Rna-Seqc Generates Error

Question

[federico.gaiti]

Hi all,

I was running RNA-SeQC software in command line. I tried withto follow the example sets provided on RNA-SeQC website 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

[Istvan Albert]

In all such cases you need to make sure your bam file is exists, it is indexed and that it does contain the samples.

Include a few lines of the command to invoke and the results of doing a samtools view

Answer 1

[federico.gaiti]

I re-did everything from the beginning and it seems the problem was in the command "AddOrReplaceReadGroups".

Now I have all the required input files:

• an indexed, coordinate-sorted BAM file with read group information

• .BAI of the BAM file in the same folder

• my FASTA ref sequence

• .FAI of the ref sequence

• .DICT of the ref sequence

• GTF annotation file

Now it's running and it's not giving me any sort of error.

Let you know if I'll be stuck again

Thanks for the comments

[Istvan Albert]

thanks for following up

Answer 2

[Chris Miller]

The relevant part of this error seems to be: "filename not available"

Are you sure your bam path is correct and that the software can access it (permissions are set properly, etc)?