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# easySFS TL;DR - easySFS is a tool for the effective selection of population size projection for construction of the site frequency spectrum. It may be used to convert VCF to dadi/fastsimcoal/momi2 style SFS for demographic analysis. ## Why is this needed? The site frequency spectrum can not be constructed in a coherent fashion on a data matrix with missing values. Missing data is a prominent feature of RADSeq-like datasets and simply removing sites with missingness would drastically throw out the majority of your data. One could also impute missing values, some people do this, but if you have lots of missing data the imputation will be unreliable. The down projection method is a sort of compromise between these two extremes. You "project down" to a smaller sample size and "average over" all possible resamplings to construct a complete data matrix. To be clear, I didn't invent this down projection strategy, I believe Marth et al 2004 get the credit here, I just made this python program for automating exploration of the projection values. ## Choosing projection values In terms of how to choose projection values Gutenkunst et al 2009 provide a heuristic mechanism, suggesting that maximizing the number of segregating sites is the best strategy. Whether this is true or not is an open question at this point, but it's the best we've got, and it's what most people do, if they are being careful. EasySFS simply counts the number of segregating sites per projection value for each population. Then you may choose the projection value based on the results it displays. ## A note on input VCF file format This is a relatively simple script. It was created for use with VCF files from RAD-style datasets. VCF file formats differ pretty dramatically so ymmv. Right now it's been tested and seems to run fine for VCF as output by both pyrad/ipyrad and tassel. # Installation and operation ## Dependencies The script assumes you have matplotlib and dadi installed. ## Install & Run * Install [miniconda for python3](https://docs.conda.io/en/latest/miniconda.html) * Create and activate a new environment: `conda create -n easySFS` & `conda activate easySFS` * Install dependencies: `conda install -c bioconda dadi pandas` * Clone this repo * `git clone https://github.com/isaacovercast/easySFS.git` * `cd easySFS` * `chmod 777 easySFS.py` * `./easySFS.py` ## General workflow Converting VCF to SFS is a 2 step process. The first step is to run a preview to identify the values for projecting down each population. The second step is to actually do the conversion specifying the projection values. It looks like this: `./easySFS -i input.vcf -p pops_file.txt --preview` Which will output something like this: ``` Pop1 (2, 45.0) (3, 59.0) (4, 58.0) (5, 49.0) (6, 41.0) (7, 35.0) (8, 27.0) (9, 20.0) (10, 13.0) (11, 8.0) (12, 8.0) (13, 5.0) (14, 2.0) (15, 2.0) (16, 1.0) pop2 (2, 68.0) (3, 96.0) (4, 106.0) (5, 110.0) (6, 108.0) (7, 89.0) (8, 76.0) (9, 66.0) (10, 56.0) (11, 49.0) (12, 42.0) (13, 39.0) (14, 34.0) (15, 29.0) (16, 27.0) (17, 26.0) (18, 24.0) (19, 23.0) (20, 21.0) (21, 22.0) (22, 20.0) (23, 19.0) (24, 16.0) (25, 16.0) (26, 15.0) (27, 15.0) (28, 13.0) (29, 13.0) (30, 14.0) (31, 14.0) (32, 14.0) (33, 13.0) (34, 12.0) (35, 9.0) (36, 9.0) (37, 8.0) (38, 8.0) (39, 8.0) (40, 6.0) (41, 6.0) (42, 6.0) (43, 5.0) (44, 5.0) (45, 5.0) (46, 4.0) (47, 4.0) (48, 4.0) (49, 3.0) (50, 3.0) (51, 3.0) (52, 3.0) (53, 3.0) (54, 3.0) (55, 2.0) (56, 2.0) (57, 2.0) (58, 2.0) (59, 2.0) (60, 2.0) (61, 2.0) (62, 0.0) (63, 0.0) (64, 0.0) (65, 0.0) (66, 0.0) (67, 0.0) (68, 0.0) (69, 0.0) (70, 0.0) (71, 0.0) (72, 0.0) (73, 0.0) (74, 0.0) (75, 0.0) (76, 0.0) ``` Each column is the number of samples in the projection and the number of segregating sites at that projection value. The dadi manual recommends maximizing the number of segregating sites, but at the same time if you have lots of missing data then you might have to balance # of segregating sites against # of samples to avoid downsampling too far. Next run the script with the