rsem-prepare-reference using UCSC annotation fails with exons from different orientations

[Margaret Starostik]

I am getting the following error upon building RSEM references using UCSC annotation:

"According to the GTF file given, transcript NM_001026033 has exons from different orientations! "rsem-extract-reference-transcripts RSEM/ucsc_ce10 0 ucsc_ce10_compiled.gtf None 1 knownIsoforms.txt ucsc_ce10_compiled.fa" failed! Please check if you provide correct parameters/options for the pipeline!"

What I did:

(1) Downloaded GTF from UCSC Table Browser

Assembly: Oct.2010 (WS220/ce10) 

Group: Genes and Gene Predictions; Track: RefSeq Genes

Table: refGene

Output format: GTF

The first few lines of the file:
chrI ce10_refGene stop_codon 8378299 8378301 0.000000 - . gene_id "NM_182066"; transcript_id "NM_182066"; 
chrI ce10_refGene CDS 8378302 8378421 0.000000 - 0 gene_id "NM_182066"; transcript_id "NM_182066"; 
chrI ce10_refGene exon 8378299 8378421 0.000000 - . gene_id "NM_182066"; transcript_id "NM_182066"; 
chrI ce10_refGene CDS 8379131 8379233 0.000000 - 1 gene_id "NM_182066"; transcript_id "NM_182066"; 
chrI ce10_refGene exon 8379131 8379233 0.000000 - . gene_id "NM_182066"; transcript_id "NM_182066"; 
chrI ce10_refGene CDS 8379700 8379809 0.000000 - 0 gene_id "NM_182066"; transcript_id "NM_182066"; 
chrI ce10_refGene exon 8379700 8379809 0.000000 - . gene_id "NM_182066"; transcript_id "NM_182066"; 
chrI ce10_refGene CDS 8380324 8380439 0.000000 - 2 gene_id "NM_182066"; transcript_id "NM_182066"; 
chrI ce10_refGene exon 8380324 8380439 0.000000 - . gene_id "NM_182066"; transcript_id "NM_182066"; 

chrI ce10_refGene CDS 8380905 8380979 0.000000 - 2 gene_id "NM_182066"; transcript_id "NM_182066"; 

(2) Created knownIsoforms.txt from the GTF above since it's not available to download for C. elegans 

 

The first few lines of the file:

NM_182066 NM_182066

NM_059873 NM_059873

NM_001129046 NM_001129046

NR_070240 NR_070240

NR_052806 NR_052806 

(3) Ran RSEM as follows: 

rsem-prepare-reference --gtf ucsc_ce10_compiled.gtf

      --transcript-to-gene-map knownIsoforms.txt \

      chrI.fa,chrII.fa,chrIII.fa,chrIV.fa,chrV.fa,chrM.fa,chrX.fa \

      RSEM/ucsc_ce10

     

I have extensively used RSEM in the past with human Ensembl annotation and had no problems, but this is the first time using C. elegans UCSC annotation.

Thanks,

Margaret