Gene annotation of the human UROD gene

Practical exercise

Enrique Blanco - eblanco@imim.es
Roderic Guigo - rguigo@imim.es
Abstract: In this exercise, a previously annotated gene will be used to measure the accuracy of different gene finding approaches. geneid, GENSCAN, FGENESH and GENEWISE will be used to annotate the sequence. Both search by signal, content and homology (protein and cDNA sequences) methods will be employed in order to improve the ab initio results. Weak conservation in Start codons and the short length of the initial exon leads to wrong prediction of this exon in most cases.
Colour legend:
  • Genomic element
  • Operations or links

    • Gene prediction (T)

    Gene structure in Eukaryotes:


    Signals and regions:
    Signals
    Start (ATG)
    STOPs (TGA,TAA,TAG)
    Donor (GT)
    Acceptor (AG)
    Exons
    Single
    First
    Internal
    Terminal
    Regions
    Exons
    Introns
    Intergenic
    5' and 3' UTRs

    Predicted exons:


    geneid structure:
    A. Gene annotation
    Step 1. Accesing EMBL database to retrieve the gene
    • Go to EMBL database

    • Select Get Nucleotide sequences

    • Type sequence name U30787

    • Press Go button

    • Click on the EMBL:U30787 (coding sequences) entry link

    • Have a look at the different entry fields: detect the mRNA and CDS exons

    • Select Text Entry to see the plain text file


    • Get back to the query results

    • Click on the EMBL:U30787 (Primary Accession) entry link

    • Look at the archive of versions of this entry.


    B. Exploring ab initio gene prediction
    Step 2. Running geneid
    • Connect to the geneid server

    • Paste the FASTA sequence

    • Choose geneid output format

    • Run geneid in diffent levels (compare with the EMBL annotations):

      1. Searching signals: Select acceptors, donors, start and stop codons. Look for them in the real annotation of the sequence

      2. Searching exons: Select All exons and try to find the real ones

      3. Finding genes: You do not need to select any option (default behaviour). Compare the predicted gene with the real gene

      Figure 1. Signal, exons and genes predicted by geneid in the sequence HS307871

    • Try to get the gene prediction again, but using the parameter file for: Drosophila melanogaster, Caenorhabditis elegans and Oryza sativa. Can you see something in common? Do you realize species-specific training is not so specific?

    Step 3. Running other genefinders

    As there are several alternative programs to analyze a DNA sequence, we can run each application and observe the common parts of the predictions.

    1. GENSCAN:
      • Connect to the GENSCAN server

      • Paste DNA sequence

      • Press Run Genscan button

      • Compare annotations and predictions

    2. FGENESH:
      • Connect to Softberry homepage

      • On the left frame, select GENE FINDING in Eukaryota

      • Select the program FGENESH

      • Paste DNA sequence

      • Press Search button

      • Compare annotations and predictions

    3. NOTE: First exon is always missed in the predictions and there are some problems to detect the donor site from exon 5. Detection of Start codons is a serious drawback in current gene finding programs (see Figure 2). However, this problem can be overcome by using homology information to complete the gene prediction.
    Figure 2. EMBL annotation and genes predicted by Grail, GENSCAN, geneid and FGENESH in the sequence HS307871


    C. Using EST/cDNA homology information
    Step 4. Using FGENESH-C + alignment of transcripts

    Let's search for ESTs supporting future predictions by FGENESH-C:

    • Open the NCBI blast server

    • Choose blastn program (genomic query versus genomic database)

    • Search the est_human database

    • Paste the genomic sequence and press the Blast! and Format!

      Filter this output in order to select those non-overlapping ESTs that could form a complete cDNA sequence (see Figure 3). Moreover, ESTs not divided into two or more pieces in the genomic sequence (containing a couple of splice sites) should be rejected.

    • Let us imagine we select the DA976126 SYNOV2

    • Search the link and go to the GenBank entry. Save it in FASTA format

    Figure 3. Best human ESTs in the alignment mapped on the genomic sequence HS307871

    • Connect to the FGENESH-C server (on Gene finding with similarity menu)

    • Paste the sequence U30787

    • Paste the DA976126 SYNOV2 EST (the last one in the first column)

    • Press the Search button

    • Analyze the prediction (compare to the annotations)


    D. Using protein homology information
    Step 5. Spliced alignment

    Spliced alignment is very useful when we have additional information (a putative homologous protein sequence) about the content of the sequence. Thus, gene prediction is guided by fitting the protein sequence into the best splice sites predicted in the genomic sequence.
    • Open the NCBI blast server

    • Choose blastx program (genomic query versus protein database)

    • Paste the genomic sequence and press the Blast! and Format!

    • Select the first protein. Go to the GenBank entry and convert it into a
      FASTA sequence (it is the real protein annotated in the genomic sequence).

    • Open genewise web server to use this protein to predict the best gene structure

    • Paste both protein and genomic sequences and run the program

    • Compare predicted gene (end of the file) and annotations: look for splice sites within introns to check exon boundaries are correct

      Figure 5. Best HSPs representing proteins homologues similar to the genomic sequence HS307871 obtained using blastx



    E. Results

    Here you can find the solutions to every exercise:

    EMBL annotation
    EMBL annotation (plain text)
    FASTA sequence
    geneid results: signals
    geneid results: exons
    geneid results: genes
    GENSCAN results
    FGENESH results
    Blastn + human ESTs results
    FGENESH-C results
    The EST SYNOV2
    Blastx + protein results
    The URO-D protein
    Genewise (human protein)


    F. Bibliography
    1. J.F. Abril and R. Guigó. gff2ps: visualizing genomic annotations. Bioinformatics 16:743-744 (2000).

    2. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215:403-410 (1990).

    3. Burge, C. and Karlin, S. Prediction of complete gene structures in human genomic DNA. J. Mol. Biol. 268, 78-94 (1997).

    4. E. Blanco, G. Parra and R. Guigó. Using geneid to Identify Genes. In A. D. Baxevanis and D. B. Davison, chief editors: Current Protocols in Bioinformatics. Volume 1, Unit 4.3. John Wiley & Sons Inc., New York. ISBN: 0-471-25093-7 (2002).

    5. Asaf A. Salamov and Victor V. Solovyev. Ab initio Gene Finding in Drosophila Genomic DNA Genome Res. 10: 516-522 (2000).

    6. D. Hyatt, J. Snoddy, D. Schmoyer, G. Chen, K. Fischer, M. Parang, I. Vokler, S. Petrov, P. Locascio, V. Olman, Miriam Land, M. Shah, and E. Uberbacher. Improved Analysis and Annotation Tools for Whole-Genome Computational Annotation and Analysis: GRAIL-EXP Genome Analysis Toolkit and Related Analysis Tools. Genome Sequencing & Biology Meeting (2000).

    7. Ewan Birney and Richard Durbin. Using GeneWise in the Drosophila Annotation Experiment. Genome Res. 10: 547-548 (2000).