Shell script pipeline for inferring ML gene trees for many loci (e.g. genomic RAD loci)
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README.md

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Shell script pipeline for inferring ML gene trees for many loci (e.g. genomic RAD loci)

LICENSE

All code within the PIrANHA repository, including MAGNET v0.1.6 pipeline code, is available "AS IS" under a generous GNU license. See the LICENSE file for more information.

CITATION

If you use scripts from this repository as part of your published research, then I require you to cite the PIrANHA repository and/or MAGNET package as follows (also see DOI information below):

Bagley, J.C. 2017. PIrANHA. GitHub repository, Available at: http://github.com/justincbagley/PIrANHA/.

Bagley, J.C. 2017. MAGNET v0.1.6. GitHub package, Available at: http://github.com/justincbagley/MAGNET.

Alternatively, please provide the following link to this software program in your manuscript:

http://github.com/justincbagley/MAGNET

Example citations using the above URL: "We estimated a gene tree for each RAD locus in RAxML v8 (Stamatakis 2014) using the MAGNET v0.1.6 pipeline (http://github.com/justincbagley/MAGNET). Each RAxML run specified the GTRGAMMA model and coestimated the maximum-likelihood phylogeny and bootstrap proportions from 500 bootstrap pseudoreplicates."

DOI

The DOI for MAGNET, via Zenodo, is as follows: DOI. Here is an example of citing MAGNET using the DOI:

Bagley, J.C. 2017. MAGNET. GitHub package, Available at: http://doi.org/10.5281/zenodo.166024.

INTRODUCTION

The estimation of species-level phylogenies, or "species trees" is a fundamental goal in evolutionary biology. However, while "gene trees" estimated from different loci provide insight into the varying evolutionary histories of different parts of the genome, gene trees are random realizations of a stochastic evolutionary process. Thus, gene trees often exhibit conflicting topologies, being incongruent with each other and incongruent with the underlying species tree due to a variety of genetic and biological processes (e.g. gene flow, incomplete lineage sorting, introgression, selection).

With the advent of recent advances in DNA sequencing technologies, biologists now commonly sequence data from multiple loci, and even hundreds to thousands of loci can quickly be sequenced using massively parallel sequencing on NGS sequencing platforms. Full-likelihood or Bayesian algorithms for inferring species trees and population-level parameters from multiple loci, such as *BEAST and SNAPP, are computationally burdensome and may be difficult to apply to large amounts of data or distantly related taxa (or other cases that complicate obtaining MCMC convergence). By contrast, a number of recently developed and widely used "summary-statistics" approaches rely on sets of gene trees to infer a species tree for a set of taxa (reviewed by Chifman and Kubatko, 2014; Mirarab and Warnow, 2015). These methods are specifically designed to estimate gene trees or use gene trees input by the user, which are treated as observed data points analyzed in a distance-based or coalescent algorithm. Moreover, summary-statistics approaches to species tree inference tend to be accurate and typically much faster than full-data approaches (e.g. Mirarab et al., 2014; Chifman and Kubatko, 2014). Examples of species tree software in this category include programs such as BUCKy (Larget et al., 2010), STEM (Liu et al., 2010), spedeSTEM, NJst (Liu and Yu, 2011), ASTRAL and ASTRAL-II (Mirarab and Warnow, 2015), and ASTRID (Vachaspati and Warnow, 2015). Phylogenetic network models implemented in recent software like SplitsTree and SNaQ also improve network and inference by analyzing sets of gene trees.

Despite the importance of gene trees in species tree and network inference, few resources have been specifically designed to aid rapid estimation of gene trees for different loci. MAGNET (MAny GeNE Trees) is a shell script pipeline within the PIrANHA (PhylogenetIcs ANd PHylogeogrAphy) repository (https://github.com/justincbagley/PIrANHA) that helps fill this gap by automating inferring a maximum-likelihood (ML) gene tree for each locus in a multilocus dataset. Here, the term "locus" is used loosely to refer to a DNA alignment of homologous nucleotide characters including both variable and invariant DNA sites. The MAGNET package was originally coded up to aid analyses of RAD loci generated by massively parallel sequencing of ddRAD-seq genomic libraries (Peterson et al. 2012). However, MAGNET can be used to estimate gene trees from any multilocus dataset in the appropriate format, and three starting file types are supported: single NEXUS, single G-PhoCS, or multiple PHYLIP files.

