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  • annotation file did not work

    I am working on Salmonella rnaSeq and using Galaxy to analyse data. Here are two sets of assemblies

    I found the annotation file from RefSeq assembly worked with Cufflink but the annotation file from GenBank assembly did not work.
    Any suggestions?
    I want to use the GenBank assembly because it contains more gene names

  • #2
    update:
    I unloaded the gff file which did not work in GFF3 online validator (http://genometools.org/cgi-bin/gff3validator.cgi) and ran it. I got the following information
    ....................................................................................................................
    Validation unsuccessful!

    GenomeTools error: more than one pseudogene attribute on line 252 in file "/var/www/servers/genometools.org/htdocs/cgi-bin/gff3/GCA_000009505.1_ASM950v1_genomic.gff.gz"
    ....................................................................................................................

    My question is what "more than one pseudogene attribute on line 252 in file" means?

    In contrast, the gff file which worked in Galaxy passed the examination of GFF3 online validator

    Comment


    • #3
      The offending lines appear to be these (and there are ~150+ more)

      Code:
      AM933172.1      EMBL    gene    58700   59680   .       +       .       ID=gene50;Name=SEN0050;gbkey=Gene;gene_biotype=pseudogene;is_ordered=true;locus_tag=SEN0050;part=1/2;pseudo=true;pseudogene=unitary
      AM933172.1      EMBL    gene    59682   59903   .       +       .       ID=gene50;Name=SEN0050;gbkey=Gene;gene_biotype=pseudogene;is_ordered=true;locus_tag=SEN0050;part=2/2;pseudo=true;pseudogene=unitary
      There is a 1 bp gap in the two part entry for this pseudogene.

      Are you sure the GenBank version has "more" genes? The main NCBI record you linked says this

      Code:
      RefSeq assembly and GenBank assembly identical:   yes
      Last edited by GenoMax; 01-27-2016, 10:09 AM.

      Comment


      • #4
        GenoMax,
        Thank you very much!
        I do not know bioinformatic too much and I do not know programming.
        I just simply searched several gene names appearing in the RefSeq gff file and found they did not appear in the GenBank gff file.
        For the "more than one pseudogene attribute" issues, I found line 252 and many other lines have either " pseudo=true; pseudogene=unitary" or " pseudo=true; pseudogene=unknown".
        I deleted "pseudogene=unitary" and "pseudogene=unknown" in 010 Editor.
        Now the file passed the the examination of GFF3 online validator.
        I have not run it with Cufflinks in Galaxy and do not know if the change will hurt something.

        Comment


        • #5
          Those two files do have different annotation. I did not look very closely but if the pseudogenes is all that is different then it may make sense to use the RefSeq file. Edits you did to genbank file should not affect your read counts.

          Comment


          • #6
            Genomax,

            I tried the file I edited, but it did not either.
            Now I am thinking the "gap" you mentioned between the two parts in one ID is the cause.
            However, I found the RefSeq gff file also has one "gap" situation.
            .....................................................................................................................
            NC_011294.1 RefSeq region 1020368 1020421 . - . ID=id51;Dbxref=PSEUDO:CAR32502.1;Note=Note the frameshift mutation following codon 18~Located within a degenerate Gifsy-2 prophage;gbkey=misc_feature;part=1/2;pseudo=true

            NC_011294.1 RefSeq region 1020127 1020366 . - . ID=id51;Dbxref=PSEUDO:CAR32502.1;Note=Note the frameshift mutation following codon 18~Located within a degenerate Gifsy-2 prophage;gbkey=misc_feature;part=2/2;pseudo=true
            ...............................................................................................................

            They are both ID=id51
            one is from 1020368 to 1020421
            The other one is from 1020127 to 1020366
            There is a gap between the two parts

            Comment

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