Project description:The aim of this experiment was to profile DNase-I accessibility at a subset of genomic regions in extremely high coverage. After DNase-I treatment, DNA fragments from specific loci were targeted using bead capture, amplified, and sequenced.
Project description:Multiplexed Chromatin Conformation Capture in Mouse Erythroid cells , from hundreds of targeted loci, using agilent oligo capture technology and high throughput sequencing.
Project description:Multiplexed Chromatin Conformation Capture in Mouse Erythroid cells , from hundreds of targeted loci, using agilent oligo capture technology and high throughput sequencing. Two erythroid Ter119+ cell replicates and a mouse ES cell control
Project description:Microarray-based enrichment of selected genomic loci is a powerful method for genome complexity reduction. Since the vast majority of exons in vertebrate genomes are smaller than 150 nt, we have explored the use of short fragment libraries (85-110bp) to achieve higher enrichment specificity by reducing carryover and adverse effects of flanking intronic sequences. These short fragment libraries were enriched for 1.69 Mb of exonic sequences, using custom 244K microarrays, and sequenced using AB/SOLiD. High enrichment specificity (60 M-bM-^@M-^S 75%) was obtained at 67-213x average coverage, with 77-92% and 90-98% of targeted regions covered with more than 25% and 10% of the average coverage, respectively. As a more appropriate measure of the evenness of coverage, which is relatively independent of sequencing depth, we introduce the evenness of coverage parameter E. E values up to 75% were achieved. To verify the accuracy of SNP/mutation detection we evaluated 384 known non-reference SNPs in the targeted regions. At ~ 200x average sequence coverage, we were able to survey 96.4% of 1.69 Mb of genomic sequence with only 4.2% false negative calls while 3.6% of targeted regions were marked as unsurveyed. A total of 1197 new variants were detected. Verification revealed only 8 false positive calls, resulting in an overall false positive rate of less than 1 per ~200,000 bp (0.0005%, equivalent to an overall phred score of 55). 4 samples + capture design file
Project description:Nuclear and mitochondrial organelles must maintain a communication system. Loci on the mitochondrial genome were recently reported to interact with nuclear loci. To determine whether this is part of a DNA based communication system we used genome conformation capture to map the global network of DNA-DNA interactions between the mitochondrial and nuclear genomes (Mito-nDNA) in Saccharomyces cerevisiae cells grown under three different metabolic conditions. The interactions that form between mitochondrial and nuclear loci are dependent on the metabolic state of the yeast. Moreover, the frequency of specific mitochondrial - nuclear interactions (i.e. COX1-MSY1 and Q0182-RSM7) showed significant reductions in the absence of mitochondrial encoded reverse transcriptase machinery. Furthermore, these reductions correlated with increases in the transcript levels of the nuclear loci (MSY1 and RSM7). We propose that these interactions represent an inter-organelle DNA mediated communication system and that reverse transcription of mitochondrial RNA plays a role in this process. Genome Conformation Capture (GCC) has been performed on exponentially growing Saccharomyces cerevisiae cultures in glucose containing media. Paired end sequencing on an Illumina Genome Analyser was performed before the sequences were analysed by the propieatry software Topography 1.19. Inter- and intra- chromosomal interactions were mapped onto the S. cerevisiae S288 genome scaffold.
Project description:Nuclear and mitochondrial organelles must maintain a communication system. Loci on the mitochondrial genome were recently reported to interact with nuclear loci. To determine whether this is part of a DNA based communication system we used genome conformation capture to map the global network of DNA-DNA interactions between the mitochondrial and nuclear genomes (Mito-nDNA) in Saccharomyces cerevisiae cells grown under three different metabolic conditions. The interactions that form between mitochondrial and nuclear loci are dependent on the metabolic state of the yeast. Moreover, the frequency of specific mitochondrial - nuclear interactions (i.e. COX1-MSY1 and Q0182-RSM7) showed significant reductions in the absence of mitochondrial encoded reverse transcriptase machinery. Furthermore, these reductions correlated with increases in the transcript levels of the nuclear loci (MSY1 and RSM7). We propose that these interactions represent an inter-organelle DNA mediated communication system and that reverse transcription of mitochondrial RNA plays a role in this process. Genome Conformation Capture (GCC) has been performed on exponentially growing Saccharomyces cerevisiae cultures in glycerol lactate or galactose media. Paired end sequencing on an Illumina Genome Analyser was performed before the sequences were analysed by the propieatry software Topography 1.19. Inter- and intra- chromosomal interactions were mapped onto the S. cerevisiae S288 genome scaffold.
Project description:Genome-wide association studies have identified over 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions; several map to gene deserts, regions of several hundred kb lacking protein-coding genes. We hypothesized that gene deserts harbour long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21 and 9q31.2. We identified interaction peaks between putative regulatory elements ("bait fragments") within the captured regions and "targets" that included both protein-coding genes and long non-coding (lnc)RNAs, over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2 and MYC; target lncRNAs included DIRC3, PVT1 and CCDC26. For two gene deserts we were able to define a set of SNPs that were correlated with the published risk variant and that clustered within the bait end of an interaction peak. Preliminary functional analyses implicate one SNP (rs12613955; 2q35) as a potentially functional variant. Capture Hi-C was carried out in BT483, SUM44, and GM06990 cell lines to investigate breast cancer risk loci 2q35, 8q24.21 and 9q31.2.
Project description:We used the nanopore Cas9 targeted sequencing (nCATS) strategy to specifically sequence 125 L1HS-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. The genome-wide profile of L1 methylation was also assessed by bs-ATLAS-seq in the same cell lines (E-MTAB-10895).