Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA147033 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR353696.fastq.gz | Fastqsanger.gz | |||
| SRR353696_1.fastq.gz | Fastqsanger.gz | |||
| SRR353696_2.fastq.gz | Fastqsanger.gz | |||
| SRR353697.fastq.gz | Fastqsanger.gz | |||
| SRR353697_1.fastq.gz | Fastqsanger.gz |
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Project description:Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long span paired-end-tag (PET) sequencing approach using 10 Kb genomic DNA inserts to study human genome structural variations (SVs). The use of 10 Kb insert size allows the identification of breakpoints within repetitive or homology containing regions of a few Kb in size and results in a higher physical coverage compared to small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and 2 non-cancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germline SVs, whereas tandem duplications, unpaired inversions, inter-chromosomal translocations, and complex rearrangements are overrepresented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA-PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures which are compatible with breakage-fusion-bridge cycles, and discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers. Structural variations of 15 human cancer samples and 2 human normal samples were identified by long span paired-end sequencing
2011-04-06 | E-GEOD-26954 | biostudies-arrayexpress
Project description:We used massively parallel DNA sequencing of paired-end ditags (DNA-PET) to identify structural genetic factors associated with disease progression and drug-resistance in representative samples from four CML patients and one CML cell line. The functional consequences of our genetic findings were evaluated in primary CML cells and cell lines, and validated in a larger CML cohort. We discovered a novel intronic deletion that correlated with imatinib-resistance, and was subsequently confirmed to be a polymorphism in normal East-Asian, but not African or Caucasian, populations. We found that the polymorphism favored expression of transcripts lacking a pro-apoptotic domain, which is critical for imatinib-induced cell death. A CML cell line containing the polymorphism also exhibited BCR-ABL-independent imatinib-resistance. Structural variations of 5 human CML samples were identified by long span paired-end sequencing
2012-02-27 | E-GEOD-28303 | biostudies-arrayexpress
Project description:We identified genomic structural alterations of six patients with signs of neurodevelopmental disorder (NDDs) that harbour chromosomal rearrangements using large-insert paired-end tag sequencing (DNA-PET). This technique allowed the refinement of chromosomal breakpoints and lead to the identification of seven disrupted genes (GNAQ, RBFOX3, UNC5D, TMEM47, NCAPG2, GTDC1 and XIAP). For one patient we filtered the entire panel of structural variations (SVs) with his parents and identified a unique SV that disrupted a single gene: GTDC1. We then validated the functional consequences of the chromosomal breakpoint disruption of GTDC1 by using patient-derived iPSCs. By differentiating these cells into neural progenitor cells (NPCs) and neurons, we interrogated the disease process at the cellular level and observed defects in the proliferation and glycosylation status of NPCs and also defects in neuronal maturation and function. We compared these results with GTDC1-deficient wild-type human NPCs and neurons, and observed similar phenotypic features as in the patient-derived cells which confirm that GTDC1 is involved in the patient’s phenotype. We show here that the combination of genomic screening with iPSCs technology provides a mechanistic insight into possible contributory effects of candidate genes implicated in NDDs and for personalized medicine. Structural variations were identified by long insert DNA paired-end tag (DNA-PET) sequencing, a mate-pair sequencing approach.
