RNA-SeQC input error

[sphil]

Hey guys,

I want to use RNA-SeQC for my Rna-seq data. Preparing it like it's done on the website I get the following error:

java.lang.IllegalStateException: Inappropriate call if not paired read
at net.sf.samtools.SAMRecord.requireReadPaired(SAMRecord.java:642)
at net.sf.samtools.SAMRecord.getMateUnmappedFlag(SAMRecord.java:669)
at org.broadinstitute.sting.utils.sam.GATKSAMRecord.getMateUnmappedFlag(GATKSAMRecord.java:307)
at org.broadinstitute.cga.rnaseq.gatk.CountReadMetricsWalker.map(CountReadMetricsWalker.java:188)
at org.broadinstitute.cga.rnaseq.gatk.CountReadMetricsWalker.map(CountReadMetricsWalker.java:37)
at org.broadinstitute.sting.gatk.traversals.TraverseReads.traverse(TraverseReads.java:103)
at org.broadinstitute.sting.gatk.traversals.TraverseReads.traverse(TraverseReads.java:51)
at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:69)
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:225)
at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:104)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:227)
at org.broadinstitute.cga.rnaseq.gatk.GATKTools.runIntronReadCount(GATKTools.java:225)
at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runRegionCounting(ReadCountMetrics.java:236)
at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runReadCountMetrics(ReadCountMetrics.java:57)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.runMetrics(RNASeqMetrics.java:207)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:158)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:127)


It proposes that the reads are :
Inappropriate call if not paired read

however i call with the '-singelEnd' flag such that it should be fine. Any guesses how to fix it?


Best,

Phil

[simonandrews]

Is this just a typo? It should be -singleEnd not -singelEnd.

[sphil]

sry, actually it is a typo....
Problem still exists, though

[aparna]

Does anybody know how to fix this? Apparently I emailed the developers - no response.

[kopi-o]

No, but I would recommend that you use RSeQC (formerly EVER-seq) instead.

[sschavan]

Hi all,

I was running RNA-SeQC software in Windows with command line options. I tried with the example sets provided on https://confluence.broadinstitute.or...Tools/RNA-SeQC and following the instructions as stated.

The command was:
"java -jar RNA-SeQC_v1.1.7.jar -n 1000 -s "TestId|ThousandReads.bam|TestDesc" -t gencode.v7.annotation_goodContig.gtf -r Homo_sapiens_assembly19.fasta -o ./testReport/ -strat gc -gc gencode.v7.gc.txt"

However I ran into the below error, and I am trying to see if anyone else faced the same error or knows the fix:

C:\example_files_RNASeQC>java -jar RNA-SeQC_v1.1.7.jar -n 1000 -s "TestId|ThousandReads.bam|TestDesc" -t gencode.v
7.annotation_goodContig.gtf -r Homo_sapiens_assembly19.fasta -o C:\example_files_RNASeQC\testReport -strat gc -gc
gencode.v7.gc.txt
RNA-SeQC v1.1.7 05/14/12
Creating rRNA Interval List based on given GTF annotations
Retriving contig names from reference
contig names in reference: 85
Loading GTF for Read Counting
Converting to refGene
Transcript objects to RefGen format: 1 s
Running IntronicExpressionReadBlock Walker ....
Arguments: [-T, IntronicExpressionReadBlock, --outfile_metrics, C:\example_files_RNASeQC\testReport/TestId/TestId.
metrics.tmp.txt, -R, Homo_sapiens_assembly19.fasta, -I, ThousandReads.bam, -refseq, C:\example_files_RNASeQC\testR
eport/refGene.txt, -l, ERROR]
org.broadinstitute.sting.utils.exceptions.UserException: Unable to read index file, for input source: C:\example_f
iles_RNASeQC\testReport\refGene.txt.idx
at org.broadinstitute.sting.gatk.refdata.tracks.RMDTrackBuilder.getFeatureSource(RMDTrackBuilder.java:224)
at org.broadinstitute.sting.gatk.refdata.tracks.RMDTrackBuilder.createInstanceOfTrack(RMDTrackBuilder.java:128)
at org.broadinstitute.sting.gatk.refdata.tracks.RMDTrackBuilder.createInstanceOfTrack(RMDTrackBuilder.java:145)
at org.broadinstitute.cga.rnaseq.gatk.CountReadMetricsWalker.initialize(CountReadMetricsWalker.java:521)
at org.broadinstitute.cga.rnaseq.gatk.IntronicExpressionReadBlockWalker.initialize(IntronicExpressionReadBlockWalker.java:40)
at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:48)
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:243)
at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runReadCountMetrics(ReadCountMetrics.java:58)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.runMetrics(RNASeqMetrics.java:220)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:166)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:135)
RNA-SeQC Total Runtime: 0 min

