Annotation of the human sequence HS307871

Practical exercise

Enrique Blanco -
Roderic Guigo -

Abstract: In this exercise, a previously annotated gene will be used to measure the accuracy of different gene finding approaches. GRAIL, GENSCAN, geneid, FGENESH, GenomeScan, GrailEXP 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 of Start codons will lead to wrong prediction of initial exons in most cases.

Colour legend:
  • Genomic element
  • Operations or links

  • A. Gene annotation

    Step 1. Accesing EMBL database to retrieve the gene
    • Go to EMBL database

    • Select Nucleotide sequences

    • Type sequence entry name HS307871

    • Press Go button

    • Click on EmblEntry link

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

    • Click on Text Entry link to see the plain text formatted output

    • This is the sequence in FASTA format

    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 with different parameters:

      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

    Step 3. Running other genefinders

    Provided that there are several alternative programs to analyze a DNA sequence, we can run every 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. GRAIL:
      • Connect to GrailEXP homepage

      • Activate Perceval Exon Candidates box

      • Paste DNA sequence

      • Press Go! button

      • Check the results

      • Compare annotations and predicted exons

    4. 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 GrailEXP
    • Connect to GrailExp homepage

    • Activate Galahad EST/mRNA/cDNA Alignments box

    • Select GrailEXP database (RefSeq/HTDB/dbEST/EGAD/Riken)

    • Activate exon assembly: Gawain Gene Models

    • Paste DNA sequence

    • Press Go! button

    • Check the results: predictions and supporting information

    • Compare annotations, ab initio GRAIL prediction and five predicted alternative spliced variants
    Figure 3. Comparison between EMBL annotation and genes predicted ab inition by Grail Vs five alternative predictions supported by ESTs information in the sequence HS307871

    Step 5. Using other gene finding programs + alignment of transcripts

    Using blastn, we can search the database est_human for ESTs supporting future predictions. Filter this output in order to select those non-overlapping ESTs that could form a complete cDNA sequence (see Figure 4). Moreover, ESTs not divided into two or more pieces in the genomic sequence (containing a couple of splice sites) should be rejected.
    • Connect to the FGENESH-C server (on Gene finding with similarity menu)

    • Paste the sequence HS307871

    • Paste the cDNA sequence or EST you have selected

    • Press the search button

    • Notice that predicted gene will necessarily supported by homology information, so it will likely mapped only in the genomic region overlapping your EST query.

    Figure 4. Best human ESTs in the alignment mapped on the genomic sequence HS307871

    D. Using protein homology information

    Step 6. 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. Display the FASTA sequence or click here. Obviously, 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

    Step 7. Spliced alignment using homologous proteins

    From blastx output, choose several homologous genes and run genewise for each one separately, again. Observe the gain of accuracy as long as the homologue is closer to the original human protein:

    Step 8. Using protein homology information: GenomeScan

    Protein homology information can also be used to enhance ab initio predicted exons supported by blastx HSPs as in the case of GenomeScan and geneid improving therefore the final prediction GenomeScan:
    • Connect to the GenomeScan web server

    • Retrieve the protein from the previous blast search

    • Paste both genomic and protein sequences

    • Press the button GenomeScan

    • Check the results. It seems that the first exon has not been detected even using homology information. This is due to the fact that blast programs have a minimal word lenght.

    Figure 6. GenomeScan output: first exon is not correctly predicted probably due to blast length restrictions

    E. Using a genome annotation browser

    Step 9. Golden path archive:
    • Open the UCSC Genome Bioinformatics Site

    • Select the blat link to locate the genomic coordinates of our sequence

    • Paste the DNA sequence in FASTA format (HS307871)

    • Submit the file

    • Click over the first hit: (browser link)

    • Compare the graphical annotation with the EMBL entry of the gene

    • Analyze these different sets of output options:
      Genes and Gene Prediction Tracks,
      mRNA and EST Tracks

    Figure 7. (a) UCSC genome browser representation of the region containing the gene uroporphyrinogen decarboxylase (URO-D) (b) UCSC genome browser representation of the contex (100Kbps) region around the gene uroporphyrinogen decarboxylase (URO-D).

    F. 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
    GRAIL results
    GrailEXP results
    Blastn + human ESTs results
    Blastx + protein results
    Genewise (human protein)
    Genewise (ovis protein)
    Genewise (mouse protein)
    Genewise (rat protein)
    Genewise (Danio rerio protein)
    Genewise (Drosophila melanogaster protein)
    Genewise (Drosophila virilis protein)
    Genewise (yeast protein)
    Genewise (fission yeast protein)
    GenomeScan results

    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. G. Parra, E. Blanco, and R. Guigó. Geneid in Drosophila. Genome Research 10:511-515 (2000).

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

    7. Yeh, R.-F., Lim, L. P. and Burge, C. B. Computational inference of homologous gene structures in the human genome. Genome Res. 11: 803-816 (2001).

    8. 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).

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