Adrian Baez-Ortega
Transmissible Cancer Group, University of Cambridge
Somatypus is an open-source pipeline that makes use of the powerful variant caller Platypus for identifying germline and somatic SNVs and indels in sequencing data coming from a set of unpaired samples. It has been designed to offer great sensitivity and specificity, even for low-frequency somatic mutations found in cancer genomes. Although this pipeline can be applied to any cancer sequence data, it is particularly useful for the processing of data obtained from many tumour samples that are closely related to each other and lack matched normal samples, such as transmissible cancer or metastasis data.
Somatypus has been tested on Ubuntu (14.04.4) systems, and it should work well on any Linux distribution. It has not been tested on Mac systems, although it may work after some minor code modifications.
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Call single nucleotide variants (SNVs) and short insertions/deletions (indels) from any number of sequence alignment files (subject to memory constraints), at a probably unmatched speed.
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Filter low-quality or ambiguous variants, while keeping those variants occurring with low frequency (in a few samples) or aberrant copy number (not fitting a diploid model).
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Allow the use of additional calling options for Platypus, as long as they do not override the ones defined in the pipeline.
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Run seamlessly from a single command.
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Resume execution after an unexpected interruption.
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Call long indels, structural variants or copy number changes.
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Manage samples in pairs, or distinguish between “tumour” and “host/normal” samples (or between germline and somatic variants). Identification of somatic variants must be performed downstream.
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Take into account cross-sample contamination. Identification of variants caused by contamination must be performed downstream.
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Allow extensive workflow customisation (unless the source code is altered).
(The following information can be found in the INSTALL.txt file.)
Somatypus has been designed as a set of Bash and Python scripts that do not require a real installation, but simply adding the software directory to the PATH environment variable, thus making set-up straightforward. However, it has some minimal software dependencies:
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Python 2.6 or later (not tested on Python 3.X), including development libraries.
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Platypus (http://www.well.ox.ac.uk/platypus), which in turn requires htslib (http://www.htslib.org), which in turn requires zlib (http://zlib.net).
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VCFtools (https://vcftools.github.io), for using the vcf-sort command.
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The tabix package (http://sourceforge.net/projects/samtools/files/tabix), for using the tabix and bgzip commands. It should come together with htslib.
For the sake of tidiness, it is advisable to create a folder called "somatypus" in the location where you usually install software, and install all the dependencies and the Somatypus pipeline itself (the somatypus-x.x folder) into it.
The following instructions assume that you have administrator privileges in the system you are using (i.e. that you can do "sudo"); otherwise, you should contact you system's administrator. Although it should be obvious, please note that you need to replace things like "path/to/" and "/FULL/PATH/TO/" with the actual path to the relevant folder. "/FULL/PATH/TO/" indicates that the absolute path (beginning at "/", e.g. /home/user/...) must be used.
The recommended installation order is:
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Python ≥2.6
Python is normally installed by default in most UNIX systems. However, in order to run Platypus, you need to install the Python development libraries.
On Debian or Ubuntu Linux, you can install them with:sudo apt-get install python-devOn RPM-based Linux distributions, you can install them with:
sudo yum install python-devel -
zlib (development files)
If you have trouble installing htslib (below), you probably need to install this first.
On Debian or Ubuntu Linux, you can install the corresponding package with:sudo apt-get install zlib1g-devOn RPM-based Linux distributions, you can install it with:
sudo yum install zlib-devel -
htslib
This is necessary in order to run Platypus.
Latest version as of April 2016: https://github.com/samtools/htslib/releases/download/1.3/htslib-1.3.tar.bz2
Once downloaded and uncompressed, you can install it with:cd path/to/htslib-x.x ./configure make sudo make installAfter this, you need to add the htslib directory to your LD_LIBRARY_PATH environment variable. You can do this either by editing your ~/.bashrc file with a text editor (e.g. nano) and adding the following line:
export LD_LIBRARY_PATH=/FULL/PATH/TO/htslib-x.x:$LD_LIBRARY_PATHOr just by appending the relevant line to the file with:
cd path/to/htslib-x.x echo "export LD_LIBRARY_PATH=$PWD:\$LD_LIBRARY_PATH" >> ~/.bashrcEither way, it is then necessary to source the .bashrc file for the changes to be applied:
source ~/.bashrc -
Platypus
This is the best and the worst part of the pipeline.
