DNAseq Variant Calling Pipeline (hg38)

A comprehensive workflow for variant calling from raw FASTQ files using GATK (or SAMtools) methodologies, aligned to the hg38 reference genome.

Workflow Overview

1. Reference genome preparation
2. Read alignment with BWA-MEM
3. Quality control with FastQC
4. Duplicate removal with Picard
5. Variant calling with:
   • GATK HaplotypeCaller
   • SAMtools
6. Variant filtering and annotation

Prerequisites

Software Requirements

• bwa (v0.7.17+)
• bcftools (v1.15+)
• FastQC (v0.11.9+)
• Picard (v2.27+)
• GATK (v4.3.0.0+)
• samtools (v1.15+)
• SnpEff/SnpSift (v5.1+)

Reference Data

• hg38 reference genome (download from UCSC)
• Corresponding BWA index files
• Known variants databases (for BQSR if needed)

Output Files

• sorted_SRR14634773.bam → Sorted BAM file
• rmdup_SRR14634773.bam → Deduplicated BAM file
• picard_output.bam → Final BAM with read groups
• GATK_variation_calling.vcf → Variants called with GATK
• bcftools_variation_output.vcf → Variants called with SAMtools/bcftools
• snpSift_output.vcf / bcftools_snpSift_output.vcf → Filtered variants

Notes

• Use GATK HaplotypeCaller for germline variant calling.
• Use GATK Mutect2 (not shown here) for somatic variant calling.
• Adjust filtering thresholds (QUAL, DP, MQ) based on your dataset.

Installation

# Clone this repository
git clone https://github.com/yourusername/DNAseq-for-variant-analysis.git
cd DNAseq-for-variant-analysis

# Download reference genome (example)
wget https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz
gunzip hg38.fa.gz
bwa index hg38.fa