An effective approach for identification of in vivo protein–DNA binding sites from paired-end ChIP-Seq data
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE16940 | GEO | 2010/06/04
SECONDARY ACCESSION(S): PRJNA117657
REPOSITORIES: GEO
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Project description:ChIP-Seq, which combines chromatin immunoprecipitation (ChIP) with high-throughput massively parallel sequencing, is increasingly being used for identification of proteinM-bM-^@M-^SDNA interactions in-vivo in the genome. In general, current algorithms for ChIP-seq reads employ artificial estimation of the average length of DNA fragments for peak finding, leading to uncertain prediction of DNA-protein binding sites. Here, we present SIPeS (Site Identification from Paired-end Sequencing), a novel algorithm for precise identification of binding sites from short reads generated from paired-end Solexa ChIP-Seq technology. SIPeS uses a dynamic baseline directly via M-bM-^@M-^Xpiling upM-bM-^@M-^Y the corresponding fragments defined by the paired reads to efficiently find peaks corresponding to binding sites. The performance of SIPeS is demonstrated by analyzing the ChIP-Seq data of the Arabidopsis basic helix-loop-helix transcription factor ABORTED MICROSPORES (AMS). The robustness of SIPeS was demonstrated in higher sensitivity and spatial resolution in peak finding compared to three existing peak detection algorithms. Keywords: transcription factors (protein-DNA interactions) Examination of protein-DNA interactions in buds of Arabidopsis anther cell
2010-06-04 | E-GEOD-16940 | biostudies-arrayexpress
Project description:CUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Size-fractionation of aligned reads revealed that the collection of long (>270 bp) DNA fragments corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that shorter (<120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of shorter fragments, which constituted only about 5% of the total reads, indicates the unique positioning of the poised RNA polymerase II before transcription initiation, which has not been detected in the steady state of transcription by standard chromatin immunoprecipitation.
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Project description:ChIP-Seq is a technique used to analyse protein-DNA interactions. The protein-DNA complex is pulled down using a protein antibody, after which sequencing and analysis of the bound DNA fragments is performed. A key bioinformatics analysis step is “peak” calling - identifying regions of enrichment. Benchmarking studies have consistently shown that no optimal peak caller exists. Peak callers have distinct selectivity and specificity characteristics which are often not additive and seldom completely overlap in many scenarios. In the absence of a universal peak caller, we rationalized one ought to utilize multiple peak-callers to 1) gauge peak confidence as determined through detection by multiple algorithms, and 2) more thoroughly survey the protein-bound landscape by capturing peaks not detected by individual peak callers owing to algorithmic limitations and biases. We therefore developed an integrated ChIP-Seq Analysis Pipeline (ChIP AP) which performs all analysis steps from raw fastq files to final result, and utilizes four commonly used peak callers to more thoroughly and comprehensively analyse datasets. Results are integrated and presented in a single file enabling users to apply selectivity and sensitivity thresholds to select the consensus peak set, the union peak set, or any sub-set in-between to more confidently and comprehensively explore the protein bound landscape. (https://github.com/JSuryatenggara/ChIP-AP).
