Pipeline to generate NGS reads from a phylogeny
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README.md

TreeToReads

Simulation pipeline to generate next generation sequencing reads from realistic phylogenies.
Can be used to test effects of model of evolution, rates of evolution, genomic distribution of mutations, and phylogenetic relatedness of samples and of reference genome on SNP calling and evolutionary inference.

TreeToReads (TTR) provides features not available in alternative simulation approaches:

  • An observed genome is a tip on the simulated tree, allowing for direct comparison of read mapping between simulated and observed data.
  • Variable sites are simulated on an input phylogeny, and next generation sequencing reads are simulated from these evolved genomes.

Together these make TTR a useful simulator for testing the effects of phylogenetic structure on read mapping and inference of variable sites.

Inputs are a phylogeny, a genome to be used as a tip in the tree, and a set of configuration parameters in a control file

Optional inputs can include a sequencing error model parameterized from empirical data, and a distribution for the distances separating pairs of mutations in the genome.

Outputs are mutated genomes representing all tips in the phylogeny, and simulated whole genome sequencing reads representing those genomes. These are are useful for testing and comparison of analysis pipelines. Mutations are currently only single nucleotide variants - no indels or rearrangements.

The code is still in development - but testing welcome, and will be supported via email ejmctavish, gmail.

Schematic of TreeToReads procedure

Requirements:

  • Seq-Gen
  • Art

(can be run without Art if you want to generate mutated genomes, but not reads)

python packages

  • Dendropy

To install requirements

Install TTR as a python module

python setup.py install

Install Dendropy (requires dendropy4)

(This step is not necessary if you installed TTR as a module)

pip install dendropy
Install seq-gen, software to simulate mutations (http://tree.bio.ed.ac.uk/software/seqgen/)

on ubuntu using apt-get:

sudo apt-get install seq-gen

on Mac or linux (using homebrew, http://brew.sh/):

brew install seq-gen
Art is optional, but is required to generate reads from simulated genomes
Install ART, software to generate short reads from simulated genomes (http://www.niehs.nih.gov/research/resources/software/biostatistics/art/)

on ubuntu:

wget http://www.niehs.nih.gov/research/resources/assets/docs/artbingreatsmokymountains041716linux64tgz.tgz
tar -xzvf artbingreatsmokymountains041716linux64tgz.tgz

add art_illumina to path (see http://askubuntu.com/questions/60218/how-to-add-a-directory-to-my-path)

on Mac (using homebrew):

brew install art

or wget http://www.niehs.nih.gov/research/resources/assets/docs/artbingreatsmokymountains041716macos64tgz.tgz tar -xzvf artbingreatsmokymountains041716macos64tgz.tgz

add art_illumina to path (see https://coolestguidesontheplanet.com/add-shell-path-osx/)

Indelible is optional, but is required to simulate insterations and deletions.

Install from indelible webpage, and add to path. http://abacus.gene.ucl.ac.uk/software/indelible/

Note: to compile indelible from source for linux installations currently requires adding this include statement to the top of MersenneTwister.h

#include <unistd.h>

(I've reported the issue and it shoud be fixed soon)
add indelible to your path


Running the simulations (quick version):

git clone https://github.com/snacktavish/TreeToReads.git
cd TreeToReads
python treetoreads.py example.config

Edit the configuration file, example.config, to fit your data.

Currently generates paired end illumina data. Alternatively, genomes can be generated, and ART may be run separately using any chosen parameters.

Full Tutorial


Expected output

The script print out the parameter values and some other useful info. If it runs successfully it will end with "TreeToReads completed successfully!"

The output files will be in the the output directory specified in the config file, e.g. example_out and will consist of:

Key files

fasta_files - a folder containing the simulated genomes for each tip in the tree
fastq - folder containing folders with the names of each tip from the simulation tree, in each of these folders is the gziped simulated fastq. sim.vcf - A vcf file for the simulated mutations with respect to the anchor genome. CURRENTLY DUE TO A BUG VCF FILES ARE NOT GENERATED WHEN INDELS ARE SIMULATED. mutsites.txt - unordered list of the locations of mutations in the genome

Other files generated by analysis (mostly useless)

analysis_configuration.cfg - a copy of the control file used for the analysis
seqgen.out - output messages form the seq-gen software
simtree.tre.bu - a backup copy of the tree
simtree.tre - the tree used for simulations: reformatted and polytomies randomly resolved with 0 length branches
analysis.sh - the bash commands run by the analysis
These folders contain the simulated read in fastq format
seqs_sim.txt - an intermediate file used for generating variable sites
SNPmatrix - a file in format SEQUENCE, BASE, POSITION describing all variable sites in the genome
art_log - log messages from ART software

Docker container

TreeToReads is also available as a Docker container:

docker pull snacktavish/treetoreads
docker run snacktavish/treetoreads example.config

to run the default example, or

docker run -v /an/example/path:/a/container/path snacktavish/treetoreads /a/container/path/my_treetoreads_config.cfg

to run on real data, where /an/example/path/ contains the file my_treetoreads_config.cfg.

(See the Docker manual for more information about mounting host directories in the container.)


Citations

This tool relies on Dendropy, ART, and Seqgen. Please cite them (as well as this repo) in any published work using this simulation pipeline (appropriate citations below)

McTavish E. J., Timme R, (2015) Tree To Reads. https://github.com/snacktavish/TreeToReads bioRxiv

Huang W., Li L, Myers J. R., Marth G. T. (2012). ART: a next-generation sequencing read simulator, Bioinformatics 28 (4): 593-594

Rambaut A. and Grassly N. C. (1997) Seq-Gen: An application for the Monte Carlo simulation of DNA sequence evolution along phylogenetic trees. Comput. Appl. Biosci. 13: 235-238

Sukumaran, J. and Mark T. Holder. 2010. DendroPy: A Python library for phylogenetic computing. Bioinformatics 26: 1569-1571.