I have been using VarScan , GATK and Mutect now for calling my somatic variations. I have used the VarScan, GATK and Mutect all on the recalibrated bam files after runnnig the Base recalibrator, but I find some discrepancies in the output when I am trying to use the baits bed file provided by the company which is used for target enrichment. When am calling the variants with Mutect for normal/tumor pair with SureSelect exome target bed file using the --interval option I am just getting 1 or 2 hits. Is this a good way to call the exonic mutations? Or should I continue with results of the mutect without the exome target kit bed file. I have already tried it without the bed files and I received over 1500 somatic high conifdence variants out of which only 50% are on the exonic regions. The problem is it is not possible to use the VarScan with the target bed file option. You can just remove the false positive calls in VarScan and nothing more and then check for the exonic mutations from the result. So I would like to have some inputs from you guys who are already using all the 3 methods for calling the somatic mutations to share your views. I know it is much likely to have more high confidence mutations using the target baits bed file while calling the mutations but VarScan is limited to that and Mutect can do that with interval option but then results are not comparable. So can someone suggest what should be the best thing to do here. The command am using in Mutect is below. Also please let me know if this is correct or not.
java -Xmx14g -jar /scratch/GT/softwares/mutect/muTect-1.1.4.jar --analysis_type MuTect --reference_sequence /scratch/GT/vdas/test_exome/exome/hg19.fa --cosmic /data/PGP/exome/mutect/hg19/hg19_cosmic_v54_120711.vcf --dbsnp /scratch/GT/vdas/test_exome/exome/databases/dbsnp_137.hg19.vcf --input_file:normal /scratch/GT/vdas/pietro/exome_seq/results/N_S8981/N_S8981.realigned.recal.bam --input_file:tumor /scratch/GT/vdas/pietro/exome_seq/results/T_S7999/T_S7999.realigned.recal.bam --out /scratch/GT/vdas/pietro/exome_seq/results/mutect/param_test/mutect_S_333soma_t_3.txt --coverage_file /scratch/GT/vdas/pietro/exome_seq/results/mutect/param_test/LG3.coverage.wig.txt --vcf /scratch/GT/vdas/pietro/exome_seq/results/mutect/param_test/mutect_S_333soma_t_3.vcf --intervals /scratch/GT/vdas/referenceBed/hg19/ss_v4/SureSelect_XT_Human_All_Exon_V4.bed --fraction_contamination 0.25
I would like to have some suggestions