Back to bioinformatics- getting to know the software is the turning point- once Graham had re-taught Clare and I the steps involved in Blasting sequences and then aligning them- we felt we were getting somewhere.
Graham is an absolute expert-he explains the role of probability and statistics in the running of MEGA and BLAST.
In a straight forward way this is how you begin to create a phylogenetic tree using DNA sequences from a bat gene:
1.DNA sample you purified is sent away for sequencing.
2. The sequence is now inputted into BLAST where it is compared against thousands of other sequences. The sequence is identified and linked to similar organisms- in this case there is a 100% match to a particular Myotis species of bat. Any other similarity to the same gene or sequence in other bat species is also indicated.
3. You want to create a phylogenetic tree- a diagram which shows the evolutionary pathway-was this sequence once the same in a common ancestor and is it possible to trace back a stage or branch at which it evolved or changed.
4. Select the search results listed in BLAST and use Clustal X to align the sequences- you are using a programme to position the sequences from each of the species of bat to gain the best fit or match possible. Too large a task without Clustal X.
5. Load the alignment file into MEGA- this will then use the aligned sequences to create a phylogenetic tree- various tools to analyse the reliability of your tree are used in MEGA- this is known as boot-strapping.
6. A tree is produced with numbers at each Node- over 50% the connection is somewhat likely but really 80% upwards is sought.
So with much ado we created our first phylogenetic trees.