Pore-C Pipeline-Toolbox: a comprehensive pipeline for Pore-C data analysis

Abstract

Three-dimensional (3D) genomic architecture is crucial for the regulation of different biological processes, and the study of multi-way chromatin structures represents a cutting-edge area in this field. Pore-C is an advanced experimental technique integrating chromosome conformation capture (3C) with Nanopore long-read sequencing, which can effectively capture complex multi-way chromatin contacts across the genome. Due to the absence of comprehensive and dedicated tools, the analysis of Pore-C data remains challenging, restricting its broader application. To address this limitation, we developed Pore-C Pipeline Toolbox (PPL-Toolbox), a specialized software for a comprehensive analysis of multi-way chromatin interactions from Pore-C data. PPL-Toolbox incorporates a suite of optimized modules, including quality control, multi-way interaction extraction, noise reduction for multi-way contacts, advanced visualization tools for multi-way contacts, and haplotype map generation. Evaluation on both simulated and real datasets demonstrates that the PPL-Toolbox outperforms existing methods by providing a more comprehensive set of results. PPL-Toolbox is expected to become a powerful and versatile tool, driving advance in multi-way 3D genomics research, and enabling new discoveries in the field. PPL-Toolbox is publicly available on GitHub (https://github.com/versarchey/PPL-Toolbox).

Keywords: 3D genomics; long-read sequencing; multi-way chromatin conformation capture.

Figure 1

Overview of the PPL-Toolbox pipeline. The blue section illustrates the workflow for extracting multi-way contacts data using PPL-Toolbox. The red, green, and purple sections represent different classifications of multi-way alignment results. The orange section highlights additional analysis functions provided by PPL-Toolbox.

Figure 2

Denoising multi-way contacts using PPL-Toolbox. (a) Multi-way contact noise reduction process based on interaction frequency. Each Pore-C concatemer is treated as a hyperedge. Each hyperedge is expanded into a fully connected graph based on the VPCs matrix, and edges with low interaction frequencies are removed from the entire graph. After identifying all the maximal connected components, new hyperedges are constructed, completing the denoising process. (b) Comparison of order distribution before and after noise reduction. (c) Comparison of cis-trans VPCs ratio before and after noise reduction with bin size 500 Kb. (d) Comparison of VPCs maps before and after noise reduction (250 Kb resolution). Chromosome 1 of the GM12878 HiPore-C data is used as an example for demonstration. The dashed boxes highlight regions where the structural features are clearer after denoising.

Figure 3

Comparison of result extraction performance. (a and b) The Pearson correlation of eigenvectors (a) and insulation scores (b) between Pore-C Snakemake results, PPL-Toolbox reanalyzed results, and high-quality Hi-C. (c) Visualization of CDK6 results from Pore-C Snakemake and PPL-Toolbox. “Common” refers to reads detected by both Pore-C Snakemake and PPL-Toolbox. “PPL-specific” and “Pore-C Snakemake-specific” represent the unique reads detected by each respective method. (d and e) Comparison of boundary distance between boundaries and restriction enzyme cutting sites. Average “CLOSER” (d) and “BOTH” (e) distance between mapping result boundaries and their neighbor restriction enzyme cutting sites on different chromosomes. The numbers in the circle, as well as those in the diagram, are measured in base pairs (bp).

Figure 4

Data quality evaluation methods implemented in PPL-Toolbox. Comparison of BOTH, CLOSER, and DVD across chromosomes. (a and b) Data in (a) are derived from HiPore-C, and data in (b) are from Pore-C. (c and d) comparison of BOTH, CLOSER, and DVD by chromosomes: HiPore-C (c) and Pore-C (d). (e and f) Distribution of mapping quality scores, with differences highlighted using elliptical dashed lines for HiPore-C in (e) and Pore-C in (f).

Figure 5

Generating haplotype-resolution contacts map using PPL-Toolbox. (a) Coverage of heterozygous SNPs on Pore-C fragments. (b) Percentage of labeled fragments during haplotype-tagging process. (c) Haplotype imbalance phenomenon in Pore-C. The regions enclosed by dashed elliptical boxes represent the boundaries of two super-TAD on the inactive paternal X chromosome. (d) Haplotype imbalance phenomenon in Pore-C (Super-Loop).

Figure 6

Data visualization implemented in PPL-Toolbox. (a) PAX5-associated central enhancer E14 and its multiple omics tracks. (b) Visualization of Pore-C tracks. Track 0 and track 1 show the multi-way contacts information of the original region. Tracks 2–5 show the results of multiple interactions intersecting the central enhancer E14 associated with PAX5 in different visualization methods.