Portcullis

Portcullis stands for PORTable CULLing of Invalid Splice junctions from pre-aligned RNA-seq data. It is known that RNAseq mapping tools generate many invalid junction predictions, particularly in deep datasets with high coverage over splice sites. In order to address this, instead for creating a new RNAseq mapper, with a focus on SJ accuracy we created a tool that takes in a BAM file generated by an RNAseq mapper of the user's own choice (e.g. Tophat2, Gsnap, STAR2 or HISAT2) as input (i.e. it's portable). It then, analyses and quantifies all splice junctions in the file before, filtering (culling) those which are unlikely to be genuine. Portcullis output's junctions in a variety of formats making it suitable for downstream analysis (such as differential splicing analysis and gene modelling) without additional work. Portcullis can also filter the original BAM file removing alignments associated with bad junctions. Both the filtered junctions and BAM files are cleaner and more usable resources which can more effectively be used to assist in downstream analyses such as gene prediction and genome annotation.

Installation

From package manager

The simplist way to install portcullis is via package manager. We support both brew and bioconda. We try to keep these reciepes as up to date as possible but sometimes there can be a lag between updating the source code and ensuring the package managers have the latest reciepes.

For brew type: brew install brewsci/bio/portcullis

For bioconda type: conda install portcullis --channel=bioconda

From source

Installing from source will ensure you have the latest version of the software. If you wish to install from source please first confirm that first you have these dependencies are installed and configured:

• GCC V4.8+
• autoconf V2.53+
• automake V1.11+
• make
• libtool V2.4.2+
• zlib
• pthreads
• samtools V1.2+
• Python3 V3.5+ (including python3 development libraries and the pandas packages)
• Sphinx-doc V1.3+ (Optional: only required for building the documentation.)

NOTE ON INSTALLING PYTHON: Many system python installations do not come with the C API immediately available, which prevents Portcullis from embedding python code. We typically would recommend installing anaconda3 as this would include the latest version of python, all required python packages as well as the C API. If you are running a debian system and the C libraries are not available by default and you wish to use the system python installation the you can install them using: sudo apt-get install python-dev. Also, if you have installed python to a custom location please verify that the bin directors on the PATH environment variable, and the lib (or lib64) directory is on the LD_LIBRARY_PATH or LD_RUN_PATH as appropriate.

Then proceed with the following steps:

• Clone the git repository (For ssh: git clone git@github.com:maplesond/portcullis.git; or for https: git clone https://github.com/maplesond/portcullis.git), into a directory on your machine.
• "cd" into root directory of the installation
• Build boost by tying ./build_boost.sh.
• Create configuration script by typing: ./autogen.sh.
• Generate makefiles and confirm dependencies: ./configure
• Compile software: make
• Run tests (optional) make check
• Install: sudo make install

The configure script can take several options as arguments. One commonly modified option is --prefix, which will install portcullis to a custom directory. By default this is "/usr/local", so the portcullis executable would be found at "/usr/local/bin" by default. Type ./configure --help for full details and available options.

NOTE: if KAT is failing at the ./autogen.sh step you will likely need to install autotools. The following command should do this on MacOS: brew install autoconf automake libtool. On a debian system this can be done with: sudo apt-get install autoconf automake libtool.

Operating Instructions

After portcullis has been installed, the portcullis executable should be available.

Typing portcullis or portcullis --help at the command line will present you with the portcullis help message.

These modes are available:

• prep - Prepares input data so that it is suitable for junction analysis
• junc - Calculates junction metrics for the prepared data
• filter - Separates alignments based on whether they are likely to represent genuine splice junctions or not
• bamfilt - Filters a BAM to remove any reads associated with invalid junctions
• full - Runs prep, junc, filter and optionally bamfilt as a complete pipeline

Typing portcullis <mode> --help will bring up help and usage information specific to that mode.

In addition to portcullis, we provide a tool-suite for manipulating junction files

An online version of the manual can be found here: https://portcullis.readthedocs.org/en/latest/.

Licensing

GNU GPL V3. See COPYING file for more details.

Authors

• Daniel Mapleson
• Luca Venturini
• David Swarbreck

See AUTHORS file for more details.

Acknowledgements

Affiliation: The Earlham Institute (EI) Funding: The Biotechnology and Biological Sciences Research Council (BBSRC)