than in controls. By randomly splitting the current sample into a discovery and validation set, we observed that both the DVCs and DMCs identified from the first set could independently predict obesity status in the second set. Furthermore, both the genes harboring DMCs and the genes harboring DVCs showed significant enrichment of genes identified by genome-wide association studies on obesity and related diseases, such as hypertension, dyslipidemia, type 2 diabetes and certain types of cancers, supporting their roles in the etiology and pathogenesis of obesity. We generalized the recent finding on methylation variability in cancer research to obesity and demonstrated that differential variability is also an important feature of obesity-related methylation changes. Future studies on the epigenetics of obesity will benefit from both statistics based on means and statistics based on variances.

AID/APOBEC cytosine deaminase induces genome-wide kataegis

Directory of Open Access Journals (Sweden)

Lada Artem G

2012-12-01

Full Text Available Abstract Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm, are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.

Comparative Analysis of the Genomic DNA Isolation Methods on Inula sp. (Asteraceae

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Emre SEVİNDİK

2016-12-01

Full Text Available Simple, fast, low-cost and high throughput protocols are required for DNA isolation of plant species. In this study, phenol chloroform isoamyl alcohol and commercial (Sigma DNA isolation kit methods were applied on some Inula species that belong to Asteraceae family. Genomic DNA amounts, A260, A280, A260/A230 and purity degrees (A260/A280 that were obtained through both methods were measured through electrophoresis and spectrophotometer. Additionally, PCR amplification was realized by primer pairs specific to nrDNA ITS, cpDNA ndhF (972F-1603R and trnL-F regions. Results showed that maximum genomic DNA in nanograms obtained by phenol chloroform isoamyl alcohol method. The study also revealed that I. macrocephala had the maximum DNA and I. heterolepis had the minimum DNA amount. A260/A280 purity degrees showed that the highest and lowest purity in gDNAs obtained through phenol-choloform isoamyl alcohol method were in I.aucheriana and I. salicina, respectively. The highest and lowest purity degrees of gDNAs obtained through commercial kit was observed in I. fragilis and I. macrocephala samples, respectively. PCR amplification results showed that while band profiles of each three regions (ITS, trnL-F and ndhF did not yield positive results in PCR amplifications using phenol-choloform isoamyl alcohol method; PCR band profiles obtained through commercial kit yielded positive results. As a result, it is fair to say that the relation of genomic DNA with PCR was found to be more efficient although the maximum amount of genomic DNA was obtained through phenol chloroform isoamyl alcohol method.