Practical - Exploring sequence database

NCBI - Interrogation of databases by Entrez

Use access number

Select Nucleotide on the NCBI website and search for the sequence NC_045512.

Exercise 1 What is it? How long is the genome?

Exercise 2 Consult the Comment field. Which bank does this sequence come from? Is it reliable? What sequencing technology was used? What can you say about this entry compared with the initial GenBank entry (1st sequence of the SARS-Cov-2 genome)?

Look for the coding region corresponding to the N protein (nucleocapsid phosphoprotein).

Exercise 3 What do you think of the composition of this protein? What problems might such a sequence pose in a similarity search?

Exercise 4 Display the genome sequence in fasta format. How does this format compare with the RefSeq format?

Search by annotation (Filters/Advanced)

Search Nucleotide for sequences containing the BRCA1 gene for Homo sapiens, using filters or the advanced search.

Exercise 5 Why are there several sequences in RefSeq? in Genbank?

Among all the results, one of the sequences is a fragment of the gene in a breast cancer patient. To find it more easily, search for KT152889 with the search bar.

Exercise 6 Quickly position the annotated gene elements on a drawing. What is the position of the variation on the sequence? What does it correspond to?

Among the features, the sequence source provides a cross-reference to NCBI’s Taxonomy bank. Follow this link.

Taxonomy bank

Exercise 7 What human subspecies are listed in the Taxonomy bank?

Exercise 8 What other organisms are listed in the Homininae ?

Exercise 9 What orders are closest to the primate order? Which has the most available proteins?

Exercise 10 How many genomes are available in the Genome Bank for Animals (Metazoa), Fungi, Viridiplantae and Eukaryotes? Comment.

Protein sequence search in UniprotKB

Go to the UniprotKB bank.

Advanced search fields

Exercise 11 How many human protein sequences are listed in UniprotKB? in SwissProt?

Exercise 12 Search for the protein associated with the BRCA1 gene in human. Compare the results. Which is the best entry?

Example of a SwissProt entry

Search for the MUC1_HUMAN protein

Exercise 13 Has the existence of this protein been established (see Protein existence at the top of the page)?

Exercise 14 Compare the default format, the text format and the fasta format (available in Download).

Exercise 15 How many isoforms are described for this protein (see Sequences & Isoforms)? Align the isoforms and see how they differ. For that, click on align isoforms and launch the alignment.

Function - GO annotations

Exercise 16 The Go ontology can be used to describe proteins in 3 ways. Which are they?

Exercise 17 Follow the link to the entry “negative regulation of intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator”. What is it? What type of GO is it? How often is it used to describe proteins?

Exercise 18 Are there any synonyms for the term GO?

Structure

Exercise 19 What can you say about the structures obtained experimentally for the protein?

Exercise 20 What can you say about the reliability of the AlphaFold prediction for this protein?

Tab External links (top of page)

Sequence databases

Exercise 21 Why are there so many more links to EMBL/GenBank/DDBJ than to RefSeq?

Families and domains in INTERPRO database

Display the graphical representation (View protein in InterPro).

Exercise 22 How many distinct protein domains are there in this protein?

Domain: IPR000082 SEA domain

Exercise 23 From which banks do the 3 signatures associated with this InterPro domain originate?

You can get more information by passing the mouse over the colored bars, to open the domain in the associated bank, click on the corresponding link on the right.

Exercise 24 Follow the link to the associated PFAM signature. Display the signature (click on the Profile HMM tab). Comment the signature.

In a new tab, search for the BRCA1_HUMAN protein in InterProScan.

Exercise 25 How many distinct protein domains are there in this protein?