Package: sisrs
# Running SISRS as a User## Install DockerFollow the instructions [here](https://docs.docker.com/install/) to installDocker CE for your operating system.There's quite a bit going on in those instructions. As an example, if you'reon Ubuntu you'll follow the specific instructions[here](https://docs.docker.com/install/linux/docker-ce/ubuntu/).Ignore anything that talks about Docker EE. They're just trying to sell youstuff.Note that SISRS currently only runson Linux.## Getting the Docker imageDownload the SISRS docker image which comes with all the dependencies forrunning SISRS.`docker pull anderspitman/sisrs`## Running SISRSFirst start up a docker container using the image obtained above:`docker run -it anderspitman/sisrs bash`Then from within the Docker container:```pip install sisrssisrs-python```# Developing SISRSSISRS=====SISRS: Site Identification from Short Read SequencesVersion 1.6.2Copyright (c) 2013-2016 Rachel Schwartz <Rachel.Schwartz@asu.edu>https://github.com/rachelss/SISRSMore information: Schwartz, R.S., K.M Harkins, A.C. Stone, and R.A. Cartwright. 2015. A composite genome approach to identify phylogenetically informative data from next-generation sequencing. BMC Bioinformatics. 16:193.(http://www.biomedcentral.com/1471-2105/16/193/)Talk from Evolution 2014 describing SISRS and its application:https://www.youtube.com/watch?v=0OMPuWc-J2E&list=UUq2cZF2DnfvIUVg4tyRH5NgLicense=======This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version.This program is distributed in the hope that it will be useful, but without any warranty; without even the implied warranty of merchantability or fitness for a particular purpose. See the GNU General Public License for more details.Requirements============* Built-In Genome Assemblers (Required if SISRS is building your composite genome)* Velvet (tested with v.1.2.10) (http://www.ebi.ac.uk/~zerbino/velvet/)* Minia (tested with v.2.0.7) (http://minia.genouest.org/)* AbySS (tested with v.2.0.2) (http://www.bcgsc.ca/platform/bioinfo/software/abyss)* Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)* Python 2.7, Biopython, and PySAM* Samtools v1.3.1 (http://www.htslib.org/)* GNU Parallel (http://www.gnu.org/software/parallel/)* MAFFT (http://mafft.cbrc.jp/alignment/software/)* BBMap [requires Java] (https://sourceforge.net/projects/bbmap/)Input=====Next-gen sequence data such as Illumina HiSeq reads.Data must be sorted into folders by taxon (e.g. species or genus).Paired reads in fastq format must be specified by _R1 and _R2 in the (otherwise identical) filenames.Paired and unpaired reads must have a fastq file extension.Running SISRS=============## Usage:sisrs command options### By default, SISRS assumes that* A reference genome is not available and a composite assembly will beassembled using Velvet* The K-mer size to be used by Velvet in contig assembly is 21.* Only one processor is available.* Files are in fastq format.* Paired read filenames end with _R1 and _R2* A site is only required to have data for two species to be included in thefinal alignment.* Folders containing reads are in the present working directory* SISRS data will be output into the present working directory* A minimum of three reads are required to call the base at a sitefor a taxon.### Commands:**sites**: produce an alignment of sites from raw reads**loci**: produce a set of aligned loci based on the most variable regions of the composite genome#### Subcommands of sites:**subSample**: run sisrs subsampling scheme, subsampling reads from all taxa to ~10X coverage across species, relative to user-specified genome size**buildContigs**: given subsampled reads, run sisrs composite genome assembly with user-specified assembler**alignContigs**: align reads to composite genome as single-ended, uniquely mapped**mapContigs**: align composite genome reads to a reference genome (optional)**identifyFixedSites**: find sites with no within-taxa variation**outputAlignment**: output alignment file of sisrs sites**changeMissing**: given alignment of sites (alignment.nex), output a file with only sites missing fewer than a specified number of samples per site#### Option Flags:* -g : the approximate genome size (**mandatory** if sisrs will be assembling a composite genome)* -p : use this number of processors *[Default: 1]** -r : the path to the reference genome in fasta format *[Optional]** -k : k-mer size (for assembly) *[Default: 21]** -f : absolute path to the directory containing the folders of reads *[Default: Current Directory]** -z : absolute path to either empty or non-existent directory where SISRS will output data *[Default: Current Directory]** -n : the number of reads required to call a base at a site  *[Default: 3]** -t : the threshold for calling a site; e.g. 0.99 means that >99% of bases for that taxon must be one allele; only recommended for low ploidy with <3 individuals  *[Default: 1 (100%)]** -m : the number of species that are allowed to have missing data at a site* -o : the length of the final loci dataset for dating* -l : the number of alleles* -a : assembler [velvet, minia, abyss, or premade; *Default: velvet*]- If using a premade composite genome, it must be in a folder named 'premadeoutput' in the same directory as the folders of read data, and must be called 'contigs.fa'* -s : Sites to analyze when running 'loci' [0,1,2]- 0 [Default], all variable sites, including singletons- 1, variable sites excluding singletons- 2, only biallelic variable sites* -c : continuous command mode for calling subcommands [1,0]- 1 [Default]: calling a subcommand runs that subcommand **and all subsequent steps in the pipeline**- 0: calling a subcommand runs **only** that subcommandOutput======Nexus file with variable sites in a single alignment. Usable in most major phylogenetics software as a concatenated alignment with a setting for variable-sites-only.Test Data=========The folder test_data (https://github.com/rachelss/SISRS_test_data) contains simulated data for 10 species on the tree found in simtree.tre . Using 40 processors this run took 9 minutes. Analysis of the alignment output by sisrs using raxml produced the correct tree.Sample commands==============1. Basic sisrs run: start with fastq files and produce an alignment of variable sites```sisrs sites -g 1745690```2. Basic sisrs run with modifications```sisrs sites -g 1745690 -p 40 -m 4 -f /usr/test_data -z /usr/output_data -t .99 -a minia```3. Run only sisrs read subsampling step```sisrs subSample -g 1745690 -f /usr/test_data -c 0```4. Produce an alignment of loci based on the most variable loci in your basic sisrs run. Note - this command will run sisrs sites if (and only if) it was not run previously.```sisrs loci -g 1745690 -p 40 -l 2 -f /usr/test_data           # Will run sites first, then locisisrs loci -g 1745690 -p 40 -l 2 -f /usr/SISRS_sites_ouput   # Will run loci from previous sites data```5. Get loci from your fastq files given known loci.first name your reference loci ref_genes.fa and put in your main folder```sisrs loci -p 40 -f /usr/test_data```