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. 2017 Jan 9;45(1):e4.
doi: 10.1093/nar/gkw809. Epub 2016 Sep 12.

ChIA-PET2: a versatile and flexible pipeline for ChIA-PET data analysis

Guipeng Li  1 , Yang Chen  1 , Michael P Snyder  2 , Michael Q Zhang  3   4
Affiliations

ChIA-PET2: a versatile and flexible pipeline for ChIA-PET data analysis

Guipeng Li et al. Nucleic Acids Res. .

Abstract

ChIA-PET2 is a versatile and flexible pipeline for analyzing different types of ChIA-PET data from raw sequencing reads to chromatin loops. ChIA-PET2 integrates all steps required for ChIA-PET data analysis, including linker trimming, read alignment, duplicate removal, peak calling and chromatin loop calling. It supports different kinds of ChIA-PET data generated from different ChIA-PET protocols and also provides quality controls for different steps of ChIA-PET analysis. In addition, ChIA-PET2 can use phased genotype data to call allele-specific chromatin interactions. We applied ChIA-PET2 to different ChIA-PET datasets, demonstrating its significantly improved performance as well as its ability to easily process ChIA-PET raw data. ChIA-PET2 is available at https://github.com/GuipengLi/ChIA-PET2.

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Figures

Figure 1.
Figure 1.
ChIA-PET2 workflow. Linkers are first trimmed for each pair of reads in different ways according to different ChIA-PET protocols. Only read pairs that satisfy certain user-defined conditions are kept. Then, each end of the read pairs is aligned to the reference genome independently using bwa. PETs with high mapping quality scores are kept to call ChIP-Seq-like peaks by MACS2. The pairing information is not used in the peak calling step. Once peaks are called, PETs that link these peaks are clustered as loops in the loop detection step. If phased genotype data are available, ChIA-PET2 will assign the PETs to parental alleles. Statistical metrics are plotted as quality controls. After the loops are detected, a significant loop calling step is applied using MICC.
Figure 2.
Figure 2.
ChIA-PET2 quality controls. (A) Statistics metrics of linker trimming. (B) Statistics metrics of read alignment. (C) Different types of PETs and their percentages. (D) Intra/Inter-chromosomal interactions percentage with at least four supported PETs. (E) PET count distribution of all candidate intra-chromosomal loops before statistical confidence estimation. (F) PET count distribution of all candidate inter-chromosomal loops before statistical confidence estimation.
Figure 3.
Figure 3.
Results of the ChIA-PET2 pipeline on real ChIA-PET datasets. (A) Number of CTCF-mediated chromatin loops detected by different methods from bridge-linker ChIA-PET data. CTCF, RAD21 and SMC3 occupancy in the anchors of loops are shown above the histogram. (B) Overlap of CTCF-mediated chromatin loops detected by ChIA-PET2 and the pipeline by Tang et al. (4). (C) Number of RNA POL2-mediated chromatin loops detected by different methods. POL2 occupancy in the anchors of loops is shown above the histogram. (D) Overlap of POL2-mediated chromatin loops detected by ChIA-PET2 and Mango. (E) Reproducibility of ChIA-PET2 and Mango.
Figure 4.
Figure 4.
Short-range chromatin loops. (A) The distribution of genomic distance between loop anchors. POL2 occupancy in the anchors of RNA POL2 mediated chromatin loops detected by different methods. Red dashed line marks the 8kb distance. (B) CTCF, RAD21 and SMC3 occupancy in the anchors of CTCF mediated short (<8 kb) chromatin loops detected by ChIA-PET2. (C) Example of short chromatin loops near the Myc gene and ChIP-Seq/RNA-Seq signal along the window. PET count number is labelled at the loop. (D) Another example of short chromatin loops near gene TP53I13.
Figure 5.
Figure 5.
Allele-specific analysis. (A) Signal of CTCF,RAD21, SMC3 ChIP-Seq, DNase-Seq and ChIA-PET peaks near the H19/IGF2 region. (B) Contact matrix generated from all valid ChIA-PET PETs. (C) Maternal contact matrix generated from maternal phased PETs (left panel) and maternal extended phased PETs (right panel). Red arrow indicates the chromatin loop and gray arrow indicates absent or attenuated peak signal. (D) Paternal contact matrix generated from paternal phased PETs (left panel) and paternal extended phased PETs (right panel). The red arrow indicates the chromatin loop and the gray arrow indicates an absent or attenuated peak signal and the blue arrow indicates that both alleles have the peak signal.
Figure 6.
Figure 6.
Comparison with Hi-C contact map. (A) Signal of CTCF,RAD21, SMC3 ChIP-Seq, DNase-Seq and ChIA-PET peaks in a 2Mb region in chromosome 4. (B) Heatmap of contact matrix directly from Rao et al. (5) with additional annotation. (C) Heatmap of the contact matrix generated from ChIA-PET data by the ChIA-PET2 pipeline. The red arrow marks the chromatin loop and the gray arrow marks the absence of a peak signal.
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