Project description:We have identified de novo copy number variations (CNVs) generated in ageing bulls. Blood samples from eight bulls were collected and SNP arrayed in a prospective design over 30 months allowing us to differentiate de novo CNVs from constant CNVs that are present throughout the sampling period. Quite remarkably, the total number of CNVs doubled over the 30-month period, as we observed an almost equal number of de novo and constant CNVs (107 vs. 111 or 49% vs. 51%, respectively). Twice as many de novo CNVs emerged during the second half of the sampling schedule as in the first part. It suggests a dynamic generation of de novo CNVs in the bovine genome that becomes more frequent, as the age of the animal progresses. In a second experiment de novo CNVs were detected through in vitro ageing of bovine fibroblasts by sampling passage #5, #15 and #25. De novo CNVs also became more frequent, but the proportion of them was only ~25% of the total number of CNVs (21 vs. 64). Temporal generation of de novo CNVs resulted in increasing genome coverage. Genes and quantitative trait loci overlapping de novo CNVs were further investigated for ageing related functions.
2017-03-03 | GSE95612 | GEO
Project description:Microsatellite loci identification from next-gen sequencing
Project description:DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase Pol IV transcribes target sequences to initiate 24-nt siRNA production and action. The Arabidopsis DTF1/SHH1 has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. Taken together, our results show that DTF1 is a core component of the RdDM pathway. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci. Examination of whole-genome DNA methylation and small RNA in 12-day-old Col-0, dtf1-2, nrpd1-3 and nrpe1-11 seedlings
Project description:Cancer development is associated with multiple genetic alterations and genomic instability either at the chromosomal or base pair level is generally thought to underlie these changes. However, it is still unknown whether genetic instability is absolutely required for tumorigenesis. Here we investigated the genomic instability status of four cytogenetically stable diploid cell lines CAL51, SK-UT-1B, A204 and CH1. We applied high resolution 500K single nucleotide polymorphism (SNP) array analysis and found that all the four cell lines have some sub-microscopic genomic copy number changes. Interestingly, there were many more sub-microscopic chromosome alterations in A204 and CH1 than in CAL51 and SK-UT-1B. Twenty-four-color fluorescence in situ hybridization (FISH) was used to analyse a large number of metaphases from CAL51, SK-UT-1B and CH1 cells. The rate of de novo chromosome rearrangements was significantly higher in CH1 than CAL51 and SK-UT-1B. Although this increased rate did not lead to many clonal cytogenetically apparent chromosome alterations in CH1 cells, it is consistent with a first step towards chromosomal instability. It is more striking that both cell lines CAL51 and SK-UT-1B which have a similar de novo chromosomal change rate to that of normal lymphocytes were microsatellite instability positive by BAT-26 microsatellite analysis. This study further strengths the current concept that genomic instability is associated with tumor development. Keywords: DNA copy number changes DNA from A204, CH1, CAL51 and SK-UT-1B were analyzed by Affymetrix Genechip Mapping 500K Set array. Data were compared with normal samples from HAPMAP database.
Project description:De novo centromeres originate occasionally from non-centromeric regions of chromosomes, providing an excellent model system to study centromeric chromatin. The maize mini-chromosome Derivative 3-3 contains a de novo centromere, which was derived from a euchromatic site on the short arm of chromosome 9 that lacks traditional centromeric repeat sequences. Our previous study found that the CENH3 binding domain of this de novo centromere is only 288 kb with a high-density gene distribution with low-density of transposons. Here we applied next generation sequencing technology to analyze gene transcription, DNA methylation for this region. Our RNA-seq data revealed that active chromatin is not a barrier for de novo centromere formation. Bisulfite-ChIP-seq results indicate a slightly increased DNA methylation level after de novo centromere formation, reaching the level of a native centromere. These results provide insight into the mechanism of de novo centromere formation and subsequent consequences. RNA-seq was carried out using material from seedling and young leaves between control and Derivative 3-3. Bisulfite-ChIP-seq was carried out with anti-CENH3 antibodies using material from young leaves in Derivative 3-3.