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Project description:ChIP-seq has become the method of choice for studying functional DNA-protein interactions on a genome wide scale. The method is based on co-immunoprecipitation of DNA binding proteins with formaldehyde cross-linked DNA, followed by deep sequencing of immunoprecipitated chromatin fragments, allowing for the identification of binding sites with high accuracy [1-5]. Traditionally, genome-wide tiling path microarrays were used for the detection of specifically immunoprecipitated DNA fragments, but current next-generation sequencers now generate sufficient data to assay multiple samples in a single sequencing run, making this method more time and cost effective compared to microarray-based approaches [2]. However, due to limitations in read length of next generation sequencing technologies (50-76bp), it is not possible to sequence the complete length of immunoprecipitated DNA fragments which can be up to 2kb long reflecting biology in case of big protein complexes. As a consequence only the ends of the immunoprecipitated DNA fragments are sequenced. Deconvolution of sequencing reads mapping to the positive and negative strand is required to identify the real DNA binding site [4-9]. This limitation is most obvious in case of large complex regions where multiple binding sites of various regulatory elements are clustered close to each other. Deconvolution of such regions and the exact identification of individual binding positions is very challenging [4]. Similar problems can occur when studying histone positioning in cases where the length of ChIP fragments is bigger than the average distance of 2 neighboring nucleosomes. In addition frequently only narrow size range of immunoprecipitated fragments is selected for sequencing and thus possible bias towards binding regions within the selected range can be expected by loosing larger fragments possible originated from protein complexes interacting with DNA. To circumvent these complications, we modified the procedure for the preparation of ChIP-seq samples by introducing an additional extensive fragmentation round after isolation of immunoprecipitated chromatin to generate 70-110 bp long DNA fragments. For testing, we choose Tcf4 protein which is a well-studied downstream element of the Wnt-pathway for which the genome wide binding site profile is known was already known from ChIP-on-CHIP experiments [10]. Using the modified approach, we were able to identify Tcf4 binding sites at near nucleotide resolution without computational deconvolution. Furthermore, high-resolution information on genome-wide binding site regions allowed for the identification of potential novel Tcf4 co-factors as well as target genes. 5 samples + 3 input samples
2010-11-01 | E-GEOD-18481 | biostudies-arrayexpress
Project description:ChIP-seq has become the method of choice for studying functional DNA-protein interactions on a genome wide scale. The method is based on co-immunoprecipitation of DNA binding proteins with formaldehyde cross-linked DNA, followed by deep sequencing of immunoprecipitated chromatin fragments, allowing for the identification of binding sites with high accuracy [1-5]. Traditionally, genome-wide tiling path microarrays were used for the detection of specifically immunoprecipitated DNA fragments, but current next-generation sequencers now generate sufficient data to assay multiple samples in a single sequencing run, making this method more time and cost effective compared to microarray-based approaches [2]. However, due to limitations in read length of next generation sequencing technologies (50-76bp), it is not possible to sequence the complete length of immunoprecipitated DNA fragments which can be up to 2kb long reflecting biology in case of big protein complexes. As a consequence only the ends of the immunoprecipitated DNA fragments are sequenced. Deconvolution of sequencing reads mapping to the positive and negative strand is required to identify the real DNA binding site [4-9]. This limitation is most obvious in case of large complex regions where multiple binding sites of various regulatory elements are clustered close to each other. Deconvolution of such regions and the exact identification of individual binding positions is very challenging [4]. Similar problems can occur when studying histone positioning in cases where the length of ChIP fragments is bigger than the average distance of 2 neighboring nucleosomes. In addition frequently only narrow size range of immunoprecipitated fragments is selected for sequencing and thus possible bias towards binding regions within the selected range can be expected by loosing larger fragments possible originated from protein complexes interacting with DNA. To circumvent these complications, we modified the procedure for the preparation of ChIP-seq samples by introducing an additional extensive fragmentation round after isolation of immunoprecipitated chromatin to generate 70-110 bp long DNA fragments. For testing, we choose Tcf4 protein which is a well-studied downstream element of the Wnt-pathway for which the genome wide binding site profile is known was already known from ChIP-on-CHIP experiments [10]. Using the modified approach, we were able to identify Tcf4 binding sites at near nucleotide resolution without computational deconvolution. Furthermore, high-resolution information on genome-wide binding site regions allowed for the identification of potential novel Tcf4 co-factors as well as target genes.
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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).
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Project description:Purpose: A method for identifying genome-wide DNA binding sites for Fosb. Methods: Alive cells were sorted from retro-Fosb-OE(over-expression) organoids. The samples were incubated with anti-Fosb antibody (Abcam, ab184938). Purified DNA was subjected to Tru-seq library construction using NEBNext Ultra II DNA Library Prep Kit and sequenced as paired-end with Illumina Novaseq 6000. HISAT2 was used to align the sequences to the mouse genome and generate bam files. bamCoverage (CPM normalized and extended reads) was used to generate bigwig files from bam files. MACS2 was used for peak calling and to generate bed files from aligned reads. HOMER annotatePeaks.pl was used to annotate the peaks. Conclusions: Target genes of Fosb through ChIP assay were consistent with predicted target genes. Thus, we concluded that Fosb, which is a key TF could regulate most of ISC signature genes to maintain Lgr5+ ISCs.
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