values for projecting for each population, like this: `./easySFS -i input.vcf -p pops_file.txt --proj 12,20` ## Input files Two input files are required, the VCF and the population specification file. VCF in the format as written out by pyrad/ipyrad is known to work well, other vcf formats may work too, but aren't guaranteed. The population assignment file is a plain text file with two columns, one for sample names and one for the population the sample belongs to, like this: ``` sample1 pop1 sample2 pop1 sample3 pop2 sample4 pop2 ``` Only samples that are in both the pop file and the vcf file will be included in the final sfs. ## Outputs By default the script generates all 1D sfs per population, all pairwise joint sfs per population pair and one multiSFS for all populations. If you specify the `-o` flag you can pass in an output directory which will be created, otherwise output files are written to the default directory `output`. There will be two directories created here `dadi` and `fastsimcoal2`. ### dadi 1D SFS will be named like this: `Pop1-<sample_size>.sfs`<br> Joint SFS will be named like this: `Pop1-Pop2.sfs Pop2-Pop3.sfs`<br> multiSFS will will be named like this: `Pop1-Pop2-Pop3.sfs` ### Fastsimcoal2 Fastsimcoal2 is pickier about file naming conventions and file format. 1D SFS will be named like this: `Pop1_MAFpop0.obs Pop2_MAFpop0.obs`<br> Joint SFS will be named like this: `prefix_jointMAFpop0_1.obs prefix_jointMAFpop0_2.obs prefix_jointMAFpop1_2.obs`<br> multiSFS will will be named like this: `prefix_MSFS.obs` ## Running example files The example files are different enough where they will give you an idea of what most of the command line options do. ### Diploid example * Diploid data * 2 populations * SFS includes all snps from w/in each locus Preview: `./easySFS.py -i example_files/wcs_1200.vcf -p example_files/wcs_pops.txt --preview -a`<br> Convert: `./easySFS.py -i example_files/wcs_1200.vcf -p example_files/wcs_pops.txt -a --proj=7,7` The `-a` flag specifies that all snps in the vcf should be used. Also, notice the `--ploidy` flag is not required since diploid is the default. ### Haploid example * Haploid data * 3 populations * SFS only includes one snp per RAD locus Preview: `./easySFS.py -i example_files/leuco_1200.vcf -p example_files/leuco_pops.txt --ploidy 1`<br> Convert: `./easySFS.py -i example_files/leuco_1200.vcf -p example_files/leuco_pops.txt --ploidy 1 --proj=8,10` Here the `--polidy` flag is required. In this example only one snp per locus will be randomly sampled for inclusion in the output sfs. ## Usage You can get usage info any time by: `./easySFS.py` ``` usage: easySFS.py [-h] [-a] -i VCF_NAME -p POPULATIONS [--proj PROJECTIONS] [--preview] [-o OUTDIR] [--ploidy PLOIDY] [--prefix PREFIX] [--unfolded] [--dtype DTYPE] [--GQ GQUAL] [-f] [-v] optional arguments: -h, --help show this help message and exit -a Keep all snps within each RAD locus (ie. do _not_ randomly sample 1 snp per locus). -i VCF_NAME name of the VCF input file being converted -p POPULATIONS Input file containing population assignments per individual --proj PROJECTIONS List of values for projecting populations down to different sample sizes --preview Preview the number of segragating sites per population for different projection values. -o OUTDIR Directory to write output SFS to --ploidy PLOIDY Specify ploidy. Default is 2. Only other option is 1 for haploid. --prefix PREFIX Prefix for all output SFS files names. --unfolded Generate unfolded SFS. This assumes that your vcf file is accurately polarized. --dtype DTYPE Data type for use in output sfs. Options are `int` and `float`. Default is `float`. --GQ GQUAL minimum genotype quality tolerated -f Force overwriting directories and existing files. -v Set verbosity. Dump tons of info to the screen ``` ## License [![License: CC BY 4.0](https://img.shields.io/badge/License-CC%20BY%204.0-lightgrey.svg)](https://creativecommons.org/licenses/by/4.0/) ## Credits `easySFS` is kind of a loose wrapper around `dadi`, which I use for internal sfs representation, and much of the grunt work.