Latest version: http://www.well.ox.ac.uk/software-download-registration
Once downloaded and uncompressed, you can install it with:cd path/to/Platypus_x.x.x ./buildPlatypus.shIf installation is successful, you will see the message "Finished building Platypus". After this, you need to make a symbolic link to the Platypus executable file in the somatypus-x.x/src folder (which will be then added to the PATH variable, see below).
cd path/to/Platypus_x.x.x ln -s $PWD/Platypus.py path/to/somatypus-x.x/src/ -
VCFtools
On Debian or Ubuntu Linux, you can install the corresponding package with:
sudo apt-get install vcftoolsOn RPM-based Linux distributions, you can install it with:
sudo yum install vcftools -
Somatypus
The last step is adding the Somatypus directory to your PATH environment variable, so that the somatypus command can be called from the command line. You can do this either by editing your ~/.bashrc file with a text editor (e.g. nano) and adding the line:
export PATH=/FULL/PATH/TO/somatypus-x.x/src:/FULL/PATH/TO/somatypus-x.x/utils:$PATHOr just by appending the relevant line to the file with:
cd path/to/somatypus-x.x echo "export PATH=$PWD/src:$PWD/utils:\$PATH" >> ~/.bashrcEither way, it is then necessary to source the .bashrc file for the changes to be applied:
source ~/.bashrc
Once all the dependencies and the pipeline have been installed, you should be able to run all of the following commands (which show the usage information of each tool):
Platypus.py callVariants -h
tabix
bgzip
vcf-sort -h
somatypus
And now you can have fun.
When the pipeline is run via the somatypus command with no options (or with -h), it displays the usage information.
| SOMATYPUS
| A Platypus-based variant calling pipeline for cancer data
| Version x.x
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| Required input:
| -i Absolute path to folder containing the input BAM files (accompanied by BAI indices).
| -g Absolute path to reference genome FASTA file (accompanied by FAI index).
| -o Absolute path to the output folder (it will be created if needed).
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| Optional input:
| -r Absolute path to file of regions to use, one per line in CHR:START-END format.
| -c Number of CPUs (processes) for Platypus *(should not exceed 8 due to a bug)*.
| -p Additional options for Platypus, within quotes and separated by spaces.
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| Options:
| -w Use windows around the variants as regions during genotyping.
| -h Print this usage information and exit.
| -v Print version and exit.
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| Usage:
| somatypus -i /path/to/bams_dir -o /path/to/out_dir -g /path/to/genome.fna -r /path/to/regions.txt -c <1-8> -p "--option=VAL --option=VAL"
It is advisable that all the input paths be absolute, rather than relative.
An optional regions file (-r option) allows the user to define a set of genomic regions (e.g. exons) wherein to perform the calling. The regions file must be a text file containing one region per line, in CHR:START-END format (e.g. 1:1028676-1028844. The chromosome names must match those in the FASTA and BAM files).
The -w option enables the use of windows of +/-200 bp around each variant as regions for genotyping. This may increase the efficiency of the genotyping process, especially in cases where no regions file is used. However, since it may cause some variants to be missed, this option should not be used unless necessary.
The number of CPUs is also optional (default is 1) but, if specified, must be at least 1, and should not exceed 8 (or even less, depending on the amount of data), due to an inveterate Platypus bug that can cause an extremely excessive memory allocation attempt (see the full documentation).
Finally, additional calling options can be passed to Platypus via the -p option. The entire additional options string must be quoted, and options must be separated by spaces. However, those options already specified in the pipeline cannot be included, namely: --logFileName, --refFile, --bamFiles, --regions, --minPosterior, --minReads, --minFlank, --trimReadFlank, --source, --getVariantsFromBAMs, --nCPU, or --output (or -o). (For obvious reasons, they should also not include --help or -h.)
A list of all the Platypus options can be consulted via: Platypus.py callVariants -h.
The full log of the pipeline execution will be stored in a file named SOMATYPUS_<date+time>.log, in the logs subfolder of the output directory, together with the logs of most of the steps. The log files and the temporary folders containing intermediate files will be numbered according to the number of the steps that interact with them.
For more information on the workflow and output files, please read the full documentation in docs/Somatypus Documentation.pdf.
Please cite Somatypus as:
A. Baez-Ortega et al. Somatic evolution and global expansion of an ancient transmissible cancer lineage. Science 365, eaau9923 (2019). DOI: 10.1126/science.aau9923.
Copyright © 2016–2017 Transmissible Cancer Group, University of Cambridge
Author: Adrian Baez-Ortega (ORCID 0000-0002-9201-4420; [email protected])
Somatypus is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 3 of the License, or (at your option) any later version.
This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.
You should have received a copy of the GNU General Public License along with this program. If not, see http://www.gnu.org/licenses.