Project description:Cancer development is associated with multiple genetic alterations and genomic instability either at the chromosomal or base pair level is generally thought to underlie these changes. However, it is still unknown whether genetic instability is absolutely required for tumorigenesis. Here we investigated the genomic instability status of four cytogenetically stable diploid cell lines CAL51, SK-UT-1B, A204 and CH1. We applied high resolution 500K single nucleotide polymorphism (SNP) array analysis and found that all the four cell lines have some sub-microscopic genomic copy number changes. Interestingly, there were many more sub-microscopic chromosome alterations in A204 and CH1 than in CAL51 and SK-UT-1B. Twenty-four-color fluorescence in situ hybridization (FISH) was used to analyse a large number of metaphases from CAL51, SK-UT-1B and CH1 cells. The rate of de novo chromosome rearrangements was significantly higher in CH1 than CAL51 and SK-UT-1B. Although this increased rate did not lead to many clonal cytogenetically apparent chromosome alterations in CH1 cells, it is consistent with a first step towards chromosomal instability. It is more striking that both cell lines CAL51 and SK-UT-1B which have a similar de novo chromosomal change rate to that of normal lymphocytes were microsatellite instability positive by BAT-26 microsatellite analysis. This study further strengths the current concept that genomic instability is associated with tumor development. Keywords: DNA copy number changes
Project description:TET protein-catalyzed 5mC oxidation not only creates novel DNA modifications such as 5hmC, but also initiates active or passive DNA demethylation. However, the TETsâ function in crosstalk with specific histone modifications is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domain underlying the methylated DNA CpG islands (CGIs). Overexpression of wild type (WT) but not catalytic inactive mutant (Mut) TET2 in TET-low-expressing cells results in increase of 5hmC level and accompanying DNA demethylation at a subset of CGIs. Importantly, this is sufficient to create de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of key developmental gene promoters, which are often located within CpG islands that are previously hyper-methylated and silenced. On the other hand, depletion of Tet1 and Tet2 in mouse ES cells results in an apparent loss of H3K27me3 at bivalent domains, which are located within CGIs and associated with a particular set of key developmental gene promoters. Collectively, these data suggest that TET proteins have a primary role in charge of regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at a subset of CpG islands associated with developmental genes. We examined H3K4me3,H3K27me3,5mC and 5hmC in 293T and mES cell types,using Illumina Hiseq2500.
Project description:Ongoing studies using genomic microarrays and next-generation sequencing have demonstrated that the genetic contributions to cardiovascular diseases have been significantly ignored in the past. The aim of this study was to identify rare copy number variants in individuals with congenital pulmonary atresia (PA). Based on the hypothesis that rare structural variants encompassing key genes play an important role in heart development in PA patients, we performed high-resolution genome-wide microarrays for copy number variations (CNVs) in 82 PA patient-parent trios and 189 controls with an Illumina SNP array platform. CNVs were identified in 17/82 patients (20.7%), and eight of these CNVs (9.8%) are considered potentially pathogenic. Five de novo CNVs occurred at two known congenital heart disease (CHD) loci (16p13.1 and 22q11.2). Two de novo CNVs that may affect folate and vitamin B12 metabolism were identified for the first time. A de novo 1-Mb deletion at 17p13.2 may represent a rare genomic disorder that involves mild intellectual disability and associated facial features.
Project description:Dependent on concise, pre-defined protein sequence databases, traditional search algorithms perform poorly when analyzing mass spectra derived from wholly uncharacterized protein products. Conversely, de novo peptide sequencing algorithms can interpret mass spectra without relying on reference databases. However, such algorithms have been difficult to apply to complex protein mixtures, in part due to a lack of methods for automatically validating de novo sequencing results. Here, we present novel metrics for benchmarking de novo sequencing algorithm performance on large scale proteomics datasets, and present a method for accurately calibrating false discovery rates on de novo results. We also present a novel algorithm (LADS) which leverages experimentally disambiguated fragmentation spectra to boost sequencing accuracy and sensitivity. LADS improves sequencing accuracy on longer peptides relative to other algorithms and improves discriminability of correct and incorrect sequences. Using these advancements, we demonstrate accurate de novo identification of peptide sequences not identifiable using database search-based approaches.