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VariantRecalibrator error

Dear GATK Team,

I'm trying to perform variant recalibration on 3 WGS sequencing data. I am following the pipeline described in Best Practices. I have generated individual g.vcf files for each patient and used GenotypeGVCFs to obtain multisample vcf for the three samples. But then I got stuck at the VQSR step. The VariantRecalibration tool throws the following error:
INFO 08:01:34,635 VariantRecalibratorEngine - Convergence after 49 iterations!
INFO 08:01:44,150 VariantRecalibratorEngine - Evaluating full set of 6602089 variants...
INFO 08:01:51,139 VariantDataManager - Training with worst 28183 scoring variants --> variants with LOD <= -5.0000.
INFO 08:01:51,140 GaussianMixtureModel - Initializing model with 100 k-means iterations...
INFO 08:01:51,535 VariantRecalibratorEngine - Finished iteration 0.
INFO 08:01:51,744 VariantRecalibratorEngine - Finished iteration 5. Current change in mixture coefficients = 0.13136
INFO 08:01:51,876 VariantRecalibratorEngine - Finished iteration 10. Current change in mixture coefficients = 0.03466
INFO 08:01:52,005 VariantRecalibratorEngine - Finished iteration 15. Current change in mixture coefficients = 0.02910
INFO 08:01:52,131 VariantRecalibratorEngine - Finished iteration 20. Current change in mixture coefficients = 0.01388
INFO 08:01:52,256 VariantRecalibratorEngine - Finished iteration 25. Current change in mixture coefficients = 0.00651
INFO 08:01:52,382 VariantRecalibratorEngine - Finished iteration 30. Current change in mixture coefficients = 0.00323
INFO 08:01:52,483 VariantRecalibratorEngine - Convergence after 34 iterations!
INFO 08:01:52,652 VariantRecalibratorEngine - Evaluating full set of 6602089 variants...

ERROR ------------------------------------------------------------------------------------------
ERROR ------------------------------------------------------------------------------------------
ERROR A USER ERROR has occurred (version 3.7-0-gcfedb67):
ERROR This means that one or more arguments or inputs in your command are incorrect.
ERROR The error message below tells you what is the problem.
ERROR If the problem is an invalid argument, please check the online documentation guide
ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
ERROR Visit our website and forum for extensive documentation and answers to
ERROR commonly asked questions https://software.broadinstitute.org/gatk
ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.
ERROR MESSAGE: NaN LOD value assigned. Clustering with this few variants and these annotations is unsafe. Please consider raising the number of variants used to train the negative model (via --minNumBadVariants 5000, for example).
ERROR -------------------------

The command I used:
java -Djava.io.tmpdir=./tmp -XX:ParallelGCThreads=1 -jar GenomeAnalysisTK.jar \
-T VariantRecalibrator \
-R GRCh37-lite.fa \
-input germline10.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 1000G_phase1.snps.high_confidence.b37.vcf \
-resource:dbsnp,known=true,training=false,truth=false,prior=2.0 dbsnp_138.b37.vcf \
-an MQ -MQCap 70 -an DP -an QD -an FS -an SOR -an MQRankSum -an ReadPosRankSum \
-mode SNP -tranche 100.0 -tranche 99.9 -tranche 99.0 -tranche 90.0 \
-recalFile recalibrate_SNP.recal -tranchesFile recalibrate_SNP.tranches -rscriptFile recalibrate_SNP_plots.R

-maxNumTrainingData 4000000 \

--minNumBadVariants 5000 \

There are more than 6million variants. Even after adjusted maxNumTrainingData and minNumBadVariants, I kept getting this message. I searched the forum and I am not sure If I need more samples or if I have a problem in my command.

Thanks a lot!



Best Answer


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