FASTQ to VCF pipeline question
I am new with programming within bioinformatics and long story short, I’m practicing writing pipeline scripts starting with the fastq to VCF pipeline. I am basically at the point where I went from fastq to sorted-bam files, and as I went to check on the status of the files using IGV, no visual mapping showed up for the bam files and am not sure why. I have 2 fastq’s (because they are paired end) per sample (one tumor and one normal), but I only used one of the two fastq’s for my own simplicity. Is this the issue on why it hasn’t show up on IGV? I’m guessing I should have used both fastq’s when using bwa mem to create the SAM file. Below is my code (yes I know, plenty of if/then statements because I’m still new to programming), let me know if anyone has a potential answer to this.
#!/bin/bash chmod +x pipeline.sh #state reference FASTA and FASTQ's refpath="/mnt/c/Users/RK/working/ref/human_g1k_v37.fasta" NFASTQ="/mnt/c/Users/RK/working/FASTQ/JS16-11705.fastqN.gz" TFASTQ="/mnt/c/Users/RK/working/FASTQ/JS16-11705.fastqT.gz" #index reference if [ ! -f /mnt/c/Users/RK/working/ref/human_g1k_v37.fasta.pac ]; then bwa index $refpath -a bwtsw; else echo "Reference file already indexed." fi #fastq to SAM if [ ! -f /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.sam ]; then bwa mem $refpath $NFASTQ > /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.sam; else echo "BWA alignment already completed for normal." fi if [ ! -f /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.sam ]; then bwa mem $refpath $TFASTQ > /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.sam; else echo "BWA alignment already completed for tumor." fi #SAM to BAM if [ ! -f /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.bam ]; then samtools view -bS /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.sam -o /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.bam; else echo "SAM to BAM already completed for normal." fi if [ ! -f /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.bam ]; then samtools view -bS /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.sam -o /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.bam; else echo "SAM to BAM already completed for tumor." fi #sort and index bam if [ -f /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.bam ] && [ -f /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.bam ]; then if [ ! -f /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.sorted.bam ]; then samtools sort /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.bam -o /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.sorted.bam samtools index /mnt/c/Users/RK/working/FASTQ/bwamem_alignN.sorted.bam; else echo "Already indexed." fi if [ ! -f /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.sorted.bam ]; then samtools sort /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.bam -o /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.sorted.bam samtools index /mnt/c/Users/RK/working/FASTQ/bwamem_alignT.sorted.bam; else echo "Already indexed." fi else echo "Error occurred, check to see if BAM file is present." fi
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