A comprehensive multi-omics bioinformatics pipeline for analyzing gene expression, ncRNA co-expression, and DNA methylation interactions in three species of stony corals. This work builds on the ncRNA landscape analysis from the deep-dive project, and is currently available as a preprint on bioRxiv:
Cross-talk among miRNAs, lncRNAs, and DNA methylation in three coral species reveal conserved epigenetic regulatory architecture Kathleen M. Durkin, Jill Ashey, Javier A. Rodriguez-Casariego, Zoe Dellaert, Zachary Bengtsson, Samuel J. White, Jose M. Eirin-Lopez, Hollie M. Putnam, Steven B. Roberts
This repository contains a complete bioinformatics pipeline for analyzing matched whole-genome bisulfite sequencing (WGBS), RNA-sequencing, and small RNA-sequencing data from three stony coral species (Acropora pulchra, Porites evermanni, and Pocillopora tuahiniensis). Analyses include:
- Gene Expression Analysis: RNA-seq data processing
- Small RNA Analysis: miRNA discovery and annotation, and target prediction
- Long Non-coding RNA: lncRNA identification and characterization
- DNA Methylation: Whole genome bisulfite sequencing (WGBS) analysis and methylation pattern characterization
- Regulatory Networks: Integration of mRNA, miRNA, lncRNA, and DNA methylation data, including identification of candidate epi-miRNAs and ceRNA triads
- Functional Annotation: GO enrichment analysis and functional characterization
- Comparative Analysis: Cross-species comparisons and orthology analysis
Here are some resources to get your started!
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DIVE genome browser - View ncRNA features across all three species in our interactive genome browser.
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CENE network viewer - explore mRNA-miRNA-lncRNA interaction networks, including epi-miRNAs and ceRNA networks, across the three species.
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OSF Project Link - Access large genomic reference files in our Open Science Framework (OSF) storage directory.
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- Access raw RNA sequences stored on NCBI.
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- Access raw small RNA sequences stored on NCBI.
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Sample metadata - per-sample colony IDs, species, collection date/site (Mo'orea, 2020-03-05), extraction & library-prep details. Colony ID prefixes:
ACR-(A. pulchra),POR-(P. evermanni),POC-(P. tuahiniensis).
Full genomic resources, sequencing data files, and count matrices can be found in the project wiki.
The repository is organized into species-specific directories and a multi-species comparison directory:
deep-dive-expression/
├── data/ # Reference files used throughout the below directories
├── D-Apul/ # Acropora pulchra analyses
│ ├── code/ # Analysis scripts (*.Rmd, *.qmd)
│ ├── data/ # Input data and references
│ └── output/ # Analysis outputs (organized by script)
├── E-Peve/ # Porites evermanni analyses
│ ├── code/
│ ├── data/
│ └── output/
├── F-Ptuh/ # Pocillopora tuahiniensis analyses
│ ├── code/
│ ├── data/
│ └── output/
├── M-multi-species/ # Cross-species comparative analyses
│ ├── code/
│ ├── data/
│ └── output/
├── manuscript_files/ # Figures and tables included in manuscript
├── references.bib # Shared bibliography
└── README.md # This file
Each species directory (D-Apul, E-Peve, F-Ptuh) follows the same structure:
code/: Contains all analysis scripts with numbered prefixes (e.g.,01-*.Rmd,02-*.Rmd)- See species-specific README files for detailed descriptions of each script
data/: Contains input data, reference genomes, and annotations- Large data files (FASTQ, BAM) are linked via the project wiki
output/: Contains subdirectories matching script names (e.g., output from01-script.Rmdgoes inoutput/01-script/)
- Linux/Unix environment (Ubuntu 20.04+ recommended)
- Minimum 32 GB RAM (64 GB+ recommended for large genomes)
- 500 GB+ available disk space
- Multi-core processor (8+ cores recommended)
The following software tools are required:
- FastQC (v0.11.9+): Quality control
- MultiQC (v1.12+): Aggregated QC reports
- fastp (v0.23+) / cutadapt (v3.5+): Adapter trimming
- HISAT2 (v2.2.1+): RNA-seq alignment
- StringTie (v2.2+): Transcript assembly and quantification
- Bismark (v0.23+): WGBS alignment and methylation calling
- ShortStack (v4.1.0): Small RNA analysis
- Bowtie / Bowtie2: Short read alignment
- SAMtools (v1.14+): BAM file manipulation
- BEDtools (v2.30+): Genomic interval operations
- miRanda (v3.3a+): miRNA target prediction
- R (v4.1+): Statistical computing
- RStudio (recommended): IDE for R
# Data manipulation and visualization
install.packages(c("tidyverse", "ggplot2", "pheatmap", "RColorBrewer"))
# Bioconductor packages
BiocManager::install(c(
"DESeq2", # Differential expression
"edgeR", # RNA-seq analysis
"WGCNA", # Co-expression networks
"methylKit", # DNA methylation analysis
"GenomicRanges", # Genomic intervals
"rtracklayer", # Genome annotations
"clusterProfiler", # GO enrichment
"biomaRt" # Annotation databases
))- CPC2: Coding potential calculator
- GffCompare: Transcript comparison
- OrthoFinder: Orthology analysis
- BLAST+: Sequence similarity searches
Refer to individual script headers for specific version requirements and conda environment configurations.
Found a bug or have a suggestion? Please open an issue with:
- Clear description of the problem or suggestion
- Steps to reproduce (for bugs)
- Expected vs. actual behavior
- System information (OS, R version, package versions)
Please be respectful and constructive in all interactions. We aim to maintain a welcoming and inclusive environment for all contributors.
If you use this pipeline or data in your research, please cite:
@misc{deepdive-expression,
title = {deep-dive-expression: Multi-omics analysis of coral gene expression and regulatory networks},
author = {E5 Coral Research Group},
year = {2026},
url = {https://github.com/urol-e5/deep-dive-expression},
note = {GitHub repository}
}- deep-dive: Predecessor project focusing on ncRNA landscape characterization
- E5 Coral Project: Broader coral resilience and epigenetics research initiative
For questions, issues, or collaborations:
- Issues: GitHub Issues
- Wiki: Project Wiki
- E5 Coral Project: Visit the main E5 project page for team information
Last Updated: August 2026
Repository maintained by: E5 Coral Research Group