# AVM BIOL368 Week 4

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## Purpose

• To continue with our class research and journal projects for the Markham et al. paper
• Continue to learn about sequencing and the similarities and differences

## Part 1

• Upload the “visit_1_S1_S9.txt” and “visit_1_S10_S15.txt” files from the BIOL368/F16 Week 4 page into the Biology Workbench
• Generate a multiple sequence alignment using ClustlW
• Choose 3 clones form 4 different subjects (as shown in table below)

• Photo of tree shown below

### Questions

1. Do the clones from each subject cluster together?
• Yes
2. Do some subjects’ clones show more diversity than others?
• Subject 1, strain 1 is more diverse than strains 2 and 3. Subject 2 strain 3 is more diverse than stains 1 and 2. Subject 3 strain 3 is more diverse than strains 1 and 2. Subject 4 strain 2 is more diverse than strains 1 and 3.
3. Do some of the subjects cluster together?
• Yes. Subjects 1 and 2 are clustered.
4. Write a brief description of the tree and how to interpret the clustering pattern with respect to the similarities and potential evolutionary relationships between subjects’ HIV sequences.
• The unrooted tree shows the similarities of the 4 subjects and clones. This tree shows that Subject 1 and 2 have very closely related viruses, and within this virus, mutations 1 and 2 of subject 2 are most closely related to mutation 3 of subject 1. All of the mutations of subject 4's virus are most closely related to each other and the mutations of subject 3 are most closely related to each other.

## Part 2

• Select a sequence from last weeks assignment and align all of the clones
• Calculate S from the alignment
• S is the number of positions where there is at least one nucleotide difference across the clones
• Save the alignment for later use
• Run an analysis for two more subjects
• Sequence differences for each subject shown at right
Subject 6
Subject 8
Subject 14
• Within the Clustdist tool generate a distance matrix for one of your alignments
• Choose the highest and lowest pairwise scores and find the % difference
• Calculate theta for each subject
• Repeat these steps for each subject
• Table below shows results from these analysis

• Create a new alignment with all the sequences from 2 different subjects
• Use Clustdist to create a pairwise distance matrix
• Find all the min and max differences
• Results shown in table below

## Conclusion

This exercise was done to determine if the HIV virus strains extracted from random test subjects originated from the same virus initially.Clones from subject 3 are the most genetically different of the subjects used. The tree shows that subjects 1 and 2 had very similar viruses, with some strains (subject 2; mutations 1 and 2, and subject 1; mutation 3) were even mores genetically similar. Subject 4 was slightly separated as well, but not as far away on the unrooted tree as subject 3. After following the protocol above and creating the unrooted tree during the exercise I understood that the extracted viruses did not stem from one original viral source. Overall, this project was helpful in helping to understand the significance of S values, theta, ClustalWdist, min and max differences and generally knowing how to understand similarities and differences between the sequences given.

## Research Project

1. What is your question?
• If HIV-1 virus evolution patterns are based on virus copy number rather than CD4 T-cell decline, how would this change the subjects classification as non-progressors, moderate progressors, and rapid progress ors? Is virus copy number correlated with diversity and divergence?
2. Make a prediction (hypothesis) about the answer to your question before you begin your analysis.
• There would be a shift in the subjects being grouped as moderate and rapid progressors. There is an overlap in virus number copies in Table 1 for subjects in both of these groups.
3. Which subjects, visits, and clones will you use to answer your question? Justify why you chose the subjects, visits, and clones you did.
• We could look at subjects 1, 3, 6 and 7 as they have very similar virus number copies. We could also choose subjects 12 and 13 to compare to those which have significantly lower virus number copies. For this specific question I think almost any of the subjects could be used and it may need to be adjusted once we actually start to work on the project.

## Acknowledgments

• Worked in collaboration with Courtney L. Merriam in class. Courtney and I agreed on the topic and details of our research in class, and wrote the section titled "Defining Our Research Project" together.
• Template followed from week 4 assignment BIOL368/F16
• Help from our professor, Dr. Dahlquist in class.
• Note: While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

Avery Vernon-Moore 14:42, 21 September 2016 (EDT)

## References

• Donovan S and Weisstein AE (2003) Exploring HIV Evolution: An Opportunity for Research. In Jungck JR, Fass MR, and Stanley ED, eds. Microbes Count! West Chester, Pennsylvania: Keystone Digital Press.
• Markham, R.B., Wang, W.C., Weisstein, A.E., Wang, Z., Munoz, A., Templeton, A., Margolick, J., Vlahov, D., Quinn, T., Farzadegan, H., & Yu, X.F. (1998). Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proc Natl Acad Sci U S A. 95, 12568-12573. doi: 10.1073/pnas.95.21.12568
• Vlahov, D., Anthony, J.C., Munoz, A., Margolick, J., Nelson, K.E., Celentano, D.D., Solomon, L., Polk, B.F. (1991). The ALIVE study, a longitudinal study of HIV-1 infection in intravenous drug users: description of methods and characteristics of participants. NIDA Res Monogr 109, 75-100.
• Week 4 Assignment Page

#### Weekly Assignments:

Assignment 1
Assignment 2
Assignment 3
Assignment 4
Assignment 5
Assignment 6
Assignment 7
Assignment 8
Assignment 9
Assignment 10
Assignment 11
Assignment 12
Assignment 14
Assignment 15


#### Individual Journals:

"Week 1: Create Wiki Page"
Individual Journal 2
Individual Journal 3
Individual Journal 4
Individual Journal 5
Individual Journal 6
Individual Journal 7
Individual Journal 8
Individual Journal 9
Individual Journal 10
Individual Journal 11
Individual Journal 12
No Week 13 Assignment
Individual Journal 14
Individual Journal 15


#### Shared Journals:

Class Journal 1
Class Journal 2
Class Journal 3
Class Journal 4
Class Journal 5
Class Journal 6
Class Journal 7
Class Journal 8
Class Journal 9
Class Journal 10
Class Journal 11
Class Journal 12
Class Journal 14
Class Journal 15