2014-09-03 | E-GEOD-51430 | biostudies-arrayexpress
Project description:We developed Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) for de novo detection of global chromatin interactions, and comprehensively mapped the chromatin interaction network bound by estrogen receptor α (ERα) in the human genome. We performed 454 and Illumina sequencing analyses. Keywords: Epigenetics Using 454, we examined 3 libraries: IHM001 (Estrogen Receptor ChIA-PET), IHM043 (Estrogen Receptor ChIP-PET) and IHM062 (IgG ChIA-PET) Using Illumina, we examined 4 libraries: IHM001 (Estrogen Receptor ChIA-PET replicate 1, Paired End Sequencing), IHH015 (Estrogen Receptor ChIA-PET replicate 2, Paired End Sequencing), H3K4me3 ChIP-Seq and RNA polymerase II ChIP-Seq
2009-09-11 | E-GEOD-18046 | biostudies-arrayexpress
Project description:Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells. RNA polymerase II (RNAPII) bound chromatin interactions were extracted with Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing, in order to study the transcription regulations with RNAPII-associated long-range chromatin interactions. Five cell lines, namely MCF7 (ATCC# HTB-22), K562 (ATCC# CCL-243), HCT116 (ATCC# CCL-247), HeLa (ATCC# CCL-2.2), and NB4 (Roussel and Lanotte, 2001) (provided by Dr. Sherman Weissman, Yale University), were grown under standard culture conditions and harvested at log phase. Harvested cells were cross-linked using 1% formaldehyde followed by neutralization with 0.2M glycine. Chromatin was isolated and subjected to ChIA-PET protocol as described in Fullwood et al (Fullwood et al: An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 2009, 462(7269):58-64). The ChIA-PET sequence reads were processed and analyzed using ChIA-PET Tool (Li et al: ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing. Genome Biol 2010, 11(2):R22).
2012-01-20 | E-GEOD-33664 | biostudies-arrayexpress
Project description:Chromosomal structural mutations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural mutations, we analyzed a representative sampling of the major types of breast tumor samples for detailed structural mutations using paired-end tag sequencing of long-insert genomic DNA (DNA-PET) with matched transcriptome ascertainment by RNA-seq. Compared with other structural mutations, tandem duplications are enriched around partners of fusion transcripts and demarcate regions of high gene expression. Moreover tandem duplications appear to be early events in tumor evolution by facilitating subsequent downstream amplification and deletion of important adjacent cancer associated genes. In a detailed reconstruction of events in chr17, we found large unpaired-inversions connect a duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently co-amplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in human normal mammary epithelial cells. Finally, using in silico approaches, we determined that genes whose expression in breast tumors are associated with either poor or good clinical prognosis appear clustered together in segments of frequent amplification or deletion, suggesting that structural abnormalities induce the loss or gain of blocks of adjacent genes with oncogenic or growth suppressor function. These analyses suggest that structural mutations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously. RNA sequencing of four primary breast cancer RNA samples (SOLiD, Applied Biosystems).
2014-07-01 | E-GEOD-57914 | biostudies-arrayexpress
Project description:Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. Genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs. The current application of PET sequencing with short insert size DNA is insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We have developed a robust procedure to generate PET sequencing data using large DNA inserts of 10 - 20 kb for the identification of SVs. We compared the characteristics of the large insert libraries with short insert (1 kb) libraries with the same sequencing depths and costs. Although short insert libraries bear an advantage in identifying small deletions, they do not provide a significantly better breakpoint resolution. Large inserts are superior to short inserts in providing higher physical genome coverage and therefore achieve greater sensitivity for the identification of the different types of SVs, including copy number neutral and complex events. Further, large inserts allow the identification of SVs within repetitive sequences which cannot be spanned by short inserts. Structural variations of three cancer cell lines using short (1 kb) and long (10 kb and 20 kb) insert size DNA fragments
2012-10-04 | E-GEOD-32674 | biostudies-arrayexpress
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Yijun Ruan mailto:ruanyj@gis.a-star.edu.sg). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track was produced as part of the ENCODE Project. It shows the locations of protein factor mediated chromatin interactions determined by Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) data (Fullwood et al., 2010) extracted from five different human cancer cell lines (K562 (chronic myeloid leukemia), HCT116 (colorectal cancer), HeLa-S3 (cervical cancer), MCF-7 (breast cancer), and NB4 (promyelocytic)). A chromatin interaction is defined as the association of two regions of the genome that are far apart in terms of genomic distance, but are spatially proximate to each other in the 3-dimensional cellular nucleus. Additionally, ChIA-PET experiments generate transcription factor binding sites. A binding site is defined as a region of the genome that is highly enriched by specific Chromatin ImmunoPrecipitation (ChIP) against a transcription factor, which indicates that the transcription factor binds specifically to this region. The protein factors displayed in the track include estrogen receptor alpha (ERa), RNA polymerase II (RNAPII), and CCCTC binding factor (CTCF). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) is a global de novo high-throughput method for characterizing the 3-dimensional structure of chromatin in the nucleus. In the ChIA-PET protocol, samples were cross-linked and fragmented, then subjected to chromatin immunoprecipitation. The DNA fragments that were brought together by the chromatin interactions were then proximity-ligated. During this proximity-ligation step, the half-linkers (created by the fragmentation) containing flanking MmeI sites (type IIS restriction enzymes) were first ligated to the DNA fragments and then ligated to each other to form full linkers. Full linkers bridge either two ends of a self-circularized fragment, or two ends of two different chromatin fragments. The material was then reverse cross-linked, purified and digested with MmeI. MmeI cuts 20 base pairs away from its recognition site. Tag-linker-tag (paired-end tag, PET) constructs were sequenced by ultra-high-throughput methods (Illumina or SOLiD paired-end sequencing). ChIA-PET reads were processed with the ChIA-PET Tool (Li et al., 2010) by the following steps: linker filtering, short reads mapping, PET classification, binding site identification, and interaction cluster identification. The high-confidence binding sites and chromatin interaction clusters were reported. Chromatin interactions identified by ChIA-PET have been validated by 3C, ChIP-3C, 4C and DNA-FISH (Fullwood et al., 2009).
