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Site Identification from Short Read Sequences.

Project description

# Running SISRS as a User

## Install Docker
Follow the instructions [here]( to install
Docker CE for your operating system.

There's quite a bit going on in those instructions. As an example, if you're
on Ubuntu you'll follow the specific instructions

Ignore anything that talks about Docker EE. They're just trying to sell you

Note that SISRS currently only runs
on Linux.

## Getting the Docker image

Download the SISRS docker image which comes with all the dependencies for
running SISRS.

`docker pull anderspitman/sisrs`

## Running SISRS

First start up a docker container using the image obtained above:

`docker run -it anderspitman/sisrs bash`

Then from within the Docker container:

pip install sisrs

# Developing SISRS


SISRS: Site Identification from Short Read Sequences
Version 1.6.2
Copyright (c) 2013-2016 Rachel Schwartz <>
More 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.

Talk from Evolution 2014 describing SISRS and its application:


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.

* Built-In Genome Assemblers (Required if SISRS is building your composite genome)
* Velvet (tested with v.1.2.10) (
* Minia (tested with v.2.0.7) (
* AbySS (tested with v.2.0.2) (
* Bowtie2 (
* Python 2.7, Biopython, and PySAM
* Samtools v1.3.1 (
* GNU Parallel (
* BBMap [requires Java] (


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 be
assembled 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 the
final 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 site
for 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 subcommand


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 ( 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 loci
sisrs 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

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