Application of the PEGASUS gene-level association test to the Drosophila Genome Reference Panel
Developed using Pegasus Version 1.3 (Nakka et al. 2016 - Genetics)
December 12, 2019
Code is modified from VEGAS source code (Liu et al. 2010 - AJHG)
Questions: Contact samuel_smith1@brown.edu
Requirements:
Perl 5
R (version 3.0.2 or higher)
Plink 1.07 (1.9 beta 3, 7 Jun is also okay)
Please note the following:
R and PLINK must be installed and in your PATH for this program to run. This program requires the R packages corpcor and CompQuadForm. If they are missing, it will return an error. This directory (PEGASUS) contains the following:
pegasus_flies.pl - source code for PEGASUS_flies program.
DGRP - directory containing genotype data from the 205 lines of the Drosophila Genome Research Project.
glist-dm5.57 - list of genes and their bounds developed from annotation file provided by the DGRP (http://dgrp2.gnets.ncsu.edu/data.html)
Usage: Commands to run this program should have the following format:
./pegasus_flies.pl [input pvalues file] [parameters]
The input p-values file is a tab-separated table of SNP names and corresponding p-values. For an example file of p-values, please see example.txt. The one required parameter is to specify which reference genotype data or custom LD file should be used for LD calculations. If using the provided DGRP data, type: -pop DGRP. If using your own genotype data to calculate LD, type -custom followed by the path to and prefix of your PLINK bed files. Alternatively, if you would like to use your own LD file containing pre-calculated values for correlation between SNPs in every gene in the dataset, type -ld-file followed by the absolute path to your LD file in PLINK format. Note that correlation values should be r and not r^2. (See below for further explanation and sample commands.)
This program uses the following flags for required LD parameter:
-custom [path + prefix of PLINK bed files for custom genotypes to use for calculating LD, must be in bed format] specify path and name of plink bed/bim/fam files with custom genotypes to calculate LD matrices (ex."path/to/bed/file/mydata")
OR
-pop DGRP use given DGRP data as reference genotypes to calculate LD
OR
-ld-file [absolute path + name of pre-calculated LD file] specify absolute path and name of pre-calculated LD file in default PLINK format (see below) containing correlation values (r) between SNPs in each gene to be evaluated.
1 10000015 X_10000015_INS 1 10000031 X_10000031_SNP 0.872127
1 10000015 X_10000015_INS 1 10000035 X_10000035_SNP 0.804592
Please note that processing the custom LD file can slow down the PEGASUS program considerably for large genome-wide SNP datasets. Parallelizing gene score computation by running each chromosome separately is highly recommended with this option.
Optional: -out [path + prefix of outfile] specify name for the .out output file with gene scores
-chr [# between 1 and 6] compute gene scores for given chromosome only. Chromosomes are labeled 1 through 6 using the following conversion: X:1, 2L:2, 2R:3, 3L:4, 3R:5, 4:6. If you wish to include genes on Y and mitochondrial, encode them as chromosomes 7 and 8, respectively.
-upper [# of bp downstream of gene to be included ex. 30000] This flag can only be used with custom genotypes or pre-calculated LD files. The default is 1kb.
-lower [# of bp upstream of gene to be included ex. 30000] This flag can only be used with custom genotypes or pre-calculated LD files. The default is 1kb.
The following is a sample command using the given example files:
./pegasus_flies.pl example.txt -pop DGRP -out test
The resulting output file test.out should match the given example.out file.
To parallelize the process for large datasets, it is useful to run each chromosome separately using the -chr flag as in the following example:
./pegasus_flies.pl example.txt -pop DGRP -chr 1 -out example_chr1
Troubleshooting:
When using custom genotypes, please make sure the length of the .bim file is the same as the length of the p-values file (they should contain the same markers).
ERROR: Could not place marker for left/right window edge - this is a plink error message that occurs when there are not enough markers in a given bp range and can be ignored.
Make sure the directory DGRP is in the same directory as pegasus_flies.pl — this directory contains files needed for the program to function.