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GenomeAnalysisTK-2.7-4-UnifiedGenotyper-Illegal base [K] seen in the allele

Hi

I am hitting with the following error when I try to use UnifiedGenotyper. Wondering if its a bug or something wrong with my bam file?
Thanks for your help in advance.

ERROR ------------------------------------------------------------------------------------------
ERROR stack trace

java.lang.IllegalArgumentException: Illegal base [K] seen in the allele
at org.broadinstitute.variant.variantcontext.Allele.create(Allele.java:205)
at org.broadinstitute.variant.variantcontext.Allele.create(Allele.java:213)
at org.broadinstitute.sting.gatk.walkers.annotator.RankSumTest.fillQualsFromPileup(RankSumTest.java:159)
at org.broadinstitute.sting.gatk.walkers.annotator.RankSumTest.annotate(RankSumTest.java:113)
at org.broadinstitute.sting.gatk.walkers.annotator.VariantAnnotatorEngine.annotateContext(VariantAnnotatorEngine.java:192)
at org.broadinstitute.sting.gatk.walkers.genotyper.UnifiedGenotyperEngine.calculateGenotypes(UnifiedGenotyperEngine.java:560)
at org.broadinstitute.sting.gatk.walkers.genotyper.UnifiedGenotyperEngine.calculateLikelihoodsAndGenotypes(UnifiedGenotyperEngine.java:234)
at org.broadinstitute.sting.gatk.walkers.genotyper.UnifiedGenotyper.map(UnifiedGenotyper.java:367)
at org.broadinstitute.sting.gatk.walkers.genotyper.UnifiedGenotyper.map(UnifiedGenotyper.java:143)
at org.broadinstitute.sting.gatk.traversals.TraverseLociNano$TraverseLociMap.apply(TraverseLociNano.java:267)
at org.broadinstitute.sting.gatk.traversals.TraverseLociNano$TraverseLociMap.apply(TraverseLociNano.java:255)
at org.broadinstitute.sting.utils.nanoScheduler.NanoScheduler.executeSingleThreaded(NanoScheduler.java:274)
at org.broadinstitute.sting.utils.nanoScheduler.NanoScheduler.execute(NanoScheduler.java:245)
at org.broadinstitute.sting.gatk.traversals.TraverseLociNano.traverse(TraverseLociNano.java:144)
at org.broadinstitute.sting.gatk.traversals.TraverseLociNano.traverse(TraverseLociNano.java:92)
at org.broadinstitute.sting.gatk.traversals.TraverseLociNano.traverse(TraverseLociNano.java:48)
at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:99)
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:313)
at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:113)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:245)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:152)
at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:91)

ERROR ------------------------------------------------------------------------------------------
ERROR A GATK RUNTIME ERROR has occurred (version 2.7-4-g6f46d11):
ERROR
ERROR This might be a bug. Please check the documentation guide to see if this is a known problem.
ERROR If not, please post the error message, with stack trace, to the GATK forum.
ERROR Visit our website and forum for extensive documentation and answers to
ERROR commonly asked questions http://www.broadinstitute.org/gatk
ERROR
ERROR MESSAGE: Illegal base [K] seen in the allele
ERROR ------------------------------------------------------------------------------------------

Cheers
Scott..

Best Answer

Answers

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    Hi Scott,

    Try checking your BAM file using ValidateSAMFile from Picard tools. That will tell you if there's anything wrong with the data. What aligner did you use for mapping?

  • newbiescottnewbiescott Member
    edited March 2014

    Thanks for your response Geraldine.

    I am using bwa for mapping, followed by GATK best practices (almost). I also used Picards ValidateSamFile just before UnifiedGenotyper. Anyway re-running ValidateSamFile again shows no errors.

    [Mon Mar 31 15:17:46 BST 2014] net.sf.picard.sam.ValidateSamFile INPUT=gatk_indel_realigner_gatk2/pe.1.reali.bam MODE=VERBOSE MAX_OUTPUT=100 IGNORE_WARNINGS=false VALIDATE_INDEX=true IS_BISULFITE_SEQUENCED=false MAX_OPEN_TEMP_FILES=8000 VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false
    [Mon Mar 31 15:17:46 BST 2014] Executing as xx on Linux 2.6.32-44-server amd64; Java HotSpot(TM) 64-Bit Server VM 1.7.0_25-b15; Picard version: 1.96(1510)
    INFO 2014-03-31 15:19:08 SamFileValidator Validated Read 10,000,000 records. Elapsed time: 00:01:21s. Time for last 10,000,000: 81s. Last read position: 2L:37,761,308
    .................
    INFO 2014-03-31 15:29:38 SamFileValidator Validated Read 100,000,000 records. Elapsed time: 00:11:51s. Time for last 10,000,000: 53s. Last read position: /
    INFO 2014-03-31 15:30:32 SamFileValidator Validated Read 110,000,000 records. Elapsed time: 00:12:46s. Time for last 10,000,000: 54s. Last read position: /
    No errors found
    [Mon Mar 31 15:32:29 BST 2014] net.sf.picard.sam.ValidateSamFile done. Elapsed time: 14.72 minutes.
    Runtime.totalMemory()=3119448064

    This is my UnifiedGenotyper command:

    /software/jre1.7.0_25/bin/java -Xmx6525m -Xms6525m -server -XX:+UseSerialGC -jar path_to/gatk/GenomeAnalysisTK-2.7-4-g6f46d11/GenomeAnalysisTK.jar -et NO_ET -K path_to_key -T UnifiedGenotyper -R path_to_fa -I path_to_bams.list -o path_to_vcf --downsampling_type BY_SAMPLE -dcov 250 --output_mode EMIT_VARIANTS_ONLY --min_base_quality_score 17 --genotyping_mode DISCOVERY --genotype_likelihoods_model BOTH --heterozygosity 0.01 --indel_heterozygosity 0.001 -stand_emit_conf 10.0 -stand_call_conf 30.0 -contamination 0.05 --annotateNDA -A AlleleBalance -A AlleleBalanceBySample -A BaseCounts -A BaseQualityRankSumTest -A ChromosomeCounts -A Coverage -A DepthPerAlleleBySample -A FisherStrand -A GCContent -A HaplotypeScore -A HardyWeinberg -A HomopolymerRun -A InbreedingCoeff -A MappingQualityRankSumTest -A MappingQualityZero -A MappingQualityZeroBySample -A QualByDepth -A RMSMappingQuality -A ReadPosRankSumTest -A SpanningDeletions -A TandemRepeatAnnotator -A VariantType

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