Analysis Options tab
Once the genotype data is loaded and all mapping files have been retrieved, Path2 will advance to the “Analysis Options” tab. The Analysis Options tab has three purposes:
If you have been following the tutorial, the Analysis options panel will look like this:
Analysis Options tab
Order of operation when the “Run” button is pressed
Performing single SNP association analyses
The “Single SNP Association Analysis Options” panel lets you specify what type of PLINK single SNP analysis you want Path2 to run. There are four tabs under the Single SNP Association Analysis Options panel, Path2 will automatically enable or disable certain options based upon the dataset imported in the Import data panel in the previous step.
For Logistic and Linear regression tests different analysis options exist. Please see the PLINK documentation for details. Here are the options included in Path2:
Once the Run button has been selected Path2 will call PLINK to perform the selected single SNP analysis. If PLINK is successfully run, the output PLINK results file should be in Path2/analysis/singlesnp/Combined/default. The original association results file generated by PLINK will be called dat.out.assoc.logistic for a Logistic regression test or dat.out.assoc.linear for a Linear regression test, for example. Regardless of the test, or if imported results are used, Path2 will copy the results file to the file: Path2/analysis/singlesnp/Combined/default/singleSNP.results.forDB . This file will be used in Pathway analysis or LD plot generation.
Ensure that the Logistic regression tab and Allelic association test option are selected then click the Run button.
Running a Logistic regression Allelic association analysis on the sample dataset
Here is an example line of a logistic association results file produced from a sample dataset:
Running Pathway/Ontology association analyses
Perhaps the most useful application of the Path2 program is in performing different Pathway- and Ontology-based association analyses. Currently, four pathway tests and one gene ontology test are built into Path2:
Please note that these Perl scripts rely on the R statistic programming language to perform the backend computational work. This is why Path2 requires Java, Perl, and R all to be installed in order to run correctly.
A particular pathway/ontology test can be run by selecting its corresponding check box in the “Pathway/Ontology Association Analysis Options” panel, then click on the Run button. As noted in the “Performing single SNP association analyses”, the tests will use the single SNP association results file at:
Path2 will always run the single SNP association test specified immediately before running the selected pathway tests.
** PLEASE NOTE! Several of the Pathway tests (for example SNP Ratio Test) can take a very long time to run depending upon the size of the dataset. Genome-wide association study level datasets must be run on a dedicated computational cluster. At this time, it is highly recommended to first run Path2 on the sample synthetic asthma dataset provided with the application in “Path2/data/sampleData” to ensure your install is set up correctly, and to familiarize yourself with the application first. Moreover, a few potential problems with running Path2 on GWAS datasets have been identified and will addressed in future versions of the application. **
Specifying the output folder for Pathway/Ontology tests
By default, you should find the pathway/ontology test results in the Path2/analysis/pathway folder. If, for example, you run the Sidak test and find no results under analysis/pathway/Sidak/Combined/default, please check the terminal output or the log file in Path2/log.txt for errors!
To change the output folder for Pathway tests, please use the file chooser in the panel “Select Output Folder for Pathway/Ontology Association Analysis Options” via the “Browse” button. This file chooser expects you to select a directory to which future Pathway/Ontology test results will be stored.
This feature is useful, for example, if you wish to run several slightly different pathway analyses on the same dataset without having to copy the Path2 output to a different folder after each analysis.
Generating LD (Linkage Disequilibrium) plots
Path2 can optionally generate LD plots for each gene in the loaded dataset if specified. SNPs are assigned to genes based on NCBI designations. This option is not available when only an analysis results file is loaded in the “Import Data” panel. The LD plots are generated by Haploview. Haploview is included in Path2 as Path2/Perl/Haploview.jar. The version of Haploview used is 4.1 built from source that can be found at the project’s SourceForge page. To run Haploview, Path2 converts its binary PLINK data to Haploview format pedigree and position files, then runs Haploview with the following options:
To generate LD plots for the sample dataset genes:
If Haploview runs successfully, the LD plots should be in: Path2/analysis/LD .
Example LD plot - gene 2205