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  • how to look at differential expression of a pseduo gene?

    Hi all

    When do RNA-Seq analysis, after I have finished the alignment by Tophat and get the accpeted_hits.bam. Now I want to looked at if it is has differential expression at the corresponding area (We called it 'pseduo gene' which haven't been annotated by reference genome ). How could I do it ?

    I think the best way is to add this pseduo gene to reference genome GTF file. And then I could play HTSeq-count or Cuffdiff to look at the differential expression of this gene .

    Here is the effort I made so far . I have add the Chr21:100000-200000 as 'exon' (a pseudo gene) to the genes.gtf of Ensembl, rename the GTF file.

    Code:
    $ head genes_hello.gtf
    [B]21 protein_coding exon 100000 200000 . + . exon_id "ENSECAE00000000001"; exon_number "1"; gene_biotype "protein_coding"; gene_id "ENSECAG00000099999"; gene_name "HELLO"; gene_source "ensembl"; p_id "P99999"; transcript_id "ENSECAT00000099999"; transcript_name "HELLO-001"; transcript_source "ensembl"; tss_id "TSS9999";[/B]
    1 protein_coding UTR 11193 11209 . + . gene_biotype "protein_coding"; gene_id "ENSECAG00000012421"; gene_name "SYCE1"; gene_source "ensembl"; p_id "P20975"; transcript_id "ENSECAT00000013004"; transcript_name "SYCE1-201"; transcript_source "ensembl"; tss_id "TSS1013";
    1 protein_coding exon 11193 11261 . + . exon_id "ENSECAE00000079002"; exon_number "1"; gene_biotype "protein_coding"; gene_id "ENSECAG00000012421"; gene_name "SYCE1"; gene_source "ensembl"; p_id "P20975"; transcript_id "ENSECAT00000013004"; transcript_name "SYCE1-201"; transcript_source "ensembl"; tss_id "TSS1013";
    However, if I run the Cuffdiff directly, the output (DE result) didn't contain any my pseudo gene information. And if I ran HTSeq-count , it gave me the error:

    Code:
    $ htseq-count -s yes aligned_sorted.sam genes_hello.gtf>gene_count.txt
    
    Error occured when processing GFF file (line 1 of file genes.gtf):
      need more than 1 value to unpack
      [Exception type: ValueError, raised in __init__.py:207]
    Error occured when processing GFF file (line 1 of file genes.gtf):
      need more than 1 value to unpack
      [Exception type: ValueError, raised in __init__.py:207]
    It seems that I didn't add the pseduo gene properly. Does anyone know how to do it?

    Thank you very much!

  • #2
    Have you tried to create a full record of your entry? If you name it "protein_coding" I'd assume at least lines for start/stop have to be specified as well, evtl also the UTR. Look at correct entries of single exon genes. Maybe the sort order is also important (put the entry in the correct place on chr21), but I'm not sure about that.

    Comment


    • #3
      A similar query has been answered: http://seqanswers.com/forums/showthread.php?t=65499

      Comment


      • #4
        @super0925: I have wondered why you chose to call this a "pseudogene" in both posts. Since the standard definition does not seem to apply (you are also seeing differential expression)?

        Comment


        • #5
          Originally posted by GenoMax View Post
          @super0925: I have wondered why you chose to call this a "pseudogene" in both posts. Since the standard definition does not seem to apply (you are also seeing differential expression)?
          Yep. I think maybe we need call it 'Anonymous gene' to explain 'the gene without annotation at the genome '

          Comment

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