Practical - Analysis of eukaryotic sequences

Ensembl genome browser

An exome from a patient with a retinal disease has been analyzed. The sequence of a mutated exon (109 bp) is available on Moodle.

Go to the Ensembl genome browser.

Mapping an exon sequence to the human genome

Open the BLAST/BLAT page.

Exercise 1 Is BLAT relevant to map our transcript on the genome? Why doesn’t use BLAST?

Perform the search.

Exercise 2 What is the localization of the exonic region (chromosome, strand)? What is the corresponding gene?

Display alignment.

Exercise 3 Which variation(s) are observed on the patient exon compared to the reference genome?

GPR179 Gene

Select the gene.

Isoforms (splice variants)

Exercise 4 How many protein coding transcripts are associated to this gene?

Exercise 5 According to the flags of the transcript table, which transcript is the more reliable?

Exercise 6 How many exons are present in this transcript?

Protein: function, GO annotation, and expression

In a new window, search for the protein encoded by this gene in SwissProt.

Exercise 7 What is the protein function according to SwissProt?

Exercise 8 GO annotations: look at the list of synonyms associated with the GO molecular function of the protein. Does standardization make sense in this case?

Exercise 9 In which tissue is the gene expressed?

Phenotype (Ensembl, Gene tab)

Exercise 10 Which phenotype(s) is linked to this gene? Is it consistent with the exome analysis?

Annotated genomic region

Display the GPR179 genomic region (region in details). Zoom out to have a good overview.

Exercise 11 To which exon does the patient sequence correspond ?

Gene prediction (ab initio + similarity)

Configure this page (left panel) to display:

  • Genes and transcripts / Prediction transcripts: Genscan predictions
  • mRNA and protein alignments
    • mRNA alignments: display human cDNAs
    • protein alignments: display Proteins (mammals) from Uniprot

Exercise 12 Compare information provided by Genscan (ab initio prediction), cDNA and protein alignments.

Repetitive elements

Configure the page to display repeats (Repeat regions/All repeats) and conserved elements using Comparative genomics:

  • Conservation regions/Constrained elements for eutherian mammals
  • BLASTz/LASTz alignments with Chimpanzee, Mouse, crocodile, Zebrafish.

Exercise 13 What is conserved in these different species?

Retrieve the genomic region with 10000 bp 5’ and 3’ flanking sequence using Export data (fasta sequence, unmasked, text) or use the genomic region available on Moodle.

Analyze this sequence with RepeatMasker using default parameters (don’t hesitate to refresh the result page).

Exercise 14 Which type of repeated element is the most represented in the genomic region?

Exercise 15 Display the annotation output and have a look at the 1st Alu repeat in the list. Can you identify a typical feature in this Alu repeat?

Exercise 16 To get a concrete idea of the importance of repetitive elements in the human genome, display the masked region.

Regulation (Optional)

Exercise 17 According to regulatory features, can we have information about GPR179 promoter? How do you explain it?

Exercise 18 Is it the same for the neighbor gene SOC7?

Display the same region in the UCSC genome browser (by following the hypertext link) and display CpG islands.

Functional analysis of a gene list

Another patient with a retinal disease exhibits several mutations including a mutation in a gene of completely unknown function. We only know that this gene is co-expressed with other genes (list available on Moodle).

GO enrichment analysis

Use the website of the Gene Ontology.

Perform a functional enrichment analysis (Biological process ontology) of this list of genes in comparison to human genes.

Exercise 19 What is the meaning of the different columns in the result table?

Exercise 20 Sort the results according to the P Value. Is the mutation a good candidate to explain the retinal disease of the patient?

Network analysis with STRING

Use the website of String.

Visualize the functional links between the genes in the list with STRING (using the “Multiple proteins” form).

Exercise 21 Which main types of links are observed between the genes of this list?

Exercise 22 Analyze the network (Analysis). Are there more links than expected in this list?

Exercise 23 Is the STRING analysis in agreement with the previous one?