HARDWARE AND SETUP

💻 MAGNET focuses on allowing users to automate the workflow necessary for quickly estimating many gene trees for many loci on their local machine.

👍 No special hardware or setup is necessary, unless the user is interested in estimating gene trees on a remote supercomputing cluster (see below).

SOFTWARE DEPENDENCIES

MAGNET v0.1.6 is composed of shell, R, and Perl scripts and also calls several software programs; thus, it relies on several software dependencies. These dependencies are described in some detail in README files for different scripts in the package. However, here I provide a list of them, with asterisks preceding those already included in the MAGNET distribution:

Users must install all software not included in MAGNET, and ensure that it is available via the command line on their local machine. On the user's local machine, Perl should be available by simply typing "Perl" at the command line; Python should be available by typing "python" at the command line; and bioscripts.convert package should be available by typing "convbioseq" at the command line. Also, RAxML should be compiled using SSE3 install commands, so that RAxML can be called by simply typing "raxmlHPC-SSE3" on the command line. For detailed instructions for setting up RAxML this way, refer to the newest RAxML user manual (available at: http://sco.h-its.org/exelixis/resource/download/NewManual.pdf).

INPUT FILE FORMAT

MAGNET assumes that you are starting from multilocus DNA sequence data in one of three formats. The First format that is supported is that of a single datafile in G-Phocs (Gronau et al. 2011) format, with the extension ".gphocs". The Second format that is supported is NEXUS format, with data in a single file having the extension ".nex". For genomic data in aligned sequence format, such as aligned RAD tags (e.g. ddRAD-seq contigs) or other SNP data derived from genotyping-by-sequencing (GBS) methods, the user should assemble the data, call SNPs, and output SNP sequence data files in .gphocs or .nex format prior to running MAGNET. This can easily be done by running pyRAD or ipyrad (Eaton 2014) while calling for output in all formats (*; you'll get .gphocs and .nex files).

However, this may not always be possible, and .gphocs format is not yet among the most popular file formats in phylogenomics/population genomics. Thus, I have added a "NEXUS2gphocs.sh" shell script utility within MAGNET (in the "shell" folder) that will convert a sequential NEXUS file into .gphocs format for you. An example NEXUS file "example.nex" is included in the distribution. Feel free to use the NEXUS2gphocs.sh utility script independently of MAGNET to convert from NEXUS to .gphocs format. However, when doing this, make sure to follow the usage guidelines below.

The Third format that is supported in MAGNET is that of DNA sequence alignments for multiple loci contained in separate PHYLIP files for each locus.

Users must specify the input fileType with the -f flag. Options are 1 for a single G-PhoCS- or NEXUS-formattted inputFile, or 2 for the multiple PHYLIP option. If -f 1, then the program will expect as standard input (stdin) the name of the inputFile. However, if -f 2, then you should supply MAGNET with the path to the desired working directory; often, this will simply be the current working directory, in which case the user can simply type "." for workingDir, but the relative or absolute path to the workingDir will also work fine.

PIPELINE

Apart from input file conversion steps, the MAGNET pipeline works by calling five different scripts, in series, each designed to conduct a task whose output is processed in the next step of the pipeline. First, the "gphocs2multiPhylip.sh" shell script is used to extract loci from the input file and place each locus in a PHYLIP-formatted file with extension ".phy". Second, a shell script named "MultiRAxMLPrepper.sh" is used to place the .phy files into separate "run folders" and prepare them to be run in RAxML. Third, a script named "RAxMLRunner.sh" is called to run RAxML on the contents of each run folder. In a "clean-up" step, MAGNET moves all .phy files files remaining in the working directory to a new folder, "phylip_files", which is created within the working directory.

