Biocomputing

Gibson Group

EMBL

The Human genome consists of 3 billion base pairs of DNA. It is hard to remember them all - especially, I find, as you get older. So it is obvious that computers are needed to store and investigate the data explosion generated by modern biology. Fortunately, the growth of the internet means that much of this data can be made accessible to many scientists who cannot afford big computers. In fact, not just scientists but anyone with a Mac or PC can access publicly available on-line resources. Today we are going to investigate both protein and genomic sequence using web servers that you could connect to when you get back home. The exercises we've chosen are based on ones that we use for teaching EMBL researchers. If they are too difficult, provide some feedback as we might be doing this again next year.

In the first part, we are going to find out about the protein responsible for the inherited disease Marfan's Syndrome. In the second part we will investigate some human genome sequence 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.

Getting Started

We are going to use PCs running the LINUX operating system but we only need to know enough to login and start netscape for browsing the web.

• Login with the Username and the password that we will supply you.
• Start the KDE Desktop by typing exec startx.
• Launch Netscape from the second desktop icon at lower left.
• Check the Netscape preferences to ensure that javascript and CSS (cascading style sheets) are enabled.
• Load this course page into the browser.

Now we are ready to start surfing...

Part 1. Using biological databases to find out about the Marfan Syndrome protein

Modern molecular genetics has revealed the genes which may get disrupted in many human inherited diseases. In some cases, knowing the gene provides helpful insight and leads to treatment, in other cases it is less helpful (so far). We'll take a look at one example using various database resources.

Getting a sequence from a database:

• In a Netscape window, go to SRS and click Start.
• Click the SWISSPROT Box and then click Continue.
• Type marfan & syndrome in a box and click Do Query.
  • How many entries have been found?
  • Which entry is for Marfan syndrome?
  • Is the other entry associated with an inherited disease?

SRS is a WWW tool to retrieve information for biological databases. We have retrieved an entry for a protein sequence from the SWISSPROT database. The entry includes the protein sequence, information about the protein and links to some other useful databases.

Examine the database entry:

• What is the name of the protein?
  • Which cellular compartment is it found in?
  • What is its function?
• Mutations (variations) which cause MFS are listed:
  • Are they restricted to a part of the protein or widespread in the sequence?
  • Is there any amino acid type that is especially often affected?
    • (Note the amino acid sequence is given in single letter notation.)
• Does the protein have a single or multidomain structure?

Using the links in the entry:

• Note: Click links with the middle button and a new window will open automatically.
• What do you get if you click on a MEDLINE link?
• What do you get if you click on a MIM link?
  • What is the difference between the MIM links?
  • Using the MIM entries, find out:
    • When was MFS first described?
    • What is the main life threatening complication?

The links took us to two other database resources, PubMed which collects abstracts of medical and biological literature and OMIM (Online Mendelian Inheritance in Man) which catalogues medically important human genes and gene disorders. These are very useful to researchers. We could have clicked on other links that would have taken us to DNA, Protein Structure and Protein Domain databases.

Checking for domains in the sequence:

• Select the protein sequence and copy it using the Netscape menu.
• Using the middle button, click to the SMART domain server.
  • Click the boxes for PFAM domains and signal peptides.
  • Now paste the sequence into the sequence box.
  • Click the Sequence Smart button to launch SMART. The calculation may take a minute or two.
• Examine the output:
  • Is there a predicted signal peptide?
    • (Signal peptides are present on proteins exported from the cell.)
  • Are there any other transmembrane segments?
  • How many types of domains are there in the protein?
  • How many times does the less common domain occur?
    • Does SWISSPROT agree?
      • Who is right?
  • How many times does the more common domain occur?
    • Does SWISSPROT agree?
      • Who is right?
  • Do you think this number of domains is typical for protein sequences?

SMART compares a sequence against a database of known protein domains. If the score for a match exceeds a probability threshold, it displays the result graphically. Matching sequences to domains is an imperfect activity: highly divergent domains may not be detected. In fact this is generally true of sequence comparisons and reflects the process of gradual sequence change driven by molecular evolution.

These exercises introduced a few databases and tools available on the web. There are plenty more available for biologists to use, especially at major sites such as the EBI, NCBI and Expasy.

Part 2. Using some human genome web servers

The human genome sequence was published with much publicity earlier this year. Only problem - it isn't quite finished yet! It should take a few more years to tidy up. Even so it is already becoming very useful to researchers. Everyone can access the genome through various academic servers. However, presenting the information available about the genome to the user is very much in its infancy. It can be hard to get at the bits of information you want. We'll have a look at two genome servers.

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.
• Set Use view to FastaSeqs.
• Set a box label from Maintext to ID and type in SRC_HUMAN then click Do Query.
  • (This entry contains the sequence for human SRC oncoprotein.)
  • The sequence is now in a suitable format.

Querying Ensembl with a protein sequence:

• Click to Ensembl.
• Click to the BLAST page.
  • (BLAST is a program to find similar sequences in a sequence database.)
  • Cut and paste the sequence into the sequence 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?
    • (There are big libraries of ESTs, which are simply randomly sequenced mRNAs.)
    • (As needed, use the middle button to click on the items).
  • What information does a red 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?
      • (Remember human genes are highly spliced to make the mRNAs: exons are spliced together while introns are removed and discarded.)
• 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?

Do you know what a tetraploid is? A normal eukaryotic genome is diploid - having two copies of the genome - while a sperm or egg cell is haploid with a single copy. It turns out that many species can easily duplicate complete genomes: they are then called tetraploids. For example wheat is a tetraploid which resulted from the fusion of two wild grass species which happened in prehistoric farming. Tetraploidy gives rise to many redundant genes (genes with overlapping functions) that can persist long after the genome fusion happened. Vertebrates, including humans, have many redundant genes. 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 (related genes) in the human genome. One of them, HCK is close to Src on chromosome 20: this gene pair was formed by a "tandem duplication" within the chromosome. 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 sets of genes.

Getting the Src locus in the Genome Browser:

• Click to the UCSF Genome Browser.
• Click to the most recent data freeze.
• Type in the genome position: chr20:37500000-37800000
• Set pixel width to 1000 and click the submit button.
• Click on coverage:
  • This expands to reveal the clones which were sequenced.
    • If they are all black they are fully sequenced.
  • You can set coverage back to dense with the track controls below.

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 in coverage 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 LYN using 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.

The gene orders for Part 2: Exercise 2 using the December 2000 release of the human genome are:

SRC, HNF4A, PKIG, MATN4, EYA2, NCOA3, BIG2, STAU

LYN, BIG1, NCOA2, EYA1, STAU2, HNF4G, PKIA, MATN2

By the way, LYN is actually a closer relative of HCK rather than SRC, but HCK is further from the rest of these genes than SRC, so we used SRC as the starting point. It will be interesting to see if this corresponds to true gene order due to a chromosomal inversion, as the sequence is improved.

You can find this page at http://www.embl-heidelberg.de/~seqanal/courses/ScienceSchool01/ScienceCourse.html