#Roary the pan genome pipeline For instructions on how to use the software, the input format and output formats, please see the Roary website.

Roary is a high speed stand alone pan genome pipeline, which takes annotated assemblies in GFF3 format (produced by Prokka) and calculates the pan genome. Using a standard desktop PC, it can analyse datasets with thousands of samples, something which is computationally infeasible with existing methods, without compromising the quality of the results. 128 samples can be analysed in under 1 hour using 1 GB of RAM and a single processor. To perform this analysis using existing methods would take weeks and hundreds of GB of RAM.

##Citation "Roary: Rapid large-scale prokaryote pan genome analysis", Andrew J. Page, Carla A. Cummins, Martin Hunt, Vanessa K. Wong, Sandra Reuter, Matthew T. G. Holden, Maria Fookes, Daniel Falush, Jacqueline A. Keane, Julian Parkhill, Bioinformatics, (2015). doi: http://dx.doi.org/10.1093/bioinformatics/btv421 Roary: Rapid large-scale prokaryote pan genome analysis

Theres are a number of dependancies required for Roary, with instructions specific to the type of system you have:

• Ubuntu/Debian
• CentOS/RedHat
• Homebrew/Linuxbrew - OSX/Linux
• Guix - Linux
• Virtual Machine - OSX/Linux/Windows
• Docker
• Installing from source - OSX/Linux

If the installation fails please contact your system administrator. If you encounter a bug please let us know by emailing [email protected] .

##Ubuntu/Debian

sudo apt-get install roary

###Ubuntu 14.04 All the dependancies can be installed using apt and cpanm. Root permissions are required.

sudo apt-get install bedtools cd-hit ncbi-blast+ mcl parallel cpanminus prank mafft fasttree
sudo cpanm -f Bio::Roary

###Ubuntu 12.04 Some of the software versions in apt are quite old so follow the instructions for LinuxBrew below.

##CentOS/RedHat To install the dependancies, the easiest way is to install LinuxBrew using the steps for Fedora, then follow the steps below for installing Roary on LinuxBrew.

##Homebrew/Linuxbrew - OSX/Linux Assuming you have homebrew (OSX) or linuxbrew (Linux) setup and installed on your system:

brew tap homebrew/science
brew install bedtools cd-hit blast mcl parallel prank mafft fasttree cpanm
sudo cpanm -f Bio::Roary

##GNU Guix Roary is not included in version in Guix 0.11.0 so guix pull is currently required before installation.

guix pull
guix package --install roary

##Virtual Machine - OSX/Linux/Windows Roary wont run natively on Windows but we have created virtual machine which has all of the software setup, including Prokka, along with the test datasets from the paper. It is based on Bio-Linux 8. You need to first install VirtualBox, then load the virtual machine, using the 'File -> Import Appliance' menu option. The root password is 'manager'.

ftp://ftp.sanger.ac.uk/pub/pathogens/pathogens-vm/pathogens-vm.latest.ova

More importantly though, if your trying to do bioinformatics on Windows, your not going to get very far and you should seriously consider upgrading to Linux.

##Docker We have a docker container which gets automatically built from the latest version of Roary in Debian Med. To install it:

docker pull sangerpathogens/roary

To use it you would use a command such as this (substituting in your directories), where your GFF files are assumed to be stored in /home/ubuntu/data:

docker run --rm -it -v /home/ubuntu/data:/data sangerpathogens/roary roary -f /data /data/*.gff

##Installing from source (advanced Linux users only) As a last resort you can install everything from source. This is for users with advanced Linux skills and we do not provide any support with this method since you have the skills to figure things out. Download the latest software from (https://github.com/sanger-pathogens/Roary/tarball/master).

Choose somewhere to put it, for example in your home directory (no root access required):

cd $HOME
tar zxvf sanger-pathogens-Roary-xxxxxx.tar.gz
ls Roary-*

Add the following lines to your $HOME/.bashrc file, or to /etc/profile.d/roary.sh to make it available to all users:

export PATH=$PATH:$HOME/Roary-x.x.x/bin
export PERL5LIB=$PERL5LIB:$HOME/Roary-x.x.x/lib

Install the perl dependancies:

sudo cpanm  Array::Utils Bio::Perl Exception::Class File::Basename File::Copy File::Find::Rule File::Grep File::Path File::Slurper File::Spec File::Temp File::Which FindBin Getopt::Long Graph Graph::Writer::Dot List::Util Log::Log4perl Moose Moose::Role Text::CSV PerlIO::utf8_strict 

Install the external dependances either from source or from your packaging system:

bedtools cd-hit blast mcl GNUparallel prank mafft fasttree

The code will not work with perl 5.8 or below (pre-modern perl). We no longer test against 5.10 (released 2007). If your running a very old verison of Linux, your also in trouble.

#Versions of software we test against

• Perl 5.14, 5.16, 5.20, 5.24
• cdhit 4.6.1
• ncbi blast+ 2.4.0
• mcl 14-137
• bedtools 2.26.0
• prank 130410
• GNU parallel 20130922, 20160722, 20150122
• FastTree 2.1.9