Question: GATK variant recal malformed vcf
0
gravatar for SemiQuant
4.6 years ago by
SemiQuant60
South Africa
SemiQuant60 wrote:

HI,

I'm attempting to do GATK variant recalibration on files i generated using HaplotypeCaller.  I keep getting the following error.

Your input file has a malformed header: We never saw the required CHROM header line (starting with one #) for the input VCF file.

I ran it though vcf-validator and it came up with no errors.  I tried removing the '<' as I read that may cause an issue and tried renaming the ':test3 H37 aln' line to contain no spaces but it had no effect.   I then tried one of the files i'm using as a reference as the input VCF and it too did not work.  Below if the top of my VCF

Any help would be appreciated.

 

##fileformat=VCFv4.1
##FILTER=<ID=LowQual,Description="Low quality">
##FORMAT=<ID=AD,Number=.,Type=Integer,Description="Allelic depths for the ref and alt alleles in the order listed">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth (reads with MQ=255 or with bad mates are filtered)">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=MLPSAC,Number=A,Type=Integer,Description="Maximum likelihood expectation (MLE) for the alternate allele count, in the same order as listed, for each individual sample">
##FORMAT=<ID=MLPSAF,Number=A,Type=Float,Description="Maximum likelihood expectation (MLE) for the alternate allele fraction, in the same order as listed, for each individual sample">
##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF specification">
##GATKCommandLine=<ID=UnifiedGenotyper,Version=3.3-0-g37228af,Date="Thu Dec 04 09:42:28 SAST 2014",Epoch=1417678948918,CommandLineOptions="analysis_type=UnifiedGenotyper input_file=[/Users/jdlim/Desktop/WGS Trial/PIPELINE TEST/Single test/SMALT only/Temp/test3 H37Rv aln BWA_sorted_dedup_realigned_sorted.bam] showFullBamList=false read_buffer_size=null phone_home=AWS gatk_key=null tag=NA read_filter=[] intervals=null excludeIntervals=null interval_set_rule=UNION interval_merging=ALL interval_padding=0 reference_sequence=./references/H37Rv/H37Rv.fa nonDeterministicRandomSeed=false disableDithering=false maxRuntime=-1 maxRuntimeUnits=MINUTES downsampling_type=BY_SAMPLE downsample_to_fraction=null downsample_to_coverage=250 baq=OFF baqGapOpenPenalty=40.0 refactor_NDN_cigar_string=false fix_misencoded_quality_scores=false allow_potentially_misencoded_quality_scores=false useOriginalQualities=false defaultBaseQualities=-1 performanceLog=null BQSR=null quantize_quals=0 disable_indel_quals=false emit_original_quals=false preserve_qscores_less_than=6 globalQScorePrior=-1.0 validation_strictness=SILENT remove_program_records=false keep_program_records=false sample_rename_mapping_file=null unsafe=null disable_auto_index_creation_and_locking_when_reading_rods=false no_cmdline_in_header=false sites_only=false never_trim_vcf_format_field=false bcf=false bam_compression=null simplifyBAM=false disable_bam_indexing=false generate_md5=false num_threads=1 num_cpu_threads_per_data_thread=1 num_io_threads=0 monitorThreadEfficiency=false num_bam_file_handles=null read_group_black_list=null pedigree=[] pedigreeString=[] pedigreeValidationType=STRICT allow_intervals_with_unindexed_bam=false generateShadowBCF=false variant_index_type=DYNAMIC_SEEK variant_index_parameter=-1 logging_level=INFO log_to_file=null help=false version=false genotype_likelihoods_model=BOTH pcr_error_rate=1.0E-4 computeSLOD=false pair_hmm_implementation=LOGLESS_CACHING min_base_quality_score=17 max_deletion_fraction=0.05 min_indel_count_for_genotyping=5 min_indel_fraction_per_sample=0.25 indelGapContinuationPenalty=10 indelGapOpenPenalty=45 indelHaplotypeSize=80 indelDebug=false ignoreSNPAlleles=false allReadsSP=false ignoreLaneInfo=false reference_sample_calls=(RodBinding name= source=UNBOUND) reference_sample_name=null min_quality_score=1 max_quality_score=40 site_quality_prior=20 min_power_threshold_for_calling=0.95 annotateNDA=false heterozygosity=0.001 indel_heterozygosity=1.25E-4 standard_min_confidence_threshold_for_calling=30.0 standard_min_confidence_threshold_for_emitting=10.0 max_alternate_alleles=6 input_prior=[] sample_ploidy=1 genotyping_mode=DISCOVERY alleles=(RodBinding name= source=UNBOUND) contamination_fraction_to_filter=0.0 contamination_fraction_per_sample_file=null p_nonref_model=null exactcallslog=null output_mode=EMIT_VARIANTS_ONLY allSitePLs=false dbsnp=(RodBinding name= source=UNBOUND) comp=[] out=org.broadinstitute.gatk.engine.io.stubs.VariantContextWriterStub onlyEmitSamples=[] debug_file=null metrics_file=null annotation=[] excludeAnnotation=[] filter_reads_with_N_cigar=false filter_mismatching_base_and_quals=false filter_bases_not_stored=false">
##INFO=<ID=AC,Number=A,Type=Integer,Description="Allele count in genotypes, for each ALT allele, in the same order as listed">
##INFO=<ID=AF,Number=A,Type=Float,Description="Allele Frequency, for each ALT allele, in the same order as listed">
##INFO=<ID=AN,Number=1,Type=Integer,Description="Total number of alleles in called genotypes">
##INFO=<ID=BaseQRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt Vs. Ref base qualities">
##INFO=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth; some reads may have been filtered">
##INFO=<ID=DS,Number=0,Type=Flag,Description="Were any of the samples downsampled?">
##INFO=<ID=Dels,Number=1,Type=Float,Description="Fraction of Reads Containing Spanning Deletions">
##INFO=<ID=FS,Number=1,Type=Float,Description="Phred-scaled p-value using Fisher's exact test to detect strand bias">
##INFO=<ID=HaplotypeScore,Number=1,Type=Float,Description="Consistency of the site with at most two segregating haplotypes">
##INFO=<ID=InbreedingCoeff,Number=1,Type=Float,Description="Inbreeding coefficient as estimated from the genotype likelihoods per-sample when compared against the Hardy-Weinberg expectation">
##INFO=<ID=MLEAC,Number=A,Type=Integer,Description="Maximum likelihood expectation (MLE) for the allele counts (not necessarily the same as the AC), for each ALT allele, in the same order as listed">
##INFO=<ID=MLEAF,Number=A,Type=Float,Description="Maximum likelihood expectation (MLE) for the allele frequency (not necessarily the same as the AF), for each ALT allele, in the same order as listed">
##INFO=<ID=MQ,Number=1,Type=Float,Description="RMS Mapping Quality">
##INFO=<ID=MQ0,Number=1,Type=Integer,Description="Total Mapping Quality Zero Reads">
##INFO=<ID=MQRankSum,Number=1,Type=Float,Description="Z-score From Wilcoxon rank sum test of Alt vs. Ref read mapping qualities">
##INFO=<ID=QD,Number=1,Type=Float,Description="Variant Confidence/Quality by Depth">
##INFO=<ID=RPA,Number=.,Type=Integer,Description="Number of times tandem repeat unit is repeated, for each allele (including reference)">
##INFO=<ID=RU,Number=1,Type=String,Description="Tandem repeat unit (bases)">
##INFO=<ID=ReadPosRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt vs. Ref read position bias">
##INFO=<ID=SOR,Number=1,Type=Float,Description="Symmetric Odds Ratio of 2x2 contingency table to detect strand bias">
##INFO=<ID=STR,Number=0,Type=Flag,Description="Variant is a short tandem repeat">
##contig=<ID=chr,length=4411532>
##reference=file:///Users/jdlim/./references/H37Rv/H37Rv.fa
#CHROM    POS    ID    REF    ALT    QUAL    FILTER    INFO    FORMAT    test3_H37Rv_aln
header malformed gatk vcf • 3.1k views
ADD COMMENTlink modified 4.6 years ago by RamRS23k • written 4.6 years ago by SemiQuant60

