I hope you are all doing well. I analyzed the FASTQ files (WES) from a patient with symptoms of deafness on a rented server. I identified the specific variant and reported it.
I no longer want to use a rented server, so I am attempting to launch it on my PC. However, the reported variant, along with several others, is missing from my Linux system. I believe they were lost during the alignment of the two FASTQ files, but I do not know why.
I set up and installed all the necessary software on my Linux machine, then analyzed the same FASTQ file on my personal computer to ensure all steps ran correctly. However, most of my variants are missing! To identify the problem, I tried several approaches and determined that the missing variants were lost after alignment. This means I aligned my FASTQ files with BWA-MEM, then converted the SAM file to BAM using SAMtools. I used the Picard command line tool to sort the BAM file and checked it in IGV to verify if the variants were present. Unfortunately, my reported variants, along with many others, are missing. However, I accidentally found only one variant that was not missing on my computer.
command lines:
IlluQC.pl \ -pe 914_1.fastq.gz 914_2.fastq.gz N A \ -l 80 \ -s 20 \ -p 3 \ -o S914 \ -z g bwa mem \ -M /home/apuser/genome-hg19/ucsc.hg19.fasta \ 914_1.fastq.gz_filtered.gz \ 914_2.fastq.gz_filtered.gz \ > 914_alined.sam samtools view \ -b 914_alined.sam \ > 914.bam PicardCommandLine SortSam \ I= 914.bam \ O= 914_sort.bam \ SO= coordinate 
