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INSTALL.md

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miRkwood

miRkwood is a computational pipeline for the identification of miRNAs and their hairpin precursors.

It is constituted of:

  • a back-end in Perl, running many third-party programs, stored in cgi-bin
  • a front-end in Perl/JavaScript, stored in html

miRkwood comes with a fair number of dependencies. It was tested on Ubuntu 12.04. For use miRkwood you can either:

  • use Docker
  • deploy miRkwood in a virtual machine (VM) with the configuration management software Ansible.
  • install miRkwood manually (for the most motivated people!)

Install

Install with Docker

The easiest way to install miRkwood is by using Docker. All you will have to install is Docker (https://docs.docker.com/engine/installation/).

A Docker image for miRkwood is stored at: https://hub.docker.com/r/iguigon/mirkwood/ You can download miRkwood image with:

sudo docker pull iguigon/mirkwood:latest

Install in a VM

  1. Create the VM

Vagrant is a tool to create and configure virtual development environments. It can be considered a wrapper around virtualization software such as VirtualBox and configuration management software such as Chef, Salt and Puppet − or Ansible in our case.

  1. Install Ansible

Ansible is an IT automation tool. It can configure systems, deploy software, and orchestrate more advanced IT tasks such as continuous deployments or zero downtime rolling updates.

  1. Install miRkwood dependencies
  • Clone the miRkwood repository on the gitHub repository

git clone https://github.com/miRkwood-RNA/miRkwood.git

  • Run Vagrant at the miRkwood repository root

vagrant up

  • Vagrant will
    • download an Ubuntu ISO
    • mount the local development directory in the virtual machine
    • provision it using Ansible
    • set up network forwarding

This step can take up to 10 minutes.

A miRkwood instance will be running at http://192.168.33.20/mirkwood

Manual install

If you prefer to install miRkwood manually or if you don't want to use a VM, you can use the script install-dependencies.sh (must be run in super-user mode) or manually install all the dependencies.

Please check that the following dependencies are installed according to your needs, and check that the paths in /cgi-bin/lib/programs.cfg are corrects. ${local_programs} refers to /{miRkwood_path}/cgi-bin/programs/.

External dependencies

  1. Mandatory dependencies

  • Perl modules
    Config::Simple
    YAML::XS
    File::Which
    Inline::CPP Inline
    Pegex
    ExtUtils::MakeMaker
    File-ShareDir-Install
    Capture::Tiny
    ExtUtils::CppGuess
    MIME::Lite (for Web version only)
    Inline::CPP (for smallRNAseq pipeline only)
    LWP::UserAgent (for smallRNAseq pipeline only)
    Bio::DB::Fasta (for smallRNAseq pipeline only)

  • Vienna package
    The ViennaRNA Package consists of a C code library and several stand-alone programs for the prediction and comparison of RNA secondary structures. See http://www.tbi.univie.ac.at/RNA/ for the installation. Please note that there is a bug with recent versions of the package. We recommand to use version 2.1.6-1 (we provide a .deb file).

Make sure that the following programs are correctly installed: b2ct
RNAfold
RNAlfold
RNAeval

Be careful that RNAfold, RNAlfold and RNAeval are usually installed in /usr/bin, and b2ct is usually installed in /usr/share/ViennaRNA/bin.

  • bedtools
    Install package 'bedtools' with your usual package manager, for instance with

sudo apt-get install bedtools

or

sudo yum install bedtools

  1. Optional dependencies for both pipelines

  • miRdup
    miRdup is a tool for the validation of pre-miRNAs predictions. Download the archive at http://www.cs.mcgill.ca/~blanchem/mirdup/ (tested with version 1.2 and 1.4) However, if you don't want or don't manage to install it, you can skip it. It will only affect the quality score of some candidates.

  • VARNA (Visualization Applet for RNA secondary structure)
    Make sure that the Java Runtime Environment is installed. Install package 'default-jre' with your usual package manager, for instance with

sudo apt-get install default-jre

or

sudo yum install default-jre

You can download VARNA jar on http://varna.lri.fr/bin/VARNAv3-91.jar and then create a symbolic link or change the corresponding line in the programs.cfg file.

