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  • pomar28
    Junior Member
    • Aug 2014
    • 1

    #1

    Trouble running rsem-prepare-reference

    I am working with non-model organism for which we have assembled draft genome, and annotated genes, in GFF3 format. I am trying to use RSEM to estimate gene/isoforms expression levels. Since RSEM does not support GFF3 format I converted GFF3 into GTF file. It looks to me that conversion worked fine, and here are the first few lines of converted GTF file:
    Scaffold1 I5K EXON 8888 9187 . + 0 gene_id "EAFF000001"; transcript_id "EAFF000001-RA";
    Scaffold1 I5K EXON 20235 20765 . + 0 gene_id "EAFF000001"; transcript_id "EAFF000001-RA";
    Scaffold1 I5K EXON 20965 21120 . + 0 gene_id "EAFF000001"; transcript_id "EAFF000001-RA";
    Scaffold1 I5K EXON 22857 22885 . + 0 gene_id "EAFF000001"; transcript_id "EAFF000001-RA";
    Scaffold1 I5K EXON 23931 24107 . + 0 gene_id "EAFF000001"; transcript_id "EAFF000001-RA";
    Scaffold1 I5K EXON 24723 24892 . + 0 gene_id "EAFF000001"; transcript_id "EAFF000001-RA";
    However my GTF is not correctly "understood" by RSEM, because rsem-prepare-reference doesn’t extract transcript reference sequences from sequence of the genome.
    When I run the ollowing command line:
    rsem-prepare-reference --gtf /EAFFGENOME/EAFF.Models_2.gtf --bowtie --bowtie-path bowtie-1.0.1 /EAFFGENOME/Eaff_11172013.genome2.fa affinis1_refs

    I’ve got the following error:

    Parsed 200000 lines
    The reference contains no transcripts!

    Any help or suggestions would be appreciated very much.

    PM
  • mikep
    Member
    • Feb 2011
    • 45

    #2
    1) Change the phase from "0" to "." (may not make a difference, but it's what I use)
    2) Do you have features in the gtf of type "transcript" (as opposed to exon)?

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