HELP: haplotypecaller doesn't call the driver oncogenic variant!
I'm using GATK haplotypecaller in order to detect varianti in a tumor sample analyzed with Illumina WES 100X2 paired end mode. I know KRAS p.G12 variant (chr12:25398284) is present in my sample (because previously seen with Sanger sequencing, KRAS is the oncogenic event in this kind of tumor). I aligned the fastq file with bwa after quality control e adapter trimmig. In my bam file I'm able to view the variant at genomic coordinate chr12:25398284 as you can see in the picture (IGV):
however GATK haplotypecaller doesn't call the variant anyway.
This is my basic command line:
java -Xmx4g -jar /opt/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar -T HaplotypeCaller -R human_g1k_v37.fasta -I my.bam -stand_call_conf 0 -stand_emit_conf 0 -o output.file
and I tryed to tune a a lot of parameter such as stand_call_conf; -stand_emit_conf; --heterozygosity; --min_base_quality_score and so on.
This is the pileup string of the chr12:25398284 coordinate in my bam file
12 25398284 C 55 ,,T.,,...,,,,,,T.......,t,t.....,,,t..,,,,,,,,,,,,.^],^],^],^], BB@ECAFCECBCBBB@DBCCCDDCABADBDBCCDD@BBEADDEDBCADBB@@BAC
The base quality is good and the mapping quality too, but the haplotypecaller does not determine any active region around the position
Any suggestion about the reason of misscalling??