Any comments are highly appreciated.

Thanks
Shweta

[kove]

I have the same question as you are!

[uqfgaiti]

Hi all,

as kove and sschavan above, I was running RNA-SeQC software in command line. I tried withto follow the example sets provided on https://confluence.broadinstitute.or...Tools/RNA-SeQC and following the instructions as stated.

The command was:
"java -jar /hox/u/uqfgaiti/Tools/picard-tools-1.84/RNA-SeQC_v1.1.7.jar -r ampQue1.fasta -s "TestID|CEL-Seq.final.sorted.bam|TestDesc" -t 2507_Intergenic+Intronic.gtf -n 1000 -BWArRNA rRNA.fasta -o RNA-SeQC_out"

However I ran into the below error, and I am trying to see if anyone else faced the same error or knows the fix:

RNA-SeQC v1.1.7 05/14/12
Retriving contig names from reference
contig names in reference: 13397
Loading GTF for Read Counting
Converting to refGene
Transcript objects to RefGen format: 2 s
Running IntronicExpressionReadBlock Walker ....
Arguments: [-T, IntronicExpressionReadBlock, --outfile_metrics, RNA-SeQC_out/TestID/TestID.metrics.tmp.txt, -R, ampQue1.fasta, -I, CEL-Seq.final.sorted.bam, -refseq, RNA-SeQC_out/refGene.txt, -l, ERROR]
net.sf.samtools.util.RuntimeEOFException: Premature EOF; BinaryCodec in readmode; streamed file (filename not available)
at net.sf.samtools.util.BinaryCodec.readBytes(BinaryCodec.java:373)
at net.sf.samtools.util.BinaryCodec.readByteBuffer(BinaryCodec.java:480)
at net.sf.samtools.util.BinaryCodec.readInt(BinaryCodec.java:491)
at net.sf.samtools.BAMFileReader.readSequenceRecord(BAMFileReader.java:429)
at net.sf.samtools.BAMFileReader.readHeader(BAMFileReader.java:403)
at net.sf.samtools.BAMFileReader.<init>(BAMFileReader.java:144)
at net.sf.samtools.BAMFileReader.<init>(BAMFileReader.java:114)
at net.sf.samtools.SAMFileReader.init(SAMFileReader.java:514)
at net.sf.samtools.SAMFileReader.<init>(SAMFileReader.java:167)
at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource$ReaderInitializer.call(SAMDataSource.java:927)
at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource$SAMReaders.<init>(SAMDataSource.java:788)
at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource$SAMResourcePool.createNewResource(SAMDataSource.java:747)
at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource$SAMResourcePool.getAvailableReaders(SAMDataSource.java:718)
at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource.<init>(SAMDataSource.java:261)
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.createReadsDataSource(GenomeAnalysisEngine.java:755)
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.initializeDataSources(GenomeAnalysisEngine.java:666)
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:227)
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:243)
at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runReadCountMetrics(ReadCountMetrics.java:58)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.runMetrics(RNASeqMetrics.java:220)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:166)
at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:135)
RNA-SeQC Total Runtime: 0 min

Any comments are highly appreciated.

Thanks
Federico