FASTQ Processing and Analysis Pipeline Overview 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: Quality Analysis using FastQC and MultiQC (Pre- and Post-trimming). Trimming and Adapter Removal using Fastp. Alignment of trimmed reads against a mitochondrial reference genome using BWA. Conversion of SAM to BAM, sorting, and duplicate marking using SAMtools and Picard. Prerequisites Ensure the following software/tools are installed and accessible in your $PATH: FastQC MultiQC Fastp BWA SAMtools Picard Java Runtime Environment (for Picard) Directory Structure 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 How to Use 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. Make Executable: chmod +x mitogenome_processing.sh Run the Script**: ./mitogenome_processing.sh Pipeline Workflow Initial Quality Check: Generates pre-trimming quality reports for raw FASTQ files using FastQC. Combines individual reports using MultiQC. Trimming with Fastp: Removes adapters and low-quality bases. Produces trimmed FASTQ files with a summary HTML report. Post-Quality Check: Generates quality reports for trimmed FASTQ files. Alignment: Aligns trimmed reads to the mitochondrial reference genome using BWA and outputs SAM files. Conversion and Sorting: Converts SAM files to BAM format using SAMtools. Sorts BAM files for downstream processing. Duplicate Marking: Marks and removes duplicates from BAM files using Picard. Outputs 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. Troubleshooting Ensure paths to tools and reference files are correct. Check the output logs for errors during alignment or duplicate marking. Acknowledgments This pipeline uses tools developed by open-source communities. Please refer to their documentation for further details: FastQC MultiQC Fastp BWA SAMtools Picard References 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