BIOL368/F14:Nicole Anguiano Week 4

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Exploring HIV Evolution In-Class Activity

Methods

Activity 1, Part 2
  • First, I opened the link to the NCBI website and searched for the Markham, et al article. Once found, I switched the search type to nucleotide and searched again using the same search criteria that had led me to the original paper (Markham RB[AUTHOR]). Then, I selected a gene from the list. I saved it in fasta format.
  • I selected 3 other files, one from subject 4, one from subject 1, and a second from subject 1, and saved all of them in fasta format.
Activity 1, Part 3
  • I navigated to the Biology Workbench site and created an account, then entered the workbench and selected "nucleic tools". I selected "Add New Nucleic Sequence" and hit "Run". I uploaded the fasta file I had created before and saved it to my workbench under the label "S2V4C2".
  • I then imported the rest of the files that I had saved, and saved them under the same labeling scheme (Subject, Visit, Clone number).
  • I selected all of the files I'd uploaded into my workbench, chose the option "ClustalW - Multiple Sequence Alignment", and ran it. I submitted it with the default settings.
  • I clicked "Session Tools" and started a new session.
Activity 2, Part 1
Activity 2, Part 2
  • I selected the four clones from Subject 13 (S13V1-1, S13V1-2, S13V1-3, S13V1-4), and ran a ClustalW on them using the default settings. I counted the number of differences in the sequence analysis. I then selected the "Import Alignment(s)" button. I selected the alignment I had just imported and ran a "CLUSTALDIST - Generate Distance Matrix with Clustal W" and ran it using the default settings. I used the values from the Clustal Distance Matrix to calculate the min and max difference by multiplying the smallest and largest numbers by the total number of base pairs, respectively.
  • I repeated the above steps with the 13 clones from subject 1, then the 10 clones from subject 7, then the combined 9 clones of subjects 2 and 4 (6 from 2, 3 from 4), then the combined 15 clones from subjects 5 and 10 (8 from 5 and 7 from 10), then the combined 7 clones from subjects 6 and 12 (3 from 6 and 4 from 12).

Results

Activity 1, Part 3

  • Figure 1: The results from the sequence alignment from the Clustal W of the 4 genes AF016768.2, AF016818.2,AF016767.2, and AF089153.2.
  • AF016767.2 and AF016768.2 come from subject 1, but AF016768.2 is slightly more similar to AF016818.2 than AF016767.2, having 6 differences with AF016767.2 and 5 differences with AF016818.2. AF016767.2 and AF016818.2 are different, having 11 differences between the two of them. AF089153.2 is the most different from all of them, having 21 locations where it is completely different from the other 3, three of them being areas that do not exist in the gene (Fig. 1).

  • Figure 2: The unrooted tree resulting from the ClustalW of the 4 genes AF016768.2, AF016818.2,AF016767.2, and AF089153.2.
  • Of the four genes used, three of the four (AF016768.2, AF016767.2, and AF016818.2) have a relatively high degree of similarity, which can be seen in the unrooted tree. Those three are clustered relatively closely together, with AF016768.2 having a closer similarity to AF016818.2 and AF016767.2 than AF016818.2 and AF016767.2 have with each other. AF089153.2 is far away from the others, indicating a large amount of dissimilarity between it and the other three genes (Fig. 2).
Activity 2, Part 1

  • Figure 3: The sequence alignment resulting from the comparison of 12 genes, 3 each from subjects 7, 15, 13, and 1 (S7V1-1, S7V1-4, S7V1-8, S15V1-3, S15V1-7, S15V1-3, S13V1-1, S13V1-2, S13V1-3, S1V1-3, S1V1-6, and S1V1-9).
  • The most similarity is seen between clones from the same subject, as expected. However, a higher degree of dissimilarity than would be expected is seen among the clones of subject 15, with 24 areas in which 1 clone is different from the rest. Conversely, the clones from subject 13 are extremely similar (which had 1 area in which 1 clone is different from the rest), as are the clones from subject 1 (which had 2 areas in which 1 clone is different from the rest. Subject 7 lies in between the group, with 14 areas in which 1 clone is different from the rest.

