##POL EVOLUTION
This collection of HyPhy (http://www.hyphy.org) scripts perform sequence analysis on collections of longitudinal HIV-1 pol (bulk) sequence isolates, sampled from multiple individuals at multiple time points. More details of the analysis can be found in this paper: http://www.ncbi.nlm.nih.gov/pubmed/23840830
This library can
- Estimate a nucleotide substitution rate for individuals
- Test whether or not the molecular clock is violated for a particular individuals
- Compare global nucleotide substitution rates between groups of patients
- Estimate dN/dS values for individuals, and groups of individuals
- Use CTL haplotype information to partition the sites for each individual into epitope and non-epitope sets (not the same for all individuals), and compare rates of evolution (and dN/dS) between them in an individual, within and between groups of individiuals
- Use ARV information to perform the same types of analyses for DRAM vs not DRAM sites
##USAGE
Assuming, you have a command line version of HyPhy installed (GUI versions are OK as well), run the following command:
HYPHYMP CPU=1 /path/to/PolEvolution/HBL/RateAnalysis.bf
And enter the following values at the prompts (use HLA information, read HLA information from data/hla.csv in the PolEvolution Directory, do Codon analyses, TN93 model, sequence data from data/clean_pol.fas, split subjects into groups based on Dual Infection status read from data/group.csv, and save the results to results/test.{csv,json})
+------------------------------+ |Include HLA or DRM information| +------------------------------+ (1):[Neither] No HLA or DRM information (2):[HLA] Provide a .csv file with HLA haplotypes for each patient ID (3):[DRAM] Provide a .csv file with ARV lists to determine relevant DRM for each patient ID Please choose an option (or press q to cancel selection):2 /Volumes/TeraMonkey/Users/sergei/Coding/PolEvolution/CTL/The CTL haplotype file in .CSV format:../data/hla.csv +----------------------+ |Perform codon analyses| +----------------------+ (1):[No] Only perform nucleotide rate analyses [FASTER] (2):[Yes] Perform codon-based rate analyses (dN/dS) in addition to nucleotide rate analyses Please choose an option (or press q to cancel selection):2 +-------------------------+ |Use this nucleotide model| +-------------------------+ (1):[JC69] Jukes Cantor 69 (all equal rates, all equal frequencies) (2):[F81] Felsenstein 81 (all equal rates, empirical frequencies) (3):[TN93] Tamura and Nei 93 (two transition rates, shared transversion rate, empirical frequencies) (4):[GTR] General Time Reversible (6 substitution rates, empirical frequencies) Please choose an option (or press q to cancel selection):3 [path]/PolEvolution/HBL/Load the combined HIV polymerase sequence file:../data/clean_pol.fas +----------------------------------------------------------------------------------------------------+ |Split subjects into two groups based on external information (e.g. dual infection, treatment status)| +----------------------------------------------------------------------------------------------------+ (1):[No] Perform a joint analysis on all subjects (2):[Yes] Split the subjects into two groups, perform two-group analyses, and compare the rates between groups Please choose an option (or press q to cancel selection):2 [path]/PolEvolution/HBL/Partitioning list of the form: pid, [0/1]::../data/group.csv Descriptive label for 'positive' patients, e.g. Treated:Dually Infected [path]/PolEvolution/HBL/Save the resulting .CSV file to::../results/test.csv [path]/PolEvolution/HBL/Save a .JSON file with divergence plots for all subjects (suitable for plotting using D3) to::../results/test.json
The results are printed to the screen and also written to the output files (see below):
#####HLA targeted epitopes evolve at different rates than the rest of the sequence in 5 subjects
## HLA-targeted epitopes nucleotide rate is significantly faster in 4 subjects
## HLA-targeted epitopes nucleotide rate is significantly slower in 1 subjects
#####HLA targeted epitopes have different dN/dS than the rest of the sequence in 2 subjects
## Subjects for whom dN/dS differs between HLA-targeted epitopes and NOT HLA-targeted epitopes = 2
## Global nucleotide substitution rate [not Dually Infected] = 0.0004, 95% CI [ 0.000, 0.001] substitutions/site/year
## Global nucleotide substitution rate [Dually Infected] = 0.0225, 95% CI [ 0.019, 0.026] substitutions/site/year
## Global nucleotide substitution rate [HLA-targeted epitopes not Dually Infected] = 0.0006, 95% CI [ 0.000, 0.002] substitutions/site/year
## Global nucleotide substitution rate [HLA-targeted epitopes Dually Infected] = 0.0285, 95% CI [ 0.023, 0.035] substitutions/site/year
## Global nucleotide substitution rate is different between HLA-targeted epitopes and NOT HLA-targeted epitopes p-value = 0.714 [LRT = 0.13]
## Global nucleotide substitution rate is different between HLA-targeted epitopes and NOT HLA-targeted epitopes in the Dually Infected group, p-value = 0.068 [LRT = 3.32]
## Global dN/dS [not Dually Infected] = 0.5455, 95% CI [ 0.091, 1.686]
## Global dN/dS [Dually Infected] = 0.1293, 95% CI [ 0.098, 0.167]
## Global dN/dS [HLA-targeted epitopes not Dually Infected] = 0.5182, 95% CI [ 0.030, 2.286]
## Global dN/dS [HLA-targeted epitopes Dually Infected] = 0.3027, 95% CI [ 0.218, 0.407]
## dN/dS is different between HLA-targeted epitopes and NOT HLA-targeted epitopes, p-value = 0.967 [LRT = 0.00]
## dN/dS is different between HLA-targeted epitopes and NOT HLA-targeted epitopes in the Dually Infected group, p-value = 0.674 [LRT = 0.18]
##DATA
Input sequences must be
- In a format readable in HyPhy (http://hyphy.org/w/index.php/DATA_FILE_PRINT_FORMAT)
- Aligned to HXB2 pol without insertions relative to HXB2
- Each sequence must be named as
ID|YYYY-MM-DD, where ID is (an abritrary) individual used to collate sequences from the same individual, and the second field is the isoltation date - There must be at least two sequences per individual ID.
See data/clean_pol.fas for an example. You can also use the HBL/PrepareSequenceFile.bf to ensure the sequences are conforming.
This file will autmatically align everything to HBX2 pol if needed.
A csv file (with a header) with a two digit haplotype for each individual (OK if some data are missing).
See data/hla.csv.
A csv file (no header) with a list of drugs (standard 3 letter abbreviations, see http://hivdb.stanford.edu).
See data/arv.csv
A csv file (with a header) with a ID,0 or 1 to assign(1) or exclude (0) each individual to a group.
See data/group.csv.
##RESULTS