Galaxy HiCExplorer: a web server for reproducible Hi-C data analysis, quality control and visualization

Abstract Galaxy HiCExplorer is a web server that facilitates the study of the 3D conformation of chromatin by allowing Hi-C data processing, analysis and visualization. With the Galaxy HiCExplorer web server, users with little bioinformatic background can perform every step of the analysis in one workflow: mapping of the raw sequence data, creation of Hi-C contact matrices, quality assessment, correction of contact matrices and identification of topological associated domains (TADs) and A/B compartments. Users can create publication ready plots of the contact matrix, A/B compartments, and TADs on a selected genomic locus, along with additional information like gene tracks or ChIP-seq signals. Galaxy HiCExplorer is freely usable at: https://hicexplorer.usegalaxy.eu and is available as a Docker container: https://github.com/deeptools/docker-galaxy-hicexplorer. INTRODUCTION Chromosome conformation capture techniques are now widely used to analyse the 3D conformation of chromatin inside the nucleus across a rising number of species, tissues and experimental conditions. In particular, the Hi-C protocol (1) has helped to uncover folding principles of chromatin, demonstrating that the genome is partitioned into active and inactive compartments (called A and B) (1) and that these compartments are further subdivided into topological associated domains (TADs) (2,3). Furthermore, Hi-C has allowed identification of chromatin loops (4,5), as well as enhancer–promoter interactions (6,7) and their influence on gene expression (8,9). However, Hi-C data processing requires tabulating hundreds of millions to billions of paired-end reads into large matrices. This poses bioinformatic challenges for efficient processing of the data and subsequent analyses. Here, we introduce Galaxy HiCExplorer, a package that aims to make Hi-C data processing, analysis and visualization available to non-bioinformaticians. Our goal is to provide a software environment able to automate the whole workflow of Hi-C data analyses from raw read mapping, filtering and correction, to the computation of topological associated domains and A/B compartments, and finally to the visualization of contact matrices, along with various other genomic features and omics data. Moreover, Galaxy HiCExplorer is easy to install, maintainable, stable and well documented. The availability of a docker container in conjunction with Bioconda (http://dx.doi.org/10.1101/207092), eliminates the need for complex software and dependency installations. Finally, HiCExplorer is transparently developed by a community of collaborators based on best practices (10) for version control, code revisions, manual and automated testing and comprehensive documentation. COMPREHENSIVE SERVER FOR HI-C ANALYSES Galaxy HiCExplorer is freely available at https://hicexplorer.usegalaxy.eu as well as a Docker container: https://github.com/deeptools/docker-galaxy-hicexplorer. Galaxy HiCExplorer was designed to provide an easily accessible data-analysis environment such that biomedical researchers can focus on critical research aspects instead of dealing with terminal-based applications that are not user-friendly. It smoothly integrates the HiCExplorer analysis toolset (8) into the Galaxy scientific analysis platform to provide web-based, easy-to-use and thoroughly tested workflows that provide pipelines for the most common Hi-C data processing steps. In contrast to other available Hi-C analysis software like HiCUP (14), HOMER (15) and TADbit (16) among others (see (17,18) for a comprehensive list of tools), Galaxy HiCExplorer provides a fully comprehensive analysis pipeline available to much broader community of researchers and is not restricted to a subset of important features. HiC-Pro (19) is one of the few packages that offers a complete pipeline; however, its visualization tools are limited and it is only available as a command line tool. Similarly, Juicer (20) offers a command line tool processing pipeline while Juicebox (21) only provides visualizations. Moreover, the integration of HiCExplorer into Galaxy offers the possibility to process and integrate other data types like ChIP-Seq or RNA-Seq into the analysis using the same interface. None of the aforementioned tools offer web server access except HiFive (22). A strong advantage of HiCExplorer is that it can take multiple matrix data formats developed by different research groups as input. Thus, it is well integrated in the landscape of Hi-C data analysis algorithms, as Hi-C matrices can be produced by other tools and visualized with HiCExplorer. Conversely, matrices can be created with HiCExplorer and then exported to be used by other software. Currently, the Galaxy HiCExplorer supports two major formats: The HiCExplorer specific h5 format and to promote standardization of Hi-C contact matrices the cooler format (23) developed within the 4D nucleome project (24). GALAXY HiCExplorer TOOLS AND WORKFLOWS Galaxy HiCExplorer provides a plethora of tools for processing, normalization, analysis, and visualization of Hi-C data (Figure 1A). Apart from HiCExplorer, the https://hicexplorer.usegalaxy.eu website and the Docker container also include the genome alignment tools BWA-MEM (25) and Bowtie2 (26), as well as additional tools for text manipulation, data import and quality control. The inclusion of deepTools (27) further facilitates the integration of ChIP-seq, RNA-seq, MNase-seq as well as other kind of datasets with Hi-C data. The analysis of Hi-C data can be divided into three steps: pre-processing (including quality control), analysis and visualization. Pre-processing and quality control hicBuildMatrix A contact matrix is the main data structure of Hi-C data analysis which is generated from the individual alignment of valid Hi-C paired-end reads. This tool filters out potentially erroneous reads, such as unmappable reads, self-ligated reads, dangling-ends, PCR duplicates or incomplete digestions (4,14) and tabulates the results based on user defined bins (either based on restriction sites or on fixed size bins). Because building the Hi-C matrix is one of the most time consuming steps in the Hi-C workflow, we developed hicBuildMatrix to be multi-processing to significantly reduce running time. A comprehensive quality report is generated as an HTML file. This report includes a number of useful quality measures including: number of valid Hi-C read pairs and the number of filtered reads per category (unmappable and non-unique pairs, duplicates, dangling ends, self-circles, etc.), number of intra-chromosomal, short-range (<20 kb) and long-range contacts, and read pair orientation. Reports from multiple samples can be integrated using MultiQC (28) or using the HiCExplorer tool hicQC. Inspection of the hicBuildMatrix quality reports helps to identify potential biases or errors in the Hi-C library preparation. For example, a high number of dangling ends is indicative of a problem with the re-lig (...truncated)