Omics-pipeline

A pipeline for DNA-seq, RNA-seq, and ATAC-seq analysis. This repository is currently under development, with pipelines being built for each analysis type.

Pipeline Structure

• Snakefile: Acts as the main blueprint, orchestrating the workflow by incorporating rules and commands from each .smk file in the rules directory.
• config/ directory: Contains YAML files with paths, parameters. Also, includes a .tsv file listing sample IDs and a configuration file specifying paths and settings.
• envs/ directory: Contains YAML files specifying dependencies and version requirements for each analysis environment.
• rules/ directory: Contains modular Snakemake (.smk) files, each defining a specific step in the workflow.

Main Workflow Steps for ATAC-seq

The pipeline consists of three primary stages, each requiring a corresponding .smk file to be executed:

1. preprocessing.smk

   • Purpose: Preprocesses raw sequencing data.
   • Description: Each sample has data split across two lanes, necessitating a merging step after trimming to produce final FASTQ files for downstream analysis.
   • Key Steps:
     • [FastQC] - Quality control on raw reads.
     • [Trimmomatic] - Trimming adapters and low-quality sequences.
     • Merging lanes to create consolidated, trimmed FASTQ files.
     • (Optional) [MultiQC] - Summarising all samples by aggregating the FastQC HTML outputs across samples (not included in the pipeline).

2. alignment.smk

   • Purpose: Aligns the merged FASTQ files to the reference genome.
   • Description: This rule handles the alignment of preprocessed reads to a combined human-virus genome reference, preparing the data for downstream analysis such as peak calling or expression quantification.
   • Key Steps:
     • [BWA MEM] - Aligns the reads to the reference genome using BWA-MEM.
     • [GATK] - (Run with Docker) Marks duplicates in the aligned BAM file to improve downstream analyses.
     • [Samtools] - Filters out mitochondrial (MT) reads and duplicates, generates a flagstat report for quality control.

3. peakcalling.smk

   • Purpose: Calls peaks from the BAM files, identifying regions of significant enrichment.
   • Description: This rule runs MACS2 to call peaks on filtered BAM files (after removing mitochondrial reads and duplicates). The results can be used for downstream analysis such as differential peak calling or visualisation.
   • Key Steps:
     • [MACS2 Call Peak] - Uses MACS2 to call peaks from BAM files, specifying the format (BAMPE for paired-end), genome size, and keeping duplicate reads.