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Variant Recalibration Input File Issue

louwslouws San FranciscoMember

Hi! I am attempting to go through the process of applying a recalibration to an input vcf. I tried following the steps outlines in the tutorial Howto: Recalibrate Variant Quality Scores .

Step 2 runs great, I used all the tranches and resources that the howto mentions. Unfortunately when I attempt to apply the recalibration (step 3) I receive the following error

Encountered input variant which isn't found in the input recal file. Please make sure VariantRecalibrator and ApplyRecalibration were run on the same set of input variants. First seen at: [VC input @ 1:14907 Q523.77 of type=SNP alleles=[A*, G] attr={AC=1, AF=0.500, AN=2, BaseQRankSum=-1.863, ClippingRankSum=-0.420, DP=86, FS=0.984, MLEAC=1, MLEAF=0.500, MQ=36.93, MQ0=0, MQRankSum=0.338, QD=6.09, ReadPosRankSum=2.064} GT=GT:AD:DP:GQ:PL 0/1:57,29:86:99:552,0,1482

When I check the input file I notice that this is the first variant call in my VCF. I tried this process with other VCF files and each time my ApplyRecalibration gets stuck on the first variant call.

Here is the command I used to generate the recalibration:

 java -Djava.io. -Xmx4g -jar GenomeAnalysisTK-3.2-2/GenomeAnalysisTK.jar \
                -T VariantRecalibrator -R /human_g1k_v37.fasta \
                -L file.intervals \
                -input input_file.vcf \

                -resource:hapmap,known=false,training=true,truth=true,prior=15.0 hapmap_3.3.b37.vcf \
                -resource:omni,known=false,training=true,truth=true,prior=12.0 1000G_omni2.5.b37.vcf \
                -resource:1000G,known=false,training=true,truth=false,prior=10.0 Mills_and_1000G_gold_standard.indels.b37.vcf \
                -resource:dbsnp,known=true,training=false,truth=false,prior=2.0 dbsnp_138.b37.vcf \

                -an DP \
                -an QD \
                -an FS \
                -an MQRankSum \
                -an ReadPosRankSum \

                -mode SNP \
                -tranche 100.0 -tranche 99.9 -tranche 99.0 -tranche 90.0 \ 
                -recalFile recal_file.recal \
                -tranchesFile tranches_file.tranches \
                -rscriptFile rscipt_file.plots.R\

And here is my command for applying the recalibration:

java -Xmx4g -jar GenomeAnalysisTK.jar
            -T ApplyRecalibration
            -R human_g1k_v37.fasta
            -input input_file.vcf
            -mode SNP
            --ts_filter_level 99.0
            -recalFile  recal_file.recal
            -tranchesFile tranches_files.tranches
            -o recalibrated_output.vcf 

Any suggestions to help me troubleshooot the issue is very welcome. I should also mention that I am using GATK 3.2.2

Thank you in advance!

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