  1. Optional dependencies for abinitio pipeline

  • tRNAscan-SE This program searches for tRNA genes in genomic sequences. Download the archive at http://lowelab.ucsc.edu/software/tRNAscan-SE.tar.gz. Extract the archive and edit the Makefile to replace '$(HOME)' by the path where you want to install it, then compile it.

  • rnammer This program predicts 5s/8s, 16s/18s and 23s/28s ribosomal RNA in genomic sequences.

    • Install hmmer

wget --directory-prefix=/tmp/ http://eddylab.org/software/hmmer/2.3.2/hmmer-2.3.2.tar.gz tar xf /tmp/hmmer-2.3.2.tar.gz --directory /opt cd /opt/hmmer-2.3.2/ ./configure --enable-threads make --directory=/opt/hmmer-2.3.2/ ln -s /opt/hmmer-2.3.2/src/hmmsearch /usr/bin/hmmsearch23

  • Install Perl dependency

sudo apt-get install libxml-simple-perl

  • Copy RNAmmer archive in /opt

cp $ROOT_PATH/provisioning/roles/mirkwood-software/files/rnammer-1.2.src.tar.Z /tmp/rnammer-1.2.src.tar.Z

  • Create RNAmmer directory

mkdir /opt/RNAmmer

  • Extract RNAmmer

tar xf /tmp/rnammer-1.2.src.tar.Z --directory /opt/RNAmmer

  • Add necessary module import to RNAmmer perl executable

sed -re 's/(use Getopt::Long;)/use File::Basename;\n\1/' -i /opt/RNAmmer/rnammer

  • Update self-path in RNAmmer perl executable

sed -re 's/"/usr/cbs/bio/src/rnammer-1.2"/dirname(FILE)/' -i /opt/RNAmmer/rnammer

  • Update paths to HMMER in RNAmmer perl executable

sed -re 's/$HMMSEARCH_BINARY\s?=.*/$HMMSEARCH_BINARY="/usr/bin/hmmsearch23"/' -i /opt/RNAmmer/rnammer

  • Make relevant user/group

chown -R www-data:www-data "/opt/RNAmmer"

  • blastX BLAST finds regions of local similarity between sequences. We use it to mask CDS regions in the sequences given by the user. Install package 'ncbi-blast+' with your usual package manager, for instance with

sudo apt-get install ncbi-blast+

or

sudo yum install ncbi-blast+

In-house programs:

  • RNAstemloop (mandatory) RNAstemloop is an in-house program that takes an output file from RNALfold and extract candidates stemloop from the secondary structures. RNAstemloop is stored in ${local_programs}. You can create a symbolic link to the program that suits your architecture, or simply change the corresponding line in the programs.cfg file.

  • piccolo (optional) piccolo is an program that performs alignments. It is stored in the ${local_programs} directory.

  • rnashuffles (optional) rnashuffles is an program that computes the thermodynamic stability. Ensuire Python pip is installed. Copy the sources where you want it to be (the sources are given in /{miRkwood_path}/provisioning/roles/mirkwood-software/files/) and then build it with pip.

pip install /path/rnashuffles

Update miRBase DB

miRkwood uses in-house program piccolo to align the sequence of the precursor with the database of mature miRNAs of plant (Viridiplantae) deposited in miRBase.

To update the miRBase database file, you can use the script construct_mirbase_fasta_file.pl (in /{miRkwood_path}/cgi-bin/bin/).

./construct_mirbase_fasta_file.pl
-species list_Viridiplantae.txt
-fasta mature.fa
-output MirbaseFile.txt

The file mature.fa can be downloaded on miRBase website: http://www.mirbase.org/ftp.shtml.

The list of species can be obtained here : http://www.mirbase.org/cgi-bin/browse.pl Expand all Viridiplantae clades and then copy/paste the corresponding lines.

Then you must replace the file MirbaseFile.txt in /{miRkwood_path}/cgi-bin/data/ with the one you just created.