Yeast GTF file needed for RNA-SeQC run

Question

[Lina F]

Hi all,

I have some yeast RNAseq data and I would like to run RNA-SeQC to get an overview of the quality of the run.

I got both the reference fasta sequence and the GTF file from Ensembl here:

# fasta 
ftp://ftp.ensembl.org/pub/current_fasta/saccharomyces_cerevisiae/dna/Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa.gz

# gtf
ftp://ftp.ensembl.org/pub/current_gtf/saccharomyces_cerevisiae/Saccharomyces_cerevisiae.R64-1-1.86.gtf.gz

However, it seems that there is no rRNA information in the GTF file. When I run RNA-SeQC, I get the following output in my log:

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: 17
Loading GTF for Read Counting
Converting to refGene
Transcript objects to RefGen format:    0 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)
...

I would like the rRNA information if possible. Does anyone know where to get a GTF file with rRNA information for yeast?

Thanks!

Answer 1

[apa@stowers]

RNA-SeQC appears to be looking for an annotation field "transcript_type" which does not exist in Ensembl GTFs, Ensembl uses "transcript_biotype" and "gene_biotype".

I think you can use the "transcript.type.field" parameter to specify which GTF field you want to use instead of "transcript_type".

Otherwise, you could run something like "perl -i -pe 's/transcript_biotype/transcript_type/' Saccharomyces_cerevisiae.R64-1-1.86.gtf" to change all instances of "transcript_biotype" to "transcript_type", then RNA-SeQC should recognize it?

[Lina F]

Ah, this makes sense -- thanks for the help!