NGS-Variant-Calling

NGS Workflow Documentation

Overview

• Goal: This project focuses on identifying somatic variants in whole-genome sequencing (WGS) data from tongue cancer samples and cell lines. The workflow leverages industry-standard tools for alignment, quality control, and variant calling, specifically targeting chromosomes 6 and 7 of the human reference genome (hg38).
• Data Source: PRJEB62494.
• Tools Used:
  
  • BWA: Burrows-Wheeler Aligner for read alignment.
  • Samtools: Processing and indexing BAM files.
  • GATK (v4): Variant calling with HaplotypeCaller.
  • SRA Toolkit: Downloading raw sequencing data.
  • FastQC: Quality control reports.
• Data:
  • Samples: Three accessions (ERR11468775, ERR11468776, ERR11468777).
  • Reference Genome: hg38 (chromosomes 6 and 7).

Key Steps

1. Data Acquisition:
   • Download raw FASTQ files from SRA (500,000 reads per sample).
   • Example command:

     fastq-dump --split-files --gzip -X 500000 ERR11468775
     

2. Reference Genome Setup:
   • Chromosomes 6 and 7 from UCSC hg38.
   • Merged and indexed with BWA.
3. Quality Control:
   • FastQC reports for raw and processed data.
4. Alignment:
   • BWA-MEM for paired-end alignment.
   • Samtools for sorting and indexing BAM files.
   • Example command:

     bwa mem ref_genome/hg38_chr6_7.fa sample_1.fastq.gz sample_2.fastq.gz | samtools sort -o sample.bam
     

5. Variant Calling:
   • GATK HaplotypeCaller for germline variants, Mutect2 for somatic variants.
6. Annotation: ENSEMBL VEP.

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