Lab Assignment 9

Lab Assignment 9
BIO 224 Laboratory
CSU, Sacramento
September 3, 2004
Lab Assignment 9
(15 pts total; due Wed Nov 17th by midnight)
1. Go to the website for Mega to install the program onto your local computer.
(http://www.megasoftware.net/ ). Go to the download for mega 4 (not the beta
version though) and enter your information for the free download. Follow the
steps to download it onto your computer.
2. Go to the “Tutorial on How to Use MEGA” and click the tutorial “Aligning
Sequences” to learn to create multiple sequence alignments. Perform Ex 1.0.2 to
Ex 1.1.5 to learn how to open up mRNA sequences. (Note: there seems to be a
misprint in their instructions- the file seems to be hsp20.meg and not hsp20.fas).
3. Once you finish with Ex 1.1.5, open up the file Drosophila_Adh.meg using the
same protocol as above (note: you are to open up the aligned data file in the
MEGA program itself- can't do the analyses in the Alignment Explorer program)
When you have opened your aligned file into the MEGA program, go to the Data
drop down menu and select "Data Explorer" to view the mRNA alignment.
Then go to the highlight drop-down menu and answer the following questions:
a. how many 0-fold degenerate sites are there?
b. how many 2-fold degenerate sites are there?
c. how many 4-fold degenerate sites are there?
d. Explain why there are so many 0-fold degenerate sites than the others
e. Explain why there are more 4-fold degenerate sites than 2-fold sites
Go to the Data drop-down menu and translate the sequences, then go back to
the highlight menu to answer the following questions:
f. How many conserved sites are there?
g. How many parsimonious informative sites are there?
h. How are parsimonious informative sites used for tree building?
4. Then, use the next Exercise within the same tutorial “Aligning Sequences” (Ex
1.2.1 and on) to upload your human mRNA sequence and the mRNAs for the
homologs you identified in question #1 of assignment #7. When you upload your
mRNA sequences (step 1.2.4), be sure to upload only the coding sequence (cds)
portion of the mRNA displayed in Fasta format. Also, translate your data into
protein sequences prior to aligning them. Then save your data as a “meg” file
extension as the tutorial states.
(note the question from assignment 7 is re-listed for you below).
[Question 1 from assign 7: Using your assigned human protein/mRNA sequence,
find one other mammalian sequence in addition to your mouse & rat sequences
(one outside of the rodent family) and also two sequences from non-mammalian
species.]
BIO 224 Laboratory
CSU, Sacramento
September 3, 2004
Save the alignment file and a mega file for you to perform the rest of the analysis.
a. How many conserved sites in the protein alignment do you have?
b. How many parsimonious sites are there?
c. Go to the Distances drop-down menu (the main program, not the
“Sequence Data Explorer” window) and “compute the pairwise
distances” for the protein using the substitution model for the pdistance (need to go to the Substitution Model part of the dialog box
and choose the amino acid setting and p-distance parameter)– paste
the table below (use the export/print function within the file drop down
tab)
[definition of p-distance (Nucleotide) from MEGA:
“This distance is the proportion (p) of nucleotide sites at which two
sequences being compared are different. It is obtained by dividing the
number of nucleotide differences by the total number of nucleotides
compared.”]
d. Perform the same analysis as part c except change the substitution
model to “Poisson correction” – paste the table below
[definition of Poisson Correction (PC) distance from MEGA:
“The Poisson correction distance assumes equality of substitution rates
among sites and equal amino acid frequencies while correcting for multiple
substitutions at the same site.”]
e. Compare the two tables you generated in c and d and explain why they
are different?
5. Tree analysis
a. Go to the Phylogeny drop down menu and then select the "Bootstrap test of
Phylogeny"and then the neighbor-joining tree analysis option. Perform the
neighbor-joining tree using the translated protein dataset and the Poisson
correction (meaning use the using the substitution model based on amino:
Poisson correction) with a bootstrap analysis (500 replicates; note that this is the
default setting). Paste the bootstrap consensus tree below. What do the
numbers at the various nodes mean?
b. Use the branch swapping function on the left of the tree and flip around
several of your branches to show yourself that these are still the same tree
although they may look different. Paste one of these and describe what these
superficial differences are between the trees.
c. Go back to the Phylogeny drop down menu and then select the "Bootstrap test
of Phylogeny" again except this time select the maximum parsimony tree
analysis. Choose the settings to be based on your protein alignment by going to
BIO 224 Laboratory
CSU, Sacramento
September 3, 2004
the "->Codon Positions" option and then select "Translated Amino Acid
Sequences" under the Data to Analyze section. Perform bootstrap analysis of
500 replicates (note: if program shuts down, then perform bootstrap with less
replicates- for example 100). Paste the consensus tree below.
d. Does the branching order match between the neighbor-joining and maximum
parsimony trees? Describe the tree differences. Why might these two
phylogenetic analyses generate different branching orders? (note: answer this
question theoretically if you trees do happen to have the same branching order)
6. Perform the relative rate test (found under the phylogeny drop-down menu) on
all three of your mammalian mRNA sequences using an appropriate outgroup.
(meaning you will need the following tests, human with mouse, human with rat,
and mouse with rat). Why did you use this outgroup? What were the results for
all three relative rate tests? Even if your relative rate tests did not show any
differences, what does it mean if you did find significant differences between
species comparisons?
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