Bioinformatics Data Integration Practicum - Final Project

Cross-Species Regulatory Element Conservation Analysis

This project analyzes the conservation of open chromatin regions (enhancers/promoters) across human and mouse tissues (Liver, Adrenal Gland) using comparative genomics tools.

Project Overview

Goal: Determine whether transcriptional regulatory element activity is more conserved across tissues or species. 1. Evaluate the quality of the datasets - The data that was given to us was pre-processed. 2. Map the mouse open chromatin regions to the human genome and map the human open chromatin regions to the mouse genome - Using Hal and HalLiftover 3. Find the percentages of the overlapped regions - Using bedtool intersect. 4. Find the regulatory regions and its functions - Using GREAT webtool. 5. Divide the open chromatin data into likely enhancers and likely promoters - Using bedtools and GENCODE/ENCODE data. 6. Find sequence patterns - Motif Analysis - Using MEMESuite, Centrimo, FIMO

Key Questions: 1. Are open chromatin regions more conserved across tissues (human Liver vs. human Adrenal) or species (human Liver vs. mouse Liver)? 2. What biological processes are regulated by conserved regions?

Setup

Dependencies

1. HALPER and HalLiftover - Clone the repository
2. bedttols - v2.30+
3. Gene Ontology Enrichment (GREAT) - To use GREAT please cut the first 3 columns from the input files

Command for keeping only the first 3 columns

      `cut -f1-3 input_bed_file  > output_file_name`

1. MEMEsuite - v MEME-suite/5.4.1 If your input files are not huge, you can also use MEME-ChIP webtool
2. Anaconda3 - For Conda environment and Python. V 2024.10-1

Installation

Create Conda environment

conda create -n conservation-env -c bioconda hal bedtools python=3.8

conda activate conservation-env

halLiftover --help # Should show usage

halStats --version # Confirm HAL is installed

bedtools --version # Ensure v2.30+

ls repos/halLiftover-postprocessing/scripts/*.py # Should list scripts

NOTE: For the installation and the entire code, the cloned github repositories are in the repos directory

If you used a conda env name other than hal, modify: sed -i 's/conda activate hal/conda activate YOURENVNAME/g' \ repos/halLiftover-postprocessing/halpermappeak_orthologs.sh - HAL Tools: For halLiftover (install via Conda). - conda create -n hal python=3.7 # Name MUST be "hal" for HALPER compatibility conda activate hal

For running the entire pipeline, use the below mentioned commands (Example run) and use the mentioned Snakefile

Snakefile

Clone our repository

git clone https://github.com/naithru/713bioinformaticspracticum.git

cd your-repo-name

If you do no have SnakeMake already

conda create -n atac_env -c bioconda -c conda-forge snakemake conda activate atac_env

Dry run

snakemake -np

Basic local run

snakemake --cores 4

Cluster/SLURM example

snakemake --jobs 20 \ --cluster "sbatch -p RM-shared --mem=8G -t 24:00:00 -c 4" \ --latency-wait 60

#Please mention more memory and cores when actually running it

Input files for pipeline

1. HALPER - idr.conservative_peak.narrowPeak
2. Overlap analysis via bedtool intersect - Bed file from the output of HALPER - mouseadrenalpeaks.bed
3. Classifying Enhancers and Promoters - Need to clean the bed files first and named as - halper_cleaned.bed
4. To find the TSS bedfile - either use the path in /ocean/project/bio230007p /ikaplow/HumanGenomeInfo/gencode.V27.annotation.protTranscript.TSSsWithStrand_sorted.bed or make the promoter bed file from gencode.v44.basic.annotation.gtf.gz (repeat the steps with proper genome for human and mouse).
5. GREAT - Take the first 3 columns of halper results - named as halpergreatfinal.bed (Performed on the webtool).
6. Motif Analysis - all the found enhancers and promoters bed files - example: enhancer_adrenal.bed

Output files pipeline

1. After Halper - Bed files. example: mouseadrenalpeaks.bed
2. Overlap - txt or output on interactive shell with overlap percentages.
3. Number of Promoters and Enhancers - Ouput on interactive shell with the number using the echo command.
4. GREAT - Results on the webtool. Can be saved as pdf, png or html.
5. Motif Analysis - Results are saved as HTML files.

Citations

1. Manuscript describing HALPER (cite this): Xiaoyu Zhang, Irene Kaplow, Morgan Wirthlin, Tyler Park, Andreas Pfenning. HALPER facilitates the identification of regulatory element orthologs across species. Bioinformatics, Volume 36, Issue 15, 1 August 2020, Pages 4339-4340.
2. Manuscript describing the Cactus alignment method (cite this if you are using a Cactus alignment): Benedict Paten, Dent Earl, Ngan Nguyen, Mark Diekhans, Daniel Zerbino and David Haussler. Cactus: Algorithms for genome multiple sequence alignment. Genome Research, Volume 21, Issue 9, 10 June 2011, Pages 1512-1528.
3. Manuscript describing HAL Format API (cite this if you are using halLiftover or halAlignmentDepth): Glenn Hickey, Benedict Paten, Dent Earl, Daniel Zerbino, and David Haussler. HAL: A Hierarchical Format for Storing and Analyzing Multiple Genome Alignments. Bioinformatics, Volume 29, Issue 10, 15 May 2013, Pages 13411342.
4. Timothy L. Bailey, James Johnson, Charles E. Grant, William S. Noble, "The MEME Suite", Nucleic Acids Research, 43(W1):W39-W49, 2015.
5. Cory Y McLean, Dave Bristor, Michael Hiller, Shoa L Clarke, Bruce T Schaar, Craig B Lowe, Aaron M Wenger, and Gill Bejerano. "GREAT improves functional interpretation of cis-regulatory regions". Nat. Biotechnol. 28(5):495-501, 2010. PMID 20436461
6. Yosuke Tanigawa, Ethan S Dyer, Gill Bejerano. "WhichTF is functionally important in your open chromatin data?" PLoS Comput Biol. 2022 Aug 30;18(8):e1010378, 2022. PMID 36040971
7. Quinlan AR and Hall IM, 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 26, 6, pp. 841842.

How to Cite our repository

Shreya Rao, S., Naithruvan, R., Jiayi, Z., & Ziyun, H. (2025). Cross Species Regulatory Elements Conservation Analysis (Version 1.0.0) [Computer software]. https://github.com/naithru/713bioinformaticspracticum

Contributors

1. Jiayi Zhao (jiayizh3@andrew.cmu.edu)
2. Shreya Rao SS (ssamaved@andrew.cmu.edu)
3. Naithruvan Rammohan (nrammoha@andrew.cmu.edu)
4. Ziyun Huang (ziyunh@andrew.cmu.edu)

Project Guide

1. Dr. Irene Kaplow - ikaplow@andrew.cmu.edu
2. Wanxing Zhang (TA) - wanxingz@andrew.cmu.edu