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Project description:Agilent whole exome hybridisation capture was performed on genomic DNA derived from Chondrosarcoma cancer and matched normal DNA from the same patients. Next Generation sequencing performed on the resulting exome libraries and mapped to build 37 of the human reference genome to facilitate the identification of novel cancer genes. Now we aim to re find and validate the findings of those exome libraries using bespoke pulldown methods and sequencing the products.
2016-05-17 | E-ERAD-37 | biostudies-arrayexpress
Project description:Targeted sequencing of genome is essential to the basic biological research and biomedicine. Therefore, various targeted enrichment methods have been developed for this end, in which various hybridization-based methods have been most widely used. However, the current hybridization-based methods (both on solid or in solution) are still restricted by the intrinsic shortcomings of nucleic acid hybridization. In this study, by combining an engineered dCas9-sgRNA with widely used magnetic isolation, we developed a new strategy to enrich target genomic DNAs in high efficiency. In this strategy, target DNA was firstly specifically recognized and bound by a complex of dCas9 and capture sgRNA (csgRNA). The DNA-dCas9-csgRNA complex was then captured on magnetic beads through the annealing of elongated 3′ end of csgRNA with single-stranded capture oligonucleotides coupled on streptavidin-coated magnetic beads. We thus named the strategy as CRISPR-assistant target enrichment (CATE). The enriched DNAs can be analyzed by the next generation sequencing (NGS). We thus named the technique as CATE-seq. Used the technique, we successfully enriched 35 target exons of 6 genes in 6 cell lines by using 54 csgRNA. We found that the target genomic DNA fragments such as exons could be efficiently enriched and analyzed by CATE-seq. The technique has several significant advantages over the current hybridization-based methods, including high simplicity, specificity, sensitivity, and throughput. This study therefore provides a new powerful tool for targeted sequencing.
2018-09-15 | GSE119994 | GEO
Project description:Panicum virgatum Exome Capture and Next Generation Sequencing
| PRJNA244250 | ENA
Project description:DNA methylation in CpG context is fundamental to the epigenetic regulation of gene expression in high eukaryotes. Disorganization of methylation status is implicated in many diseases, cellular differentiation, imprinting, and other biological processes. Techniques that enrich for biologically relevant CpG-rich genomic regions are desired since, depending on the size of an oragnism's methylome, the depth of sequencing required to cover all CpGs can be prohibitively expensive. Currently, restriction-enzyme based Reduced Representation Bisulfite Sequencing and its modified protocols are widely used to study methylation differences. Recently, Agilent Technologies and Roche NimbleGen have aimed to both reduce sequencing costs and capture CpGs of known biological relevance by marketing in-solution custom-capture hybridization platforms. These three methods target approximately 10-13% of the human methylome. For each platform - restriction-enzyme based enhanced reduced representation (ERRBS), capture based Agilent SureSelect Methyl-seq (SSMethylseq), and capture based Roche NimbleGen SeqCap Epi CpGiant (CpGiant) - we used human lung fibroblast cell line IMR90 DNA to make libraries according to each protocol and sequenced to equivalent depth. Overall, SSMethylSeq and CpGiant covered >95% of their designed capture regions whereas ERRBS covered 70% of its expected MspI regions. Methylation levels were concordant across the platforms. The concordance of annotations of CpG units for genomic features, displayed roughly the same proportions of genomic features. SSMethylSeq and CpGiant are most similar and cover marginally more annotated regions than ERRBS. However, the number of CpG units shared by all methods was low, ~26% of any platform. We conclude that captured based methods are largely consistent in terms of covered CpG loci although ERRBS provides comparable data at a significantly reduced price. Furthermore, library preparation for ERRBS can be performed with as little as 75ngs of starting material, whereas micrograms are needed for the capture hybridization techniques. Libraries were made from human lung fibroblast cell line IMR90 DNA for each protocol of ERRBS, Agilent SureSelect Methyl-seq, Roche NimbleGen SeqCap Epi CpGiant, and WGBS, then sequenced as paired-end 100bp on an Illumina HiSeq 2500.
2016-06-22 | E-GEOD-83595 | biostudies-arrayexpress
Project description:Through whole-exome sequencing we identified somatic missense mutations in DICER1 and DROSHA in Wilms tumor, a childhood kidney cancer. DICER1 and DROSHA are key enzymes in the microRNA biogenesis pathway. To determine the effect of these mutations on microRNA expression, we prepared small RNAs from Wilms tumors and used next-generation sequencing to determine the expression levels of microRNAs in the tumors. Comparison of miRNA expression in tumors with and without mutations in DICER1 or DROSHA.
2015-04-21 | E-GEOD-56955 | biostudies-arrayexpress
Project description:Studies have shown that tumors from the same patient may respond very differently to the same therapeutic agents. This study aims to investigate the genetic basis of tumors that respond abnormally well or poorly to therapeutic agents in an effort to understand the fundamental genetic basis of this response. The present protocol seeks to retrospectively perform Exome, next-generation (DNA) sequencing and/or other molecular techniques on tumor samples to identify the genetic basis of a patient’s exceptional response to chemotherapy.
| 2289468 | ecrin-mdr-crc
Project description:Recently, a number of advances have been implemented into the core ChIP-seq (chromatin immunoprecipitation coupled with next-generation sequencing) methodology to streamline the process, reduce costs or improve data resolution. Several of these emerging ChIP-based methods perform additional chemical steps on bead-bound immunoprecipitated chromatin, posing a challenge for generating similarly treated input controls required for artifact removal during bioinformatics analyses. Here we present a versatile method for producing technique-specific input controls for ChIP-based methods that utilize additional bead-bound processing steps. This reported method, termed protein attached chromatin capture (PAtCh-Cap), relies on the non-specific capture of chromatin-bound proteins via their carboxylate groups, leaving the DNA accessible for subsequent chemical treatments in parallel with chromatin separately immunoprecipitated for the target protein. Application of this input strategy not only significantly enhanced artifact removal from ChIP-exo data, increasing confidence in peak identification and allowing for de novo motif searching, but also afforded discovery of a novel CTCF binding motif.
2016-08-24 | GSE79563 | GEO
Project description:5-hydroxymethylcytosine (5hmC) is the first oxidative product of the TET-mediated 5-methylcytosine (5mC) demethylation pathway. It is a key intermediate in cytosine demethylation, and have potential regulatory functions with emerging importance in mammalian biology. In this work, we used a chemical capture-based technique that coupled with next-generation sequencing to investigate the global 5hmC methylation in five brain subregions (cerebellum, cortex, hippocampus, hypothalamus and thalamus) and liver tissues from female and male adult mice. We also performed total RNA sequencing to study the association between 5hmC and gene expression. The enriched 5-hmC library was sequenced on a HiSeq2500 by paired-end sequencing with 100 bp read length.
2017-01-31 | E-MTAB-5167 | biostudies-arrayexpress
Project description:Comparison of Custom Capture for Targeted Next Generation DNA Sequencing
| PRJNA263214 | ENA