mtDNA variant calling pipeline

This Bash script automates the processing of paired-end FASTQ files, including quality assessment, trimming, alignment, and duplicate marking. It leverages tools like FastQC, MultiQC, Fastp, BWA, SAMtools, and Picard to perform the following steps:

1. Quality Analysis using FastQC and MultiQC (Pre- and Post-trimming).
2. Trimming and Adapter Removal using Fastp.
3. Alignment of trimmed reads against a mitochondrial reference genome using BWA.
4. Conversion of SAM to BAM, sorting, and duplicate marking using SAMtools and Picard.

Ensure the following software/tools are installed and accessible in your $PATH:

• FastQC
• MultiQC
• Fastp
• BWA
• SAMtools
• Picard
• Java Runtime Environment (for Picard)

Before running the script, create and organize your directories as follows:

• Input Directory: Contains raw FASTQ files (*_R1.fastq and *_R2.fastq).
• Output Directories: The script will automatically create these:
  • Processed FASTQ files
  • Quality reports (pre- and post-trimming)
  • SAM/BAM files and final sorted BAM files
  • Marked duplicate files and metrics

1. Set Variables: Update the following paths in the script to match your file structure:

   • input_dir: Path to raw FASTQ files.
   • output_dir: Directory for trimmed FASTQ files.
   • fastqc_dir_pre: Directory for pre-trimming quality reports.
   • fastqc_dir_post: Directory for post-trimming quality reports.
   • multiqc_dir: Directory for consolidated MultiQC reports.
   • alignment_dir: Directory for alignment (SAM) files.
   • reference_mtDNA: Path to the reference genome file (FASTA format).
   • picard_output_dir: Directory for Picard-marked BAM files.

2. Make Executable:

   chmod +x mitogenome_processing.sh

    

3. Run the Script**:

   ./mitogenome_processing.sh

    

1. Initial Quality Check:

   • Generates pre-trimming quality reports for raw FASTQ files using FastQC.
   • Combines individual reports using MultiQC.

2. Trimming with Fastp:

   • Removes adapters and low-quality bases.
   • Produces trimmed FASTQ files with a summary HTML report.

3. Post-Quality Check:

   • Generates quality reports for trimmed FASTQ files.

4. Alignment:

   • Aligns trimmed reads to the mitochondrial reference genome using BWA and outputs SAM files.

5. Conversion and Sorting:

   • Converts SAM files to BAM format using SAMtools.
   • Sorts BAM files for downstream processing.

6. Duplicate Marking:

   • Marks and removes duplicates from BAM files using Picard.

• Quality Reports: Available in the directories for both pre- and post-trimming.
• Processed Files:
  • Trimmed FASTQ files in output_dir.
  • Aligned SAM files in alignment_dir.
  • Sorted BAM files in sorted_bam.
  • Marked BAM files and metrics in picard_output_dir.

• Ensure paths to tools and reference files are correct.
• Check the output logs for errors during alignment or duplicate marking.

This pipeline uses tools developed by open-source communities. Please refer to their documentation for further details:

• FastQC
• MultiQC
• Fastp
• BWA
• SAMtools
• Picard

• FastQC: Andrews, S. FastQC: A Quality Control Tool for High Throughput Sequence Data. Available at: FastQC

• MultiQC: Ewels, P., Magnusson, M., Lundin, S., & Käller, M. (2016). "MultiQC: summarize analysis results for multiple tools." Bioinformatics, 32(19), 3047–3048. https://doi.org/10.1093/bioinformatics/btw354

• Fastp: Chen, S., Zhou, Y., Chen, Y., & Gu, J. (2018). "fastp: an ultra-fast all-in-one FASTQ processor." Bioinformatics, 34(17), i884–i890. https://doi.org/10.1093/bioinformatics/bty560

• BWA: Li, H., & Durbin, R. (2009). "Fast and accurate short read alignment with Burrows-Wheeler transform." Bioinformatics, 25(14), 1754–1760. https://doi.org/10.1093/bioinformatics/btp324

• SAMtools: Li, H., Handsaker, B., Wysoker, A., & Fennell, T. (2009). "The Sequence Alignment/Map format and SAMtools." Bioinformatics, 25(16), 2078–2079. https://doi.org/10.1093/bioinformatics/btp352

  Picard: Broad Institute. (2020). Picard Tools. Available at: Picard