Analysing and editing sequence chromatographs with Geneious - Answers

Question 1: You will notice that the sequence stops abruptly after about 340 bases. Why is this?
> When DNA is amplified using PCR a fragment (or amplicon) is generated of a specific length. In this case the H15149 and L14841 generated an amplicon of aproximatly 340bp. When this amplicon undergoes Dye Terminator Sequencing (using the H15149 primer as a sequencing primer) bases will be added to the 3' terminal of the primer as it extends. However, bases cannot be added once the polymerase reaches the "end" of the amplicon - as there is no DNA left to act as a template. This is why the chromatograph stops abruptly. Another way to think of this is that you are copying a page of text and once you reach the end of the text it is impossible to copy anymore.

Question 2: How many bases were edited in your alignment? - Give a brief justification as to why these edits were required.
> Once you have removed the primer sequences and ambigious base reads at the start of the sequence - then aligned your sequences, two bases will stand out in the alignment that don't "agree" when the two strands of the DNA are sequenced independently. The exact position of these bases depends on how much you trimmed the chromatographs - but there is one at either end of the alignment. If you study the chromatograph traces carefully you will see in both cases that the computer program that "calls" the bases has made an error (due to peak spacing) and inserted two bases that are not real. In both cases the bases should be deleted so that the sequence of the forward and reverse sequences "match". The manual editing of "raw" sequence data such as this is an important step in ensuring that you have generated a sequence free of errors and also emphasises the imporatnce of sequencing PCR products in both directions.

Question 3: From information in the GenBank file, what is the common name of the closest GenBank match to the sequence we have obtained from the bear bile crystals?
> The sequence "blasts" 100% as a subspecies of black bear - if you downloaded the Genbank file EF076773 and if you click on the text view tab witihn you the file you will see that the this sequence is a 100% match with Ursus thibetanus formosanus, the common name for which is the formosan Black Bear. Other subspecies of black Bear are also 100% matches and the sequence we obtained from the Bear Bile flakes could just as likely originated from one of these subspecies.

Question 4: The WikiPedia link for Ursus thibetanus formosanus can be found here. From the information on this page - what is the conservation status of this subspecies?
> The formosan Black Bear is subspecies of the Asiatic Black Bear - it's conservation status is "endangered".

Question 5: What is the closest relative to the DNA sequence isolate from the bear bile sample based on your phylogeny? (Note: Selenarctos thibetanus and Ursus thibetanus are the same species that has undergone a taxonic change).
> Selenarctos thibetanus is the closest relative on the UPGMA tree (the Asiatic Black Bear) - you will note that the match is not exact (otherwise the tips of the tree would be closer) - this is due to the fact that we are likely comparing two different subspecies of Black bear - as evidenced by the Blastn results.

Question 6: Bootstrap values (or consensus support) on nodes give a measure of how stable the phylogentic tree is. Display the consensus support values on the tree (this can be located under the display options in the "Show node labels" menu. What is the % support that groups the bear bile sequence with its closest match on the tree?
> Since bootstrapping randomly resamples sites within the bear alignment the answer to this question will vary slightly do to the stochastic nature of resampling data - however the number should be approximately 99%.