2012-07-19 | E-GEOD-39495 | biostudies-arrayexpress
Project description:Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long span paired-end-tag (PET) sequencing approach using 10 Kb genomic DNA inserts to study human genome structural variations (SVs). The use of 10 Kb insert size allows the identification of breakpoints within repetitive or homology containing regions of a few Kb in size and results in a higher physical coverage compared to small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and 2 non-cancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germline SVs, whereas tandem duplications, unpaired inversions, inter-chromosomal translocations, and complex rearrangements are overrepresented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA-PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures which are compatible with breakage-fusion-bridge cycles, and discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers.
2011-04-06 | GSE26954 | GEO
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Yijun Ruan mailto:ruanyj@gis.a-star.edu.sg). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track is produced as part of the ENCODE Transcriptome Project. It shows the starts and ends of full length mRNA transcripts determined by GIS paired-end ditag (PET) sequencing using RNA extracts (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=rnaExtract) from different sub-cellular localizations (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=localization) in different cell lines (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=cellType). The RNA-PET information provided in this track is composed of two different PET length versions based on how the PETs were extracted. The cloning-based PET (18 bp and 16 bp) is an earlier version and detailed information can be found from reference (Ng et al. 2006). The cloning-free PET (25 bp and 25 bp) is a recently modified version which uses Type II enzyme EcoP15I to generate a longer length of PET (unpublished), which results in a significant enhancement in both library construction and mapping efficiency. Both versions of PET templates were sequenced by Illumina platform at 2 x 36 bp Paired End sequencing. See the Methods and References sections below for more details. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). Two different GIS RNA-PET protocols were used to generate the full length transcriptome PETs: one is based on a cloning-free RNA-PET library construction and sequencing strategy (unpublished), and the other is a cloning-based library construction (Ng et al. 2005) and recent Illumina paired end sequencing. Cloning-free RNA-PET (50 bp reads, 25 bp and 25 bp tag for each of the 5' and 3' ends)--Method: The cloning-free RNA-PET libraries were generated from polyA mRNA samples and constructed using a recently modified GIS protocol (unpublished). Total RNA in good quality was used as starting material and purified through MACs polyT column to obtain full length polyA mRNAs. Approximately 5 micrograms of enriched polyA mRNA were used for reverse transcription to convert polyA mRNA to full length cDNA. The obtained full length cDNA was modified and ligated with specific linker sequences, followed by circularization through ligation to generate circular cDNA molecules. The 25 bp tag from each end of the full length cDNA was extracted by type II enzyme EcoP15I digestion. The resulting PETs were ligated with sequencing adaptors at the both ends, amplified by PCR, and further purified as complex templates for paired end (PE) sequencing using Illumina platforms. Data: The sequenced RNA-PETs are unified in 25/25 bp length from each end of a cDNA. After filtering out redundant and noise tags, the unique PETs will proceed to analysis pipeline. Initially, the orientation of each tag will be screened out by the barcode built in the sequencing-template, then paired into a given orientation-PET. The orientation-determined RNA-PET is mapped onto reference genome allowing up to two mismatches. Majority of PETs are mapped on the known transcripts, or splice variants. A small portion of misaligned PETs, defined as discordant PETs, are mapped either too far from each tag, have wrong orientations, or mapped in different