Biocomputing

Gibson Group

EMBL

The aim of this practical is to get hands-on experience with three web servers that can help to investigate eukaryotic genomic sequence - especially human. We are focussing particularly on the human genome servers since the genome is largely completed now. The Ensembl server is a collaboration between the EBI and the Sanger Centre to provide "real time" human genome annotation as the sequence is generated. The annotation is automatically generated from a combination of gene prediction, encoded protein homology and EST matches: obviously it is not going to be perfect. The Human Genome Browser developed in Santa Cruz is another interface useful for getting an overview of the genome. Ensembl and the Genome Browser have links to each other. The third server that we want to introduce is one that we gave developed at EMBL. The Gene2EST server will allow you to submit gene-size queries (if need be >100,000 bp) to search the EST databases and provides both graphical and alignment output: in favourable cases - i.e. plentiful ESTs - Gene2EST can rapidly give a good description of a gene structure, including alternaitve splicing.

Exercise 1. Querying Ensembl with a protein sequence

Ensembl can be queried by keyword or by sequence similarity, depending what your needs are. It provides quite a flexible combination of protein/DNA query/databases. For example, it is well suited to probing with ESTs of interest. Today we are going to use a protein sequence as query.

• In a Netscape window, go to SRS and click Start.
• Click the SWISSPROT Box and then click Continue.
• Type SRC_HUMAN in an ID box and click Do Query.
• Click on the SWISSPROT:SRC_HUMAN entry. This entry contains the sequence for human SRC oncoprotein.
• Click on the Save button.
• Set Use view to FastaSeqs then click SAVE.
  • The sequence is now in a suitable format.

Querying Ensembl

• Click to Ensembl.
• Click to the BLAST page.
  • Cut and paste the sequence into the box.
  • Select the database of Ensembl Confirmed peptides.
  • Check that the protein version of BLAST will run.
• Click the search button.
  • The search should take a few seconds to run.
• Examine the BLAST output:
  • Which chromosome has the top hit?
  • What happens when you point the mouse at a coloured chromosomal band?
    • Now click on one with the middle button.
      • What do you get?
  • Is the top hit the Src sequence?
  • Check the second best hit (use the middle button):
    • Is it less than 70% identical to the query?
    • Is it on chromosome 6?
• Click on the link from the alignment of the top hit. Ensembl will give a graphical representation.
  • Which part of the overview provides the detailed view?
    • Try zooming in and out.
• Look at the sources of annotation data:
  • Which of (a) gene prediction, (b) protein sequence, (c) EST match, are used to find genes?
    • (As needed, use the middle button to click on the items).
  • What information does a Transcript link provide?
  • How many known genes are within 50,000 bases either side of Src?
  • Is Src on the + or - DNA strand?
  • How consistent are the different methods to find genes?
    • Do they agree well or badly?
    • Are any exons missing in some of the methods?
  • Are there many repeat elementsin this region?
• Click on an Ensembl gene.
  • Review the information supplied.
• Click on Transcript > supporting evidence:
  • Are there any database proteins that matched the gene?
  • Are all the exons supported?
  • Are the 5' exons better supported than the 3' exons?

Notes:

Exercise 2. Using the Genome Browser to evaluate whether two chromosomes are related

The Human Genome Browser is another useful interface to the human genome. We can easily click between Ensembl and the Browser and back again: You may find that some things are easier in one of these browsers than the other - though this may change over time as they are both being rapidly developed. The exercise we are going to do now could be done in Ensembl too but was easier in the Browser as we tried it - but next year who knows?

There is currently some controversy whether the vertebrate genome underwent tetraploidy or even octaploidy a long time ago in a common ancestor: We know that there are eight Src paralogues in the human genome. One of them, HCK is close to Src on chromosome 20: this gene pair was formed by a tandem duplication. Another, LYN, is found on chromosome 8 so might have arisen by genome duplication. We are going to see whether there are other genes close to Src and LYN that exhibit synteny. Syntenic chromosomal regions possess similar genes.

Going from Ensembl to the Genome Browser:

• From Ensembl, click on Jump to UCSC.
  • We will get a new window with another graphic of the Src genomic region.
• Click on coverage:
  • This expands to reveal the clones
    • If they are all black they are fully sequenced.
  • You can set coverage back to dense with the controls below.
• Now adjust the figure size:
• Set pixel width to 1000 and click jump.
  • If need be fine tune the size some more so it fills your screen nicely.

Finding a set of genes linked to Src:

• Use the zoom out buttons to get a view with about 20-30 genes.
  • As needed, customise the plot to minimise unwanted info such as RNA genes.
• Work rightward of Src using the move buttons to establish the gene order for:
  • BIG2, EYA2, HNF4A, MATN4, NCOA3, PKIG, SRC and STAU
    • Draw out the gene order on paper.
    • Are any of these genes fragmented?
  • Also note the position of any major gaps that are present between the genes.
    • Mark these on the paper.

Finding a set of genes linked to LYN:

• Open the Genome Home link with the middle mouse button.
• Click on the Browser link.
• Type LYN into the genome position box and submit.
• Click on the top matching link to get the graphical display.
• Set up the figure as before to view about 20-30 genes.
  • From the coverage and gap summaries, is the sequence as good as for the SRC region?
• Work rightward of LYNusing the move buttons to establish the gene order for the paralogous genes to the list above.
  • (The genes will have similar names with different numbers.)
  • Also note the position of any major gaps that are present between the genes.
    • Mark these on the paper.
• We can now compare the two regions:
  • Is the gene order conserved?
  • What is the longest conserved gene order?
  • Are there any inversions of gene order?
  • Do you think the inversions are real or due to error in the assembly and mapping?

Notes:

This exercise shows quite well the current state of the human genome sequence. There are some nearly finished regions and some less good ones. Also there seem to be some gaps between clone contigs - so the contigs could invert, changing gene order. Also the sequence contig order within a clone is not always correct - although the project does try to order them - so could mess up local gene prediction. I.e. the fine mapping is not reliable in the rough sequence. It will be interesting to see how much the gene order changes in these two regions in the future and if they get a bit more like each other.

Exercise 3. Using ESTs to reveal gene structure with Gene2EST

The BLAST2 program is well suited to EST detection. Because it tolerates small gaps in alignments, it deals well with sequencing errors in the ESTs. However it is painfully time-consuming to work through the output, especially if there are many 100s of matches. Furthermore, most BLAST2 servers will not allow the user to submit large query sequences. We have tried to address these deficiencies with Gene2EST. The Gene2EST server presents BLAST2-derived output in a way that allows the user to rapidly evaluate the results. A graphical display (viewed with Artemis) provides an overview of the results, while an alignment of the ESTs on the query sequence lets the user follow up the exons in detail.