ViennaNGS is a Perl distribution for rapid development of next-generation sequencing analysis pipelines. It comes with a set of modules and Moose-based classes for accomplishing standard and non-standard tasks in NGS processing. ViennaNGS' feature-richness, however, comes at some cost: dependencies on third party Perl modules and external tools and libraries, which can be tedious to install. I have therefore compiled this little ViennaNGS Installation HOWTO to help prospective users getting the software up and running quickly on their systems.

Let’s get started. The installation process provided here has been prepared for Linux systems, however the steps should be reproducible on MacOS X given that you have the common GNU tools and Perl 5.10.0 or higher available, e.g. via MacPorts.

First things first: third party dependencies

ViennaNGS depends on set of third party bioinf tools and libraries, which are required either for specific filtering and file format conversion tasks or for building internally used Perl modules. Before continuing with the ViennaNGS installation, you will need to download and install the following software and ensure that all executables are accessible to the Perl interpreter:

The last dependency is in fact a bit peculiar. Bio::DB::Sam up to version 1.41, which we use for all kind of direct BAM manipulation within ViennaNGS, for some reason will not compile with recent, HTSlib-based versions of samtools. samtools 0.1.19 seems to be the last version that is compatible with current versions of Bio::DB::Sam. So let’s download samtools 0.1.19 to a local folder, say /scratch/software, uncompress the tarball and compile the samtools library:

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mkdir -p /scratch/software
cd /scratch/software
wget https://github.com/samtools/samtools/archive/0.1.19.tar.gz
tar xvf 0.1.19.tar.gz
cd samtools-0.1.19
make CFLAGS="-g -O2 -Wall -Wno-unused -Wno-unused-result -fPIC"

The extra -Wno* CFLAGS options prevent some unused variable warnings with recent versions of gcc, whereas the -fPIC CFLAGS option prohibits a relocation error on x64_64 architectures (see also the Bio::DB::Sam README). If everything works out fine, samtools is now compiled, and a static library file libbam.a will be created in the current working directory. Remember the absolute path to libbabm.a (in our example this is /scratch/software/samtools-0.1.19), since we will need this later for compiling Bio::DB::Sam.

Setting up a local Perl module directory with cpanminus

Once all thrid-party dependencies are installed, we can now focus on the Perl dependencies of ViennaNGS, e.g. the BioPerl suite, the Moose object framework and many more. Fortunately, installation of custom Perl modules (i.e. those that are not considered Perl core modules or shipped with a Linux distribution) is a fairly trivial task these days, thanks to a tool called cpanminus. cpanminus renders installation of a custom Perl module as easy as typing cpanm and the module’s name on the command line. Moreover, it provides automatic dependency resolution, which comes in handy when you’re installing Perl modules or distributions that depend on other modules, which again depend on a bunch of modules etc. Forget about the CPAN shell or cpanplus, use cpanminus.

Before starting with cpanminus we’ll need to set a few environment variables that tell cpanm and the Perl interpreter where to keep and look for locally installed Perl modules. We’ll keep up with the cpanminus default location, ${HOME}/perl5 and add the following lines to the end of .bashrc:

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export PERL_MM_OPT="INSTALL_BASE=${HOME}/perl5"
export PERL_MB_OPT="--install_base ${HOME}/perl5"
export PERL5LIB=${HOME}/perl5/lib/perl5:${PERL5LIB}

We will also modify our PATH variable to include ${HOME}/perl5/bin, where executable scripts that are shipped with custom Perl modules will be installed:

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export PATH=${HOME}/perl5/bin:${PATH}

We can now install cpanminus, either the version shipped with our Linux distribution (example given for Debian/Ubuntu must be executed with root privileges)

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apt-get install cpanminus

or directly from https://cpanmin.us

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curl -L https://cpanmin.us | perl - App::cpanminus

Installing the ViennaNGS Perl dependencies

After all we are now ready to install the ViennaNGS Perl dependencies. Let’s start with Bio::DB::Sam

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cpanm Bio::DB::Sam

Depending on your system setup, cpanm will now start to download and install all dependencies of Bio::DB::Sam and will then ask for the location of the static libbam.a library:

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Configuring Bio-SamTools-1.41 ... Please enter the location of the bam.h and compiled libbam.a files:

Our samtools folder is /scratch/software/samtools-0.1.19 (see above), so let’s enter this here:

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/scratch/software/samtools-0.1.19

This was basically the only module that we had to install manually due to the samtools location constraint. Actually, we could have installed Bio::ViennaNGS directly and cpanm would have asked for the samtools location, but I thought this was a good chance to demonstrate cpanm usage for didactic reasons.

Installing Bio::ViennaNGS

Here comes the easiest part. Since we’ve got cpanm up and running on our system, all we need to type is

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cpanm Bio::ViennaNGS

and wait for cpanm to install all dependencies and finish. Beware that ViennaNGS depends on Statistics::R, which requires a running installation of the R Statistics Package on your system. If for any reason cpanm fails (e.g. R is not available), it will stop and report an error. You can then install the missing component and call the above command again. Moreover, whenever there is a new ViennaNGS release available on CPAN, just type cpanm Bio::ViennaNGS and you’ll get the latest version in just a couple of seconds.

Congratulations! You now have a running installation of Bio::ViennaNGS. If you sticked with the paths in our example, the modules/classes are located in ${HOME}/perl5/lib/perl5/Bio/ViennaNGS/lib and the ViennaNGS utilities can be found in ${HOME}/perl5/bin.

The ViennaNGS authors are looking forward to getting your feedback. Please use the ViennaNGS GitHub Issue Tracker for reporting bugs.