Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions

Abstract

Summary Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a robust method to capture genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution for detecting chromatin interactions, whereas 3C/Hi-C approaches could not achieve. The original ChIA-PET protocol generates short paired-end tags (2×20 bp) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end-tags is increased (up to 2×250 bp). The longer PET reads not only improve the tag mapping efficiency but also increase the probability of covering phased SNPs, which allows haplotype-specific chromatin interactions identification. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with bridge linker, Tn5 tagmentation, PCR amplification, and high-throughput sequencing. To a well-trained molecular biologist, it typically takes six days from cell harvesting to the completion of library construction, up to a further 36 hours for DNA sequencing, and less than 20 hours for processing raw sequencing reads.

Chromatin interaction analysis with updated ChIA-PET Tool (V3)

“…These DNA products were paired-end sequencing (2×150 bp) using Illumina Hi-Seq 2500. ChIA-PET Tool V3 is a computational package to process the DNA sequence data generated from ChIA-PET experimental method, and it consists of 7 steps ( Figure 3): (1) linker filtering, (2) mapping the paired-end reads to a reference genome, (3) purifying the mapped reads, (4) dividing the mapped reads into different categories, (5) peak calling from self-ligation PETs, (6) interaction calling from inter-ligation PETs, and (7) visualizing the results. It was originally published in the journal Genome Biology in 2010.…”

“…With the advance in sequencing technology, the cost of sequencing is reduced, the amount of data increases at the same time. In the long-read ChIA-PET, the length of the paired-end tags is up to 2 × 250bp [5]. Therefore, we modify ChIA-PET Tool which includes integrating the pipeline of processing short-read data and long-read data, rewriting the shell script of the processing flow with Java, revising the linker filtering with multithreading, adjusting the step of mapping to reduce running time, generating the statistics of the result, and evaluating the quality of the data.…”

ChIA-PIPE: A fully automated pipeline for ChIA-PET data analysis and visualization

“…The first version ChIA-PET protocol extracted short (2 x 21 bp) paired tags for two contacting chromatin sites (Fullwood et al, 2009). Subsequently, an improved version for long read (2 x 150 bp) ChIA-PET was developed with increased mapping accuracy and robustness (Li, X. et al, 2017). ChIA-PET has been applied to study long-range chromatin interactions in human and mouse cells, and provided key insights into the roles of a number of chromatin architecture proteins and transcriptional factors including CTCF and RNAPII in human 3Dgenome organization (Li et al, 2012;Dowen et al, 2014;Tang et al, 2015;Ji et al, 2016;Weintraub et al, 2017).…”

“…Pairs of DNA fragments in each chromatin complex are joined via bridge linker ligation (proximity ligation). The ligation products are then purified for DNA library construction and high-throughput paired-end-tag (PET) sequencing (Li X. et al, 2017).…”

“…While computational pipelines have been developed in the past for short-reads of ChIA-PET data (Li, G. et al, 2010;Phanstiel et al, 2015) and for long-read ChIA-PET (Li, X. et al, 2017; that meet these baseline requirements, there are now significant unmet needs in the high-throughput processing and analysis of ChIA-PET data because of rapid advances in the field. First, it is now desirable to have a single pipeline with the flexibility to process either short-read (2 x 21 bp) or long-read (2 x 150 bp) data.…”