These reads are ChIA-PET single linker 2 tags, meaning two qualified genomic sequences connected with a linker, where both genomic sequences is larger than or equal to 18bp. Produced by trimming sequencing adaptors, identifying the ChIA-PET bridge linker sequence from input reads, collecting singlelinker 2 tags reads, mapping these filtered reads to the reference genome, quality filtering, deduplicating, and sorting. If multiple fastq files are generated, then fastq files are merged. ChIA-PIPE also supports the analysis of other related chromatin-mapping data types. To enable structural interpretation, ChIA-PIPE calls chromatin contact domains, resolves allele-specific peaks and loops, and annotates enhancer-promoter loops. To enable visualization, ChIA-PIPE generates input files for two-dimensional contact map viewing with Juicebox and HiGlass and provides a new dockerized visualization tool for high-resolution, browser-based exploration of peaks and loops. ChIA-PIPE performs linker filtering, read mapping, peak calling, and loop calling and automates quality control assessment for each dataset. The full ChIA-PET pipeline code is available on Github.ĬhIA-PIPE is a fully automated pipeline for ChIA-PET data processing, quality assessment, visualization, and analysis. The ChIA-PET pipeline (ChIA-PIPE) was developed by the Jackson lab. Long-read ChIA-PET for base-pair resolution mapping of haplotype-specific chromatin interactions. Haplotype-resolved chromatin interactions can be deduced if phased SNP information is available for the appropriate reference genome. The final tags (from all three categories of read pairs) are used to identify genomic binding sites of the protein of interest via peak calling. The final PETs are used to generate a 2D contact-map file for visualization and also to identify clusters of overlapping intrachromosomal loops. Each category is then aligned to a reference genome and only uniquely mapped and non-redundant tags are retained for further analysis. To analyze the data, reads pairs are first partitioned into three categories: (i) read pairs with no linker sequence, (ii) read pairs with a linker sequence and one usable genomic tag, or (iii) read pairs with a linker sequence and paired end tags (PETs). These fragments are subjected to PCR amplification with minimal cycles, size selection of the DNA fragments, and high-throughput paired-end sequencing. Streptavidin beads are then used to enrich for DNA fragments containing ligation junctions (i.e., containing the biotinylated bridge linker). The ligated DNA fragments are released by reverse cross-linking, and Tn5 transposase is used to simultaneously fragment the DNA and add sequencing adaptors. Next, pairs of DNA fragments are joined by proximity ligation with a “bridge linker” – a short double-stranded DNA sequence containing an internal biotinylated nucleotide and T overhangs on each end. Chromatin complexes immobilized on antibody beads are then subjected to DNA end repair and A-tailing. Immunoprecipitation (IP) is performed using a specific antibody to enrich for chromatin complexes involving a protein of interest. Then, nuclei are released by cell lysis and are sonicated to generate chromatin complexes containing DNA fragments. First, protein-DNA interactions are stabilized by dual-cross-linking in cells. ChIA-PET is a method for capturing genome-wide chromatin interactions that involve a protein of interest.
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