Issues running RNAseqc on my bam file

Question

[vineelagangalapudi]

Hello , I have been using rseqc for a while now. I wanted to try out RNAseqc and I am running into multiple errors. I have a genome bam sorted by coordinate from STAR. I also have a indexed ref genome fasta file and also its dictionary ready. Later I added the read group information using picard AddOrReplaceReadGroups. I ran the following command.

java -jar /usr/local/apps/rnaseqc/1.1.8/RNA-SeQC_v1.1.8.jar  -r ucsc.hg19.fasta -o xyz -s "xyz|xyz.bam|xyz" -t hg19.gtf

I got the following error.
[-] Unloading BWA 0.7.15 ...
[-] Unloading samtools 1.5 ...
[-] Unloading java 1.7.0_25 ...
[+] Loading BWA 0.7.15 ...
[+] Loading samtools 1.5 ...
[+] Loading java 1.7.0_25 ...
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: 93
Loading GTF for Read Counting
Converting to refGene
Transcript objects to RefGen format:    1 s
java.lang.RuntimeException: No rRNA found in GTF transcript_type field
    at org.broadinstitute.cga.rnaseq.TranscriptList.toRRNAIntervalList(TranscriptList.java:414)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.createRefGeneAndRRNAFiles(RNASeqMetrics.java:1306)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.prepareFiles(RNASeqMetrics.java:196)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:170)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:139)
No information for rRNA available. Continuing without rRNA calculations. (Using the -BWArRNA flag for best results)
Running IntronicExpressionReadBlock Walker ....
Arguments: [-T, IntronicExpressionReadBlock, --outfile_metrics, xyz/xyz/xyz.metrics.tmp.txt, -R, ucsc.hg19.fasta, -I, xyz.bam, -refseq, xyz/refGene.txt, -l, ERROR]
/var/spool/slurm/slurmd/job51966571/slurm_script: line 6: 18690 Killed                  java -jar /usr/local/apps/rnaseqc/1.1.8/RNA-SeQC_v1.1.8.jar -r ucsc.hg19.fasta -o xyz -s "xyz|xyz.bam|xyz" -t $1

I have seen a similar post on biostars but there was no solution. I would really appreciate any inputs to my problem.

Thanks.

[WouterDeCoster]

Does hg19.gtf contain rRNA in the GTF transcript_type field?

[vineelagangalapudi]

no, that is why this is bypassing the rRNA calculation step and moving on to the next steps and then it is failing in the intronicexpressionreadblock walker step.

[anoo]

Hello,

I am working on RNASeQC and came across the same error.

RNA-SeQC v1.1.9 06/26/16 Creating rRNA Interval List based on given GTF annotations Retriving contig names from reference contig names in reference: 639 Loading GTF for Read Counting Converting to refGene Transcript objects to RefGen format: 9 s java.lang.RuntimeException: No rRNA found in GTF transcript_type field at org.broadinstitute.cga.rnaseq.TranscriptList.toRRNAIntervalList(TranscriptList.java:318) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.createRefGeneAndRRNAFiles(RNASeqMetrics.java:1167) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.prepareFiles(RNASeqMetrics.java:184) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:161) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:133) No information for rRNA available. Continuing without rRNA calculations. (Using the -BWArRNA flag for best results) Running IntronicExpressionReadBlock Walker .... Exception in thread "main" java.lang.ExceptionInInitializerError at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.<init>(GenomeAnalysisEngine.java:160) at org.broadinstitute.sting.gatk.CommandLineExecutable.<init>(CommandLineExecutable.java:53) at org.broadinstitute.sting.gatk.CommandLineGATK.<init>(CommandLineGATK.java:54) at org.broadinstitute.cga.rnaseq.gatk.GATKTools.runIntronReadCount(GATKTools.java:203) at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runRegionCounting(ReadCountMetrics.java:220) at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runReadCountMetrics(ReadCountMetrics.java:59) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.runMetrics(RNASeqMetrics.java:211) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:162) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:133) Caused by: java.lang.NullPointerException at org.reflections.Reflections.scan(Reflections.java:220) at org.reflections.Reflections.scan(Reflections.java:166) at org.reflections.Reflections.<init>(Reflections.java:94) at org.broadinstitute.sting.utils.classloader.PluginManager.<clinit>(PluginManager.java:77) ... 9 more

It would be great if any inputs provided to solve the error.

Thanks.