chromosomes, indicating exist some transcription variations which could be caused by genome structure variations: such as fusion, deletion, insertion, inversion, tandem repeat and translocation; or RNA trans-splicing etc. Cloning-based RNA-PET (34 bp reads, 18 bp and 16 bp tag for each of the 5' and 3' ends)--Method: The cloning-based RNA-PET (GIS-PET) libraries were generated from polyA RNA samples and constructed using the protocol described by Ng et al. (2005). Total RNA in good quality was used as starting material and further purified through MACs polyT column to enrich polyA mRNA and remove any contaminants (e.g., rRNA, tRNA, DNA, protein etc). Approximately 10 micrograms of polyA mRNA were then used for reverse transcription to convert polyA mRNA into full length cDNA. The obtained full length cDNA was modified with specific linker sequences, then, ligated to a GIS-developed (pGIS4) vector to form a complex full length cDNA library, which was cloned into E. coli. The plasmid DNA was then isolated from the library, followed by MmeI (a type II enzyme) digestion to generate a final length of 18 bp/16 bp ditags from each end of the full length cDNA. The single ditag (or called PET) was then ligated to form a diPET structure (a concatemer with two unrelated PET linked by a linker sequence) to facilitate Illuminaa Paired End sequencing. Data: The cloning-based RNA-PETs are unified in 18 bp and 16 bp length, respectively extracted from 5' and 3' end of each cDNA. The redundant reads were filtered out initially and unique ones were included for analysis. PET sequences were then mapped to (GRCh37, hg19, excluding mitochondirion, haplotypes, randoms and chromosome Y) reference genome using the following specific criteria (Ruan et al. 2007): A minimal continuous 16 bp match must exist for the 5' signature; the 3' signature must have a minimal continuous 14 bp match. Both 5' and 3' signatures must be present on the same chromosome. Their 5' to 3' orientation must be correct (5' signature followed by 3' signature). The maximal genomic span of a PET genomic alignment must be less than one million bp. PETs mapping to 2-10 locations are also included and may represent duplicated genes or pseudogenes in the genome. A majority of PETs mapped on the known transcripts or splice variants. A small portion of misaligned PETs, defined as discordant PETs, were mapped either too far from each other, mapped in the wrong orientation, or mapped to different chromosomes, indicating that some transcription variations exist which could be caused by genome structure variations: such as fusion, deletion, insertion, inversion, tandem repeat and translocation; or RNA trans-splicing etc. Clusters: To cluster the PETs the following procedure was applied: the mapping location of the 5' and 3' tag of a given PET was extended by 100 bp in both directions creating 5' and 3' search windows. If the 5' and 3' tags of a second PET mapped within the 5' and 3' search window of the first PET then the two PETs were clustered and the search windows were adjusted so that they contained the tag extensions of the second PET. PETs which subsequently mapped with their 5' and 3' tags within the adjusted 5' and 3' search window, respectively, were also assigned to this cluster and search window readjusted. This iterative process continued till no new PET was found to fall within the search window, at which stage all the found PETs are classified as belonging to a single cluster. This process is repeated till all PETs are assigned to a cluster. Verification: To assess overall PET quality and mapping specificity, the top ten most abundant PET clusters that mapped to well-characterized known genes were examined. Over 99% of the PETs represented full-length transcripts, and the majority fell within 10 bp of the known 5' and 3' boundaries of these transcripts. The PET mapping was further verified by confirming the existence of physical cDNA clones represented by the ditags. PCR primers were designed based on the PET sequences and amplified the corresponding cDNA inserts either from full length cDNA library (cloning-based PET) or from total RNA isolate (cloning-free PET) for sequencing confirmation.
2011-11-10 | E-GEOD-33600 | biostudies-arrayexpress