Project description:Genome editing was conducted on a t(3;8) K562 model to investigate the effects of deleting different modules or CTCF binding sites within the MYC super-enhancer. To check mutations after targeting with CRISPR-Cas9 we performed amplicon sequencing using the Illumina PCR-based custom amplicon sequencing method using the TruSeq Custom Amplicon index kit (Illumina). The first PCR was performed using Q5 polymerase (NEB), the second nested PCR with KAPA HiFi HotStart Ready mix (Roche). Samples were sequenced paired-end (2x 250bp) on a MiSeq (Illumina).
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
Project description:Chromosomes are folded into highly compacted structures to accommodate physical constraints within nuclei and to regulate access to genomic information. Recently, global mapping of pairwise contacts showed that loops anchoring topological domains (TADs), are highly conserved between cell types and species. Whether pairwise loops synergize to form higher order structures is still unclear. Here we develop a conformation capture approach to study higher order organization using chromosomal walks (C-walks) that link multiple genomic loci together into proximity chains. The data captured a hierarchical chromosomal structure at varying scales. Inter-chromosomal contacts constitute only 7-10% of the pairs and are restricted by the TAD structure of the interfacing chromosomes. About half of the C-walks stay within one chromosome, and almost half of these are restricted to intra-TAD spaces. Analysis of nested topological motifs suggests hierarchical chromosomal structure is present also within TADs. Targeted analysis of thousands of 3-walks anchored at strongly expressed genes support nested, rather than hub-like, chromosomal topology at active loci. Polycomb-repressed HOX domains are shown by the same approach to form synergistic hubs. Together, the data suggest that chromosomal territories, TADs, and intra-TAD loops are primarily driven by nested, possibly dynamic, pairwise contacts.
2016-02-04 | GSE77553 | GEO
Project description:A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
Project description:Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution single cell DNA methylation sequencing in prostate cancer, we identify 40 core hypomethylation domains, consistently hypomethylated across tumor cells and arising at early stages of malignancy. Transcriptionally silenced genes within these domains are enriched for immune-related genes; nested among repressive domains are small loci with preserved methylation, encoding cell proliferation genes that escape silencing. Prominent among hypomethylation-silenced genes is a gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells, and four IFI16-related interferon-inducible genes implicated in innate immunity. Re-expression of CD1 or IFI16 murine orthologs in immunocompetent mice abrogates prostate tumorigenesis, accompanied by activation of anti-tumor immunity. Thus, early epigenetic changes in cancer may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for circulating tumor cells.
Project description:We report a novel high-throughput method to empirically quantify individual-specific regulatory element activity at the population scale. The approach combines targeted DNA capture with a high-throughput reporter-gene expression assay. As demonstration, we have measured the activity of more than 100 putative regulatory elements from 95 individuals in a single experiment. We found that, in agreement with previous reports, most genetic variants have weak effects on distal regulatory element activity. Because haplotypes are typically maintained within but not between assayed regulatory elements, the approach can be used to identify likely causal regulatory haplotypes that contribute to human phenotypes. Finally, we demonstrate the utility of the method to functionally fine map causal regulatory variants in regions of high linkage disequilibrium identified by expression quantitative trait loci (eQTL) analyses. 104 candidate regulatory elements from 95 individuals were resequenced using Illumina custom amplicon sequencing. We then cloned the resulting DNA fragments into a massively parallel reporter assay to quantify allele-specific regulatory activity from that population. SNP-fdr.txt contains output of significance evaluation haplotype.fasta.gz contains the reference used to generate alignment files