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RNA-SeQC giving Badly formed genome loc error

SidiZSidiZ JapanMember, Broadie

Hello,

I'm trying to run RNA-SeQC by:

java -jar RNA-SeQC_v1.1.8.jar -o RNA-SeQC -r ../References/hg19_mt_virus_rDNA.fa -s "MT9-1_RNA|/Users/atgu/Documents/glioma_research/test_data_rna_seq/in_chr1.bam|TestRun" -t ../References/gencode.v19.startMinus1.annotation.gtf

Java version 1.7.0_80
RNA-SeQC version 1.1.8
BAM file is RNA-seq with paired-end reads

And it keeps giving this "Badly formed genome loc" error below. But when I checked my reference files none of them contain the chr1:566235-566236 interval so I'm confused. Also tried using the gencode gtf file without start position -1 and the same error occurred. Also tried removing any reads overlapping this interval from the BAM file with bedtools intersect, and got exactly the same error with the same interval. Any idea what might the problem be? Thank you!!!

RNA-SeQC v1.1.8.1 07/11/14
Creating rRNA Interval List based on given GTF annotations
Retriving contig names from reference
contig names in reference: 32
Loading GTF for Read Counting
Converting to refGene
Transcript objects to RefGen format: 3 s
Running IntronicExpressionReadBlock Walker ....
Arguments: [-T, IntronicExpressionReadBlock, --outfile_metrics, /Users/atgu/Documents/glioma_research/RNA-SeQC/MT9-1_RNA/MT9-1_RNA.metrics.tmp.txt, -R, ../References/hg19_mt_virus_rDNA.fa, -I, /Users/atgu/Documents/glioma_research/test_data_rna_seq/in_chr1.bam, -refseq, /Users/atgu/Documents/glioma_research/RNA-SeQC/refGene.txt, -l, ERROR]
org.broadinstitute.sting.utils.exceptions.UserException$MalformedGenomeLoc: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:The stop position 566235 is less than start 566236 in contig chr1
at org.broadinstitute.sting.utils.GenomeLocParser.vglHelper(GenomeLocParser.java:324)
at org.broadinstitute.sting.utils.GenomeLocParser.validateGenomeLoc(GenomeLocParser.java:290)
at org.broadinstitute.sting.utils.GenomeLocParser.createGenomeLoc(GenomeLocParser.java:265)
at org.broadinstitute.sting.utils.GenomeLocParser.createGenomeLoc(GenomeLocParser.java:260)
at org.broadinstitute.sting.utils.GenomeLocParser.createGenomeLoc(GenomeLocParser.java:251)
at org.broadinstitute.cga.rnaseq.gatk.CountReadBlockMetricsWalker.getBlocksForRead(CountReadBlockMetricsWalker.java:125)
at org.broadinstitute.cga.rnaseq.gatk.IntronicExpressionReadBlockWalker.makeRefSeqDerviedCounts(IntronicExpressionReadBlockWalker.java:91)
at org.broadinstitute.cga.rnaseq.gatk.CountReadMetricsWalker.map(CountReadMetricsWalker.java:265)
at org.broadinstitute.cga.rnaseq.gatk.CountReadMetricsWalker.map(CountReadMetricsWalker.java:38)
at org.broadinstitute.sting.gatk.traversals.TraverseReads.traverse(TraverseReads.java:106)
at org.broadinstitute.sting.gatk.traversals.TraverseReads.traverse(TraverseReads.java:52)
at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:72)
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:248)
at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:113)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:236)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:146)
at org.broadinstitute.cga.rnaseq.gatk.GATKTools.runIntronReadCount(GATKTools.java:226)
at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runRegionCounting(ReadCountMetrics.java:244)
at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runReadCountMetrics(ReadCountMetrics.java:59)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.runMetrics(RNASeqMetrics.java:225)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:171)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:139)
RNA-SeQC Total Runtime: 0 min

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Answers

  • SidiZSidiZ JapanMember, Broadie

    Thank you @shlee I'll look into GenePattern....I assume it's something with the GTF file (although the GTF file doesn't actually contain these loci, which seems odd)

  • shleeshlee CambridgeMember, Broadie ✭✭✭✭✭

    In this case @SidiZ, these positions must refer to the reads in your BAM. Did you remember to coordinate sort, e.g. using Picard SortSam, before running the data through RNA-SeQC?

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