Exercise solutions

 

Exercise 1 – Orthologues, paralogues and gene trees for the human BRAF gene

(a) Go to www.ensembl.org, choose human and search for BRAF. Click through to the Gene tab view.

On the gene tab, click on Orthologues in the menu on the left to see all orthologues.
Of the 23 primates in Ensembl, there are 1-to-1 orthologues in 20 primates and 1-to-many orthologues in one (chimpanzee).

The percentage of identical amino acids in the Tarsier protein (the orthologue) compared with the gene of interest, i.e. human BRAF (the target species/gene), is 95.26%. This is known as the Target %id. The identity of the gene of interest (human BRAF) when compared with the orthologue (Tarsier BRAF, the query species/gene) is 94.52% (the query %id).

Note that the difference in the values of the Target and Query %id  reflects the different protein lengths for the human and tarsier BRAF genes.

(b) There is more than one way to get to the answer.

Option 1: Go to the Orthologues page and click on the marmoset orthologue (ENSCJAG00000013536) to open the Gene tab. 
Click Genomic alignments at the left, then the Select an alignment button.
Select All Alignments -> Pairwise -> Primates -> Human and click Apply. Choose Block 1 to get the largest block of aligned sequence.
Click on Display full alignment at the bottom of the page.
The red sequence is present in exons, so there is a gene in both species in this region. You can find where the start and stop codons are located if you Configure this page and select Codons: START/STOP codons

Option 2: Go to location tab of the marmoset BRAF gene and then click on Region Comparison view at the left. Click on the Select species or regions button and select All Divisions ->Primates -> Human, then click Apply. You should see an alignment between the human BRAF gene region and the BRAF gene region for the marmoset. 

(Note: To see a blue line connecting homologous genes in the Region Comparison view page, click on Configure this page and under Comparative features, tick the box Join genes and close the menu. Zoom out on the location view to see blue lines connecting all homologous genes between marmoset and human genes in that region).

Exercise 2 – Zebrafish orthologues

(a) Start in the Location tab (region in detail) for sardh. Click on Alignments (Image) at the left, and select the 11 fish EPO_LOW_COVERAGE alignment in the view and click Apply.

The zebrafish, cave fish, cod, stickleback, medaka, amazon molly, platyfish, tilapia, fugu, tetradon and spotted gar are shown in this region. All species except the medaka show a gene in the aligned region. This can also be seen in the Alignments (text) page (the exons are highlighted in red).

(b) You can export the alignments from the Alignments (text) pages in the Location tab. Click on the blue Download alignment button at the top of the image, and choose CLUSTALW.

(c) Click on Region in detail in the left hand menu. Click on Configure this page and turn on the Multiple alignments, Constrained elements and Conservation score for 11 fish EPO_LOW_COVERAGE, all under Comparative genomics.

The 11 fish EPO_LOW_COVERAGE just shows that the region for the sardh gene can be aligned for these eleven species of fish. The 11 way GERP elements and scores tracks show where the conserved sequence is located in the alignment.

Regions with higher conservation match up with exonic regions (exons tend to be highly conserved) of the gene.

Click on the track name 11 way GERP elements, then the  (information button) and the link GERP conservations scores to read more about constrained elements (or any other data track).

Exercise 3 – Synteny

(a) Change the species to Dog using the drop-down menu below the image and click Go.

Yes, there are multiple syntenic regions in dog to human chromosome 3, which is in the centre of this view. Dog chromosomes 6, 20, 23, 31, 33, and 34 have syntenic regions to human chromosome 3.

(b) Scroll down to the bottom of the page.

There is a homologue in dog of human RHO. Click Centre on gene RHO to compare the genes between human and dog in this syntenic block.

Exercise 4 – Whole genome alignments

(a) Go to the Ensembl homepage www.ensembl.org.
Select Search: Human and type brca2 in the search box.
Click Go and then on 13:32315474-32400266:1 below BRCA2 (Human Gene). This link takes you to the Region in detail view of the gene location.

You may want to turn off all tracks that you added to the display in the previous exercises as follows:
Click Configure this page in the side menu.
Click Reset configuration.
Close the menu.

(b) Click Configure this page in the side menu.
Click on Alignments (text) under the Comparative Genomics menu. Select All alignments -> Multiple -> 32 amniota vertebrates Mercator-Pecan and click Apply.

Yes, the degree of conservation does reflect the evolutionary relationship between human and the other species. E.g. the highest degree of conservation is found within the primates cluster, while the degree of conservation between human and birds is low.

Especially the exonic sequences of BRCA2 seem to be highly conserved between the various species. This is to be expected because these are likely to be under higher selection pressure than intronic and intergenic sequences.

(c) Click on the Location tab to go back to the main view.
Click Configure this page in the side menu.
Click on Conservation regions under the Comparative genomics menu.
Select Conservation score and Constrained elements for 70 eutherian mammals EPO-Low-Coverage, Conservation score and Constrained elements for 32 amniota vertebrates Mercator-Pecan.
Close the menu.

Both the 32 way GERP elements and scores track largely correspond with the data seen in the pairwise alignment; all exons of the BRCA2 gene show a high degree of conservation. Note the UTRs which are not conserved).

(d) Click on a constrained element (brown block).
Click on View alignments (text) in the pop-up menu.
Click Configure this page in the side menu.
Select Show conservation regions.
Close the menu.

The conserved regions in the alignment at the bottom of the page will be highlighted in light blue.

Extra Exercise 5 – Pan-taxonomic Compara

(a) Go to bacteria.ensembl.org and start typing Neisseria meningitidis Z2491 in the search box to find the genome. Select Neisseria meningitidis Z2491, (TaxID 122587) from the drop-down menu. Then type NMA2179 in the search box on the homepage of Neisseria meningitidis Z2491 to find the gene. Click on the Gene ID NMA2179 in the search results to open the Gene tab.

This gene encodes for the dihydrofolate reductase. You can find this information in the description of the gene at the top of the page and under GO: Molecular function in the menu on the left.

(b) Click on Orthologues under Pan-taxonomic Compara in the side menu to see all orthologues.

There are 68 orthologues in bacteria. Tick the box in the table at the top to show details for bacteria only.

Now click on the header Target %id of the table at the bottom to sort the table. The Target %id is defined as the percentage of the orthologous sequence matching the Neisseria meningitidis Z2491 sequence.

The maximum identity is 48.47% and found between the protein of Aeromonas hydrophila subsp. hydrophila ATCC 7966 and Neisseria meningitidis Z2491. 

Likewise, Query %id is defined as the percentage of the Neisseria meningitidis Z2491 sequence matching the sequence of the orthologue. The maximum identity is 50.00% for Azotobacter vinelandii DJ.

(c) Thirteen vertebrate species have predicted orthologues for NMA2179. Tick the box in the table at the top to show details for vertebrates only.

Now type human in the box above the table at the bottom to find the orthologues in human.

There are two orthologues predicted in human, both with a 1-to-many relationship with NMA2179.

You can click on ENSG00000228716 next to DHFR to open the Gene tab for this human gene. Note that DHFR indeed encodes for the human dihydrofolate reductase.

(d) Click on Gene Tree in the side menu to view the image of the tree.

Click on the rightmost button above the image (it will display Download data from this image when you mouse over it) to open a pop-up window.

Select Newick format and click the Download button.