To discover the role of this mysterious sequence, we will search for proteins in a database that show high similarity to a translation of Sequence R.

To do this we will use a sequence search engine, BLAST, to search sequences in the database at the National Center for Biotechnology Information (NCBI).

BLASTX uses the genetic code to translate Sequence R, then compares it with every protein in the sequence database.

The BLASTX search may take a few minutes, during which time “Status” on the Web page is “Searching”.

Results will be shown in a long Web page. Scroll down to see a table of results. The best-matching sequence from the database is listed first.

We will now perform another BLAST search to see if Sequence R has a match in the human genome.

This time, we will use BLASTN, which compares a DNA query with a DNA sequence database.

In your results, you will see a good match between Sequence R and the human genomic DNA. However, the mouse (Sequence R) and human sequences differ, due to mutations.

Figure 1 shows evidence of substitution and frameshift mutations in BLASTN results.

A frameshift mutation is an insertion or deletion which disrupts the reading frame of a protein-coding sequence. From this point on, any protein sequence would be scrambled.

A frameshift mutation is strong evidence that the DNA no longer codes for a functional protein.

We typically interact with a computer using a graphical user interface (GUI). This involves using a mouse or a touch-screen, perhaps with some typing.

An alternative is the command line. This involves commands typed into a terminal.

The command line may be more difficult to use at first. However, it has some advantages. We can easily keep a copy of the commands we ran, making it easier to document our actions.

In bioinformatics, both GUI and command line are used every day. For scientific computing, we often use the command line on a Linux operating system.

First, we will use the nano text editor to create a file.

To start editing a new file called test.txt, type the following text, then ENTER

You now see something like a basic word processor. Type some text, for example “hello world”.

To find the file you created, we now will list files on the Linux server. Type the following and press ENTER

You should be able to see the name of the file you created, test.txt, and others. You can display the contents of a text file by using the cat command. Type the text below and press ENTER

DNA sequences are stored in text files. The DNA sequence we used in Task A and B is stored in the file sequence_r.fa
To display the contents of sequence_r.fa type the following text and then press ENTER

Working in the command line allows us to work with very large data files. The file chromosome8.fa contains the DNA sequence of human Chromosome 8.

To display the sequence of Chromosome 8, type the following text and press ENTER

An advantage of working at the command line is we can combine commands into a short program (script), and run them all at once.

We provide a script mutation_checker.sh, which allows you to search for GULO in animal genomes.

Type the following text (all in one line), then press ENTER

Then, select the animal(s) you would like to analyse from the numbered list, for example if you would like to analyse human and dog type:

and press ENTER

mutation_checker.sh will run BLAST to compare Sequence R to the animal(s) you choose.

Then, mutation_checker.sh checks if the BLAST alignment contains any insertions or deletions (indels).

If there are no indels, we can be confident that the GULO gene is coding for a functional enzyme, and that this species can produce it's own Vitamin C.

If there are indels, it is likely that there has been a frameshift mutuation, and that this species does not have a function GULO gene, and that this species can't produce it's own Vitamin C.