In this section we use several gene prediction programs on a particular genomic DNA sequence. For each of these programs we obtain a prediction of a candidate gene and we will analyze the differences between predictions and the annotation of the real gene.

The programs we are going to use are geneid, genscan and fgenesh, which are available through a web interface. In these, and in many other tools in the web, we access a form where we can paste, or submit, the sequence we want to analyze, and then we press a button in the form that starts the computing process in some computer where the program runs. Once this process is finished, we get a new page in our browser with the results, which in this case should be a predicted gene.

We are going to work with the sequence HS307871, which is stored in FASTA format. This sequence contains one gene, annotated in the following EMBL and NCBI records. Try to identify in these records the different pieces of information related to the annotation of the gene.

In order to use geneid follow these steps:

1. Connect to the geneid server by following this link.
   
   
2. Paste the DNA sequence.
   
   
3. Select organism (human).
   
   
4. Run geneid with different (output) parameters:
   • Searching signals: Select acceptors, donors, start and stop codons. For each type of signal, try to find the real ones.
   • Searching exons: Select All exons and try to find the real ones.
   • Finding genes: You do not need to select any option (default behavior).
     
     
5. Compare the prediction with the real annotation.
   • By taking a look to the graphical representation of the predicted sites and exons.
   • By inspection of the output and the EMBL/NCBI record.
   • By taking a look to the graphical representation of both, the output and the EMBL/NCBI annotation in this link.
     
     

In order to use genscan follow these steps:

1. Connect to the genscan server by following this link.
   
   
2. Paste the DNA sequence.
   
   
3. Select organism (vertebrate).
   
   
4. Compare the prediction with the real annotation.
   • By inspection of the output and the EMBL/NCBI record.
   • By taking a look to the graphical representation of both, the output and the EMBL/NCBI annotation in this link.
     
     

In order to use fgenesh follow these steps:

1. Connect to the fgenesh server by following this link.
   
   
2. Paste the DNA sequence.
   
   
3. Select organism (human).
   
   
4. Compare the prediction with the real annotation.
   • By inspection of the output and the EMBL/NCBI record.
   • By taking a look to the graphical representation of both, the output and the EMBL/NCBI annotation in this link.
     
     

Improve the prediction from some confirmed evidence.

1. Below the text box where we pasted the DNA sequence, we can find a text box where we can paste evidences, which should consist of one or more exons (in GFF format) that are, e.g., experimentally confirmed.
2. In this case we are going to paste as evidence the first exon which has not been predicted. Select and copy the GFF line corresponding to this exon contained in this file.
3. Paste the line into the evidences text box, and run again geneid on the sequence.
4. Compare the result with the real annotation. What has changed from the previous prediction?
   
   

1. Connect to the MetaGene server
2. Fill in sequence name and e-mail address (not compulsory)
3. Paste DNA sequence
4. Run MetaGene
5. Wait for a couple of minutes, monitoring the search process
6. Check Current Search Results

We can see the annotation of the gene together with the three predicted genes by geneid, genscan and fgenesh by following this link.

Go to the page where we saw the NCBI record, click on the link CDS, and, next to the Display button, unroll the menu box and select the display option FASTA. Now press the button Display, and we will obtain the protein-coding DNA sequence of this gene in FASTA format.

Select the entire sequence (first line is not necessary) and go to the UCSC genome BLAT search by following this link. In the big text box, paste the coding sequence we just copied, and press the Submit button on the top-right corner of this page.

The result is a single match, where we find two links, browser and details. Visit first the details link and try to understand the the information provided there. Then go backwards and visit the browser link where we will see where this gene is located within the Human genome, as well as other annotated information as EST spliced alignments, etc.