{1. Find a suitable family of sequences.}

{2. Align your sequences using PILEUP.}

{3. Repeat using different gap penalties.}

{4. Generate UPGMA and neighbor joining trees from your alignment}

{B. Neighbor joining based progressive alignment with CLUSTALW.}

{1. Find out about the CLUSTAL W program.}

{2. Align your sequences using CLUSTAL W, using both default parameters and the "&Toggle Slow/Fast pairwise alignments " option. Report these trees in your notebook and compare them with the ones generated in A4 above. }

{3. Examine the alignment and manually correct poorly aligned regions, remove very large gaps, etc. What changes did you make to your alignment and why?}

{5. Write out your final tree in Phylip (.ph) and GCG (.msf)(alignment) formats for use in the next two sections of this exercise.}

{C. Parsimony trees using the GCG version of PAUP.}

{1. Read the CLUSTAL alignment into the GCG PAUPSEARCH program. Construct a tree using the "Exhaustive tree search" and "Parsimony" options. Make a plot of this tree using the PAUPDSPLAY program. }

{2. Construct a tree using the "Heuristic tree search" and "Parsimony" options. How does this tree compare to the tree in C1?}

{3. Perform a bootstrap analysis of the parsimony tree using the "Bootstrap analysis using branch-and-bound search" and "Parsimony" options. Based on the partition analysis, what are the most likely alternatives to your tree? }

{4. Perform a bootstrap analysis using the "Bootstrap analysis using neighbor-joining distance" option and compare this to the CLUSTALW analysis.}

{D. Parsimony and maximum likelihood trees using Phylip.}

{1. Familiarize yourself with the Phylip PROTPARS program by reading the document in /software/nonrdist/phylip-3.6/doc/protpars.html. Run PROTPARS (/home/solaris/nsci/bi141s/protpars) and compare the tree to those of PAUPSEARCH. }

{2. Familiarize yourself with the Phylip PROML program by reading the document in /software/nonrdist/phylip-3.6/doc/proml.html. Run PROML (/home/solaris/nsci/bi141s/proml) and compare to the other trees you have constructed.}

{E. Questions.}

1. Explain what is meant by progressive alignment.
2. Why are progressive alignments the only practical multiple alignment techniques?
3. Explain the basic steps used by PILEUP in making a progressive alignment.
4. What type of tree is used as a guide tree by PILEUP?
5. What are the main enhancements in CLUSTAL with respect to PILEUP?
6. What are the two most serious drawbacks common to all progressive alignments?
7. Describe two ways one can evaluate the correctness of a phylogenetic tree based on sequence data?
8. What is an outgroup? Suggest a species that would be an appropriate outgroup for a tree linking humans, cows, raccoons, dogs, and elephants.
9. What is an outgroup used for in phylogenetic analysis?
10. What is meant by "once a gap, always a gap"?
11. What are the main differences between the Fitch-Margoliash method, the neighbor joining method, and the UPGMA method?
12. Bootstrapping is a resmapling method. What is it that is resampled and how is this done?
13. Why does one need to "correct" distance used in making trees?
14. Describe two problems with parsimony approaches to constructing trees.
15. How does the "Occams razor" principle relate to parsimony trees.
16. What kind of tree would be best for classifying enzymes according to function. Why?