  • Figure 4: The tree resulting from the comparison of the 12 genes S7V1-1, S7V1-4, S7V1-8, S15V1-3, S15V1-7, S15V1-3, S13V1-1, S13V1-2, S13V1-3, S1V1-3, S1V1-6, and S1V1-9.
  • The tree confirms what we saw in the sequence alignment. Subject 15 has the highest degree of difference, and its branches are quite far apart. The clones from subject 1 and the clones on subject 13 are extremely similar and are almost entirely overlapping due to their similarity. The clones from subject 7 are not as similar as those from subjects 1 and 13, but not as different as the clones from subject 15.
Activity 2, Part 2

Interpretations

Activity 1, Part 2
Activity 1, Part 3
Activity 2, Part 1
Activity 2, Part 2

Questions

Activity 1, Part 2
  1. What was the accession number of the sequence you chose?
    • The Accession number was AF016818.
  2. Which subject of the study was that HIV sequence from? Which section of the record contains information about who the HIV was collected from?
    • The sequence was from subject 2. The "Definition" section of the record contained the information about where the HIV was collected from.
Activity 2, Part 1
  • As a preliminary analysis you should generate a multiple sequence alignment and distance tree for 12 of these sequences (3 clones from each of 4 subjects).
    • See figures 3 and 4 above in results.
  • Use the data table below (Table 2) to keep a record of the data you analyzed.
Subject Clone #
1 3
1 6
1 9
7 1
7 4
7 8
13 1
13 2
13 3
15 3
15 7
15 11
  1. Do the clones from each subject cluster together?
    • The clones from each subject are in clusters that range from very loose to tight. The clones from subjects 1 and 13 are clustered very close together. The clones from subject 7 are a little farther apart than those seen in 1 and 13, with clone numbers 1 and 4 being more close together than clone 8. However, the clones from subject 15 are very far apart from each other, indicating that they are only very loosely related to one another.
  2. Do some subjects clones show more diversity than others?
    • Yes. Subject 7's clones shows more diversity than subjects 1 and 13. Subject 15's clones show more diversity than all of the other clones, with each of the branches being very long and far apart.
  3. Write a brief description of your tree and how you interpret the clustering pattern with respect to the similarities and potential evolutionary relationships between the subject's HIV sequences.
    • My tree contains a relatively short central trunk, with branches of varying length extending off. The branch with subject 13's clones is very distant from the rest, indicating it is the most different from all of the other subject's clones. However, the clones from subject 13 are extremely close together, with clone 2 being located at the branching point in which the rest separate off. This indicates that it may be a common ancestor of the two, or that it may be equally related to both of them. Clone 1 is a very short distance from clone 2, and is located on an extremely tiny branch that is so small that it is hard to see, indicating that it is very closely related to the other clones. Clone 3 is the furthest up the branch, indicating the most diversity. Overall, the clones from subject 13 are very closely related and not very diverse. The next longest branch of the main trunk is Subject 7. Subject 7's clones are much more loosely related than subject 13's. However, the clones are relatively close together, indicating that they are closely related. Clones 1 and 4 are more closely related than clone 8, as clone 8 has a branch to itself while clones 1 and 4 stem off of the same terminal point. The next longest branch off the trunk is Subject 1. Like subject 13, subject 1's clones are very closely related. Clone number 3 is located on the branching point that splits off into clones 6 and 9, indicating that it may be the common ancestor of 6 and 9, and 6 and 9 are also closely related. The final branch is for subject 15, the most dissimilar of the subjects. Each of the clones of subject 15 is located on a longer branch from those around it, indicating that they are not very similar and are much more diverse. Clone 3 is the most diverse of the clones, having diverged first and being on a branch of its own. Clones 7 and 11 split off the same branching point, but their respective branches are very long, indicating that they are not very similar.
  4. Copy and paste your tree from the Biology Workbench into a word processor and print it out to share with the class.
    • See figure 4 above.
Activity 2, Part 2

Conclusions

Activity 1, Part 2
Activity 1, Part 3
Activity 2, Part 1
Activity 2, Part 2

Overall Conclusions

Electronic Lab Notebook

Links

Nicole Anguiano
BIOL 368, Fall 2014

Assignment Links
Individual Journals
Class Journals
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