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miRkwood_installation.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!)

Usage with Docker

If you choose to use Docker you won't have to deal with installing miRkwood dependencies. 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

Usage:

  1. Abinitio pipeline:

Here is an example of command line for abinitio pipeline:

sudo docker run -v /your/mapping/repertory/:/MAPPING/
iguigon/mirkwood
mirkwood.pl
--input /MAPPING/input/sequenceSomething.fas
--output /MAPPING/output/results_abinitio
--filter-rrna --filter-trna --varna

Type the following command for detailed help on the command options: sudo docker run iguigon/mirkwood mirkwood.pl --help

  1. SmallRNAseq pipeline:

miRkwood smallRNAseq pipeline takes as input a BED file with the following syntax:

1 18092 18112 AAACGTGTAGAGAGAGACTCA 1 - 1 18094 18118 GATTCTTTTGTTTGCCACT 2 + 1 18096 18119 TCGATAGGATCAAGTACATCT 1 + 1 18100 18124 AAGAAGAAAAAGAAGAAGAAGAAG 9 +

In this file, each line is a unique read. The fields are, from left to right: name of the chromosome, starting position, ending position, read identifier, number of occurrences of the read in the data, strand. Positions follow the BED numbering convention: the first base of the chromosome is considered position 0 (0-based position) and the feature does not include the stop position.

You can convert a BAM file into the needed BED format with our custom script:

sudo docker run -v /your/mapping/repertory/:/MAPPING/
iguigon/mirkwood
mirkwood-bam2bed.pl
--in MAPPING/input.bam
--bed MAPPING/output.bed
--min 18
--max 25

Type the following command for detailed help on the command options: sudo docker run iguigon/mirkwood mirkwood-bam2bed.pl --help

Here is an example of command line for smallRNAseq pipeline:

sudo docker run -v /your/mapping/repertory/:/MAPPING/
iguigon/mirkwood
mirkwood-bed.pl
--input /MAPPING/input/sample.bed
--output /MAPPING/output/results_smallRNAseq
--genome /MAPPING/input/my_genome.fasta
--mirbase /MAPPING/input/my_mirbase_file.gff3
--gff /MAPPING/input/my_annotations_file.gff
--min-read-positions-nb 0 --max-read-positions-nb 5 --align

Type the following command for detailed help on the command options: sudo docker run iguigon/mirkwood mirkwood-bed.pl --help

With the -v /your/mapping/repertory/:/MAPPING/ parameter, Docker will mount your local folder /your/mapping/repertory/ into the container under /MAPPING/. Make sure you have stored all your needed input files in this folder. Results files will be stored here as well.

Installation 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.

Congratulations! A miRkwood instance is now running at http://192.168.33.20/mirkwood

Manual installation

If you prefer to install miRkwood manually or if you don't want to use a VM, 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/.

  1. Mandatory dependencies
  • Perl modules
    Config::Simple
    YAML::XS
    File::Which
    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)

Inline::CPP may sometimes be difficult to install. In that case, please install first the following modules:
Inline::C
Inline
Pegex
ExtUtils::MakeMaker
File-ShareDir-Install
Capture::Tiny
ExtUtils::CppGuess

  • RNAstemloop
    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.

  • 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. Tested with version 2.1.6-1.

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) 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.

  • rnashuffles
    rnashuffles is an in-house 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

  • 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.

  • piccolo piccolo is an in-house program that performs alignments. It is stored in the ${local_programs} directory.
  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+