Could you maybe also mention the exact command you are trying to execute? Maybe that will shed some light on this bizarre mystery!

ADD REPLYlink written 4.6 years ago by RamRS23k

Hi RamRS, heres my command

java -jar /Library/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -T VariantRecalibrator \
    -R "$Reference" \
    -input "$Variants" \
    -resource:iSNP,known=true,training=true,truth=true,prior=15.0 "$dbSNP" \
    -resource:iSNP2,known=false,training=false,truth=false,prior=15.0 "$dbSNP2" \
    -an QD -an HaplotypeScore -an MQRankSum -an ReadPosRankSum -an FS -an MQ \
    -mode BOTH \
    -recalFile "$outdir/output.recal" \
    -tranche 100.0 -tranche 99.9 -tranche 99.0 -tranche 90.0 \
    -tranchesFile "$outdir/output.tranches" \
    -rscriptFile "$outdir/output.plots.R"
java -jar /Library/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
        -T ApplyRecalibration \
        -R "$Ref_name" \
        -input "$Variants" \
        -mode BOTH \
        -tranchesFile path/to/output.tranches \
        -recalFile path/to/output.recal \
        -o "$outdir/output.recalibrated.filtered.vcf" \
        --ts_filter_level 99.0 
   

Any ideas?

ADD REPLYlink modified 4.6 years ago by RamRS23k • written 4.6 years ago by SemiQuant60
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