Overview A comprehensive analysis pipeline for processing tumor sequencing data from raw FASTQ files to annotated variants and copy number variations (CNVs). This pipeline integrates multiple tools for quality control, variant calling, annotation, and prioritization. Pipeline Workflow Step 1: FASTQ to VCF Conversion Input: Paired-end FASTQ files (fq1, fq2) Process: Aligns reads to reference genome, produces BAM file, and calls variants using HaplotypeCaller Output: Sorted BAM file and VCF file with raw variants Step 2: SNV Filtering Input: Raw VCF file from Step 1 Process: Filters low-quality variants using quality metrics and custom criteria Output: Filtered variant list in text format Step 3: Variant Annotation with CLEVER Input: Filtered variant file Tool: CLEVER (https://zenodo.org/records/15534528) Process: Annotates variants with functional and clinical information Output: Comprehensive variant annotation in TSV format Step 4: Coverage Calculation Input: Aligned BAM file Process: Calculates read depth across target regions Output: Coverage files for autosomes and chromosome X Step 5: Coverage File Preprocessing Gender Adjustment: Modifies chromosome X coverage for male samples (due to hemizygosity) Target Region Filtering: Focuses analysis on predefined target genes using BEDTools Batch Processing: Merges coverage files when analyzing multiple samples Step 6: CNV Calling CANOES: Identifies CNVs using read-depth approach (https://github.com/ShenLab/CANOES) HMZDelFinder: Detects homozygous deletions in exome data Output: CNV calls from both methods Step 7: CNV Annotation with PICNIC Input: CNV calls from Step 6 Process: Annotates CNVs and prioritizes potentially pathogenic variants Reference: Detailed in PMID: 10.1136/jmedgenet-2019-106377 Step 8: Phenotype-Based Prioritization (Optional) Tool: PhenoPro (http://omics.fudan.edu.cn/phenopro/) Process: Ranks variants based on provided HPO terms to assist in clinical interpretation Input Requirements FASTQ files: Paired-end sequencing data Sample information: Sample name and gender (Male/Female) Reference genome: hg19 (GRCh37) Target regions: Predefined gene panels for autosomes and chromosome X