Answering the question: Do clones from a particular subject's group (rapidly progressing, moderately progressing, and nonprogressing), share any genetic similarity with one another? Are clones from subjects in the same group the most similar, or are they as dissimilar as clones from other groups? Does the amount of variation and similarity, if any, change from the time of the first visit to a visit after about 2 years of infection with the virus?
Below are the clones chosen and the subjects/visits they are from:
Progressor Group
Subject
Visit #
Clones
Visit #
Clones
Rapid
3
1
1, 2, 3
3
2, 7, 9
Rapid
11
1
3, 5, 7
3
3, 6, 9
Rapid
15
1
3, 6, 12
4
1, 3, 4
Moderate
6
1
1, 2, 3
5
2, 4, 8
Moderate
8
1
1, 3, 5
4
4, 5, 6
Moderate
14
1
2, 3, 6
5
1, 6, 7
Nonprogressor
2
1
3, 4, 5
3
5, 6, 7
Nonprogressor
12
1
1, 2, 3
4
2, 5, 10
Nonprogressor
13
1
1, 3, 4
3
1, 2, 4
Methods and Results
First, I compiled the sequences of each of the clones that I'd specified last week (also listed above) from the bioquest web site. I went to the Biology workbench and created a new session, called "Research". In this session, I uploaded the file I just created with all of the sequences.
I began by running a CLUSTALW on each of the first visit clones. I ran one on the rapid progressors, then the moderate progressors, then the nonprogressors, importing the alignments each time so that further analysis could be performed on each alignment through CLUSTALDIST.
Figure 1: The sequence alignment for subjects 3, 11, and 15 (the rapid progressors), generated by CLUSTALW. The S value in Table 1 was calculated by counting how many places there was a difference between the sequences.Figure 22: The tree for subjects 3, 11, and 15 (the rapid progressors), generated by CLUSTALW.Figure 3: The sequence alignment for subjects 6, 8, and 14 (the moderate progressors), generated by CLUSTALW. The S value in Table 1 was calculated by counting how many places there was a difference between the sequences.Figure 4: The tree for subjects 6, 8, and 14 (the moderate progressors), generated by CLUSTALW.Figure 5: The sequence alignment for subjects 2, 11, and 12 (the nonprogressors), generated by CLUSTALW. The S value in Table 1 was calculated by counting how many places there was a difference between the sequences.Figure 6:The tree for subjects 2, 11, and 12 (the nonprogressors), generated by CLUSTALW.
After running the CLUSTALW, I ran a CLUSTALDIST on each group to find the clustal distance matrix, and calculate the theta, min difference, and max difference (Table 1).
Figure 7: The clustal distance matrix for subjects 3, 11, and 15 (the rapid progressors), generated by CLUSTALDIST. The min and max difference in Table 1 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 291.Figure 8: The clustal distance matrix for subjects 6, 8, and 14 (the moderate progressors), generated by CLUSTALDIST. The min and max difference in Table 1 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 288.Figure 9: The clustal distance matrix for subjects 2, 12, and 13 (the nonprogressors), generated by CLUSTALDIST. The min and max difference in Table 1 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 285.
Group
S
θ
Min Difference
Max Difference
Rapid Progressors
83
-
39
52
Moderate Progressors
49
-
14
32
Nonprogressors
55
-
22
44
Table 1
After comparing each group with itself, I then compared across groups. I ran a CLUSTALW and CLUSTALDIST on the first visit clones from the three rapid progressors and the three moderate progressors, then the three rapid progressors and the three nonprogressors, then the three moderate progressors and the three nonprogressors. After the comparison, I ran a CLUSTALW and CLUSTALDIST on all of the visit 1 clones.
Figure 11:The tree for the rapid and moderate progressors, generated by CLUSTALW.Figure 12:The clustal distance matrix for the rapid and moderate progressors, generated by CLUSTALDIST. The min and max difference in Table 2 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 291.Figure 14:The tree for the rapid and nonprogressors, generated by CLUSTALW.Figure 15:The clustal distance matrix for the rapid and nonprogressors, generated by CLUSTALDIST. The min and max difference in Table 2 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 291.Figure 17:The tree for the moderate and nonprogressors, generated by CLUSTALW.Figure 18:The clustal distance matrix for the moderate and nonprogressors, generated by CLUSTALDIST. The min and max difference in Table 2 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 288.Figure 19:The tree for the all three progressor groups for visit 1, generated by CLUSTALW.Figure 20:The clustal distance matrix for all three progressor groups for visit 1, generated by CLUSTALDIST. The min and max difference in Table 2 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 291.
Groups Being Compared
Min Difference
Max Difference
Rapid and Moderate
19
56
Rapid and Nonprogressor
24
54
Moderate and Nonprogressor
21
48
All
21
52
I moved from the first visit clones to the mid-visit clones, and ran a CLUSTALW on each of the mid-visit clones. I ran one on the rapid progressors, then the moderate progressors, then the nonprogressors, importing the alignments each time so that further analysis could be performed on each alignment through CLUSTALDIST.
Figure 21: The sequence alignment for subjects 3, 11, and 15 (the rapid progressors), generated by CLUSTALW. The S value in Table 3 was calculated by counting how many places there was a difference between the sequences.Figure 222: The tree for subjects 3, 11, and 15 (the rapid progressors), generated by CLUSTALW.Figure 23: The sequence alignment for subjects 6, 8, 14 (the moderate progressors), generated by CLUSTALW. The S value in Table 3 was calculated by counting how many places there was a difference between the sequences.Figure 242: The tree for subjects 6, 8, 14 (the moderate progressors), generated by CLUSTALW.Figure 25: The sequence alignment for subjects 2, 12, 13 (the nonprogressors), generated by CLUSTALW. The S value in Table 3 was calculated by counting how many places there was a difference between the sequences.Figure 262: The tree for subjects 2, 12, 13 (the nonprogressors), generated by CLUSTALW.
After running the CLUSTALW, I ran a CLUSTALDIST on each group to find the clustal distance matrix, and calculate the theta, min difference, and max difference (Table 3).
Figure 27: The clustal distance matrix for subjects 3, 11, and 15 (the rapid progressors), generated by CLUSTALDIST. The min and max difference in Table 3 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 291.Figure 28: The clustal distance matrix for subjects 6, 8, and 14 (the moderate progressors), generated by CLUSTALDIST. The min and max difference in Table 3 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 288.Figure 29: The clustal distance matrix for subjects 2, 12, and 13 (the nonprogressors), generated by CLUSTALDIST. The min and max difference in Table 3 was calculated by comparing across subjects and seeing what the highest and lowest values were, then multiplying by 288.
I repeated all of the above steps with the clones taken from the 2-year visit instead of the first visit. After that, I ran a CLUSTALW and CLUSTALDIST on all of the clones over all of the visits.