After running the MAGNET pipeline, the shell script "getGeneTrees.sh" automates post-processing of the gene trees output by RAxML, including organizing all inferred gene trees into a single "gene_trees" folder in the working directory, and combining the individual 'best' gene trees resulting from each run into a single file named "besttrees.tre". Also, if bootstrap pseudoreplicates were performed and the bootstrap tree files are detected, then the "getBootTrees.sh" script conducts similar processing on the bootstrap trees for each loucus, which are collated, renamed, and given a list file containing the name of each file. Given the directory of bootstrap trees resulting from a MAGNET run ("bootstrap_trees") can take up substantial disk space (>200 MB), users may wish to compress this directory to a zip file, for example using $ zip -r bootstrap_trees.zip bootstrap_trees/ at the conclusion of a run.

USAGE

Additional input file and usage information is available in the usage or help texts. To get regular usage info for MAGNET, type $ ./MAGNET.sh, $ ./MAGNET.sh -h ., or ./MAGNET.sh -help while in the MAGNET directory. However, it is more useful (particularly when running for the first time) to get verbose usage info for MAGNET, including detailed descriptions of each option; do this by typing $ ./MAGNET.sh -H . or ./MAGNET.sh -Help (capital "H" flag) at the command line while in the MAGNET directory. The verbose usage text is as follows:

$ ./MAGNET.sh
Usage: $(basename "$0") [Help: -h H] [Options: -f e b r s g m o] [stdin:] inputFile or workingDir
 ## Help:
  -h   help text (also: -help)
  -H   verbose help text (also: -Help)

 ## Options:
  -f   fileType (def: 1; 1 = single inputFile, 2 = multiple PHYLIP files) starting file
       type; if 1, script expects as stdin a single NEXUS or G-PhoCS inputFile in the
       current directory; if 2, then script expects workingDir with multiple PHYLIP files
  -e   executable (def: raxmlHPC-SSE3) name of RAxML executable, accessible from command line
       on user's machine
  -b   numBootstraps (def: 100) RAxML bootstrap pseudoreplicates
  -r   raxmlModel (def: GTRGAMMA; other: GTRGAMMAI, GTRCAT, GTRCATI)
  -s   simpleModel (def: NULL; other: JC69, K80, HKY85) specifies simple substitution model
       that will override any other model and apply to all DNA partitions
  -g   gapThreshold (def: 0.001=essentially zero gaps allowed unless >1000 
       individuals; takes float proportion value)
  -m   indivMissingData (def: 1=allowed; 0=removed)
  -o   outgroup (def: NULL) outgroup given as single taxon name (tip label) or comma-
       separted list

 OVERVIEW
 The goal of MAGNET is to infer a maximum-likelihood (ML) gene tree in RAxML for each of 
 multiple loci, starting from one or multiple input files containing aligned DNA sequences.
 If supplied with a single G-PhoCS ('*.gphocs') or NEXUS ('*.nex') data file (using -f 1
 option), then this script splits each locus into a separate PHYLIP-formatted alignment file, 
 and sets up and runs RAxML (Stamatakis 2014) to infer gene trees for each locus. If a NEXUS 
 datafile is supplied, it is converted into G-PhoCS format (Gronau et al. 2011) while splitting
 loci into separate interleaved sequence blocks based on information provided in a sets
 block at the end of the NEXUS file (e.g. defined using 'charset' commands), which is mandatory. 
 However, if -f 2, then the program expects as standard input the name of a working directory 
 (e.g. the relative or absolute path) containing multiple PHYLIP-formatted alignment files. 
 Under this scenario, MAGNET will skip directly to running the PHYLIP files in RAxML using 
 user-specified options. Sequence names may not include hyphen characters, or there could be 
 issues. For detailed information on MAGNET and its various dependencies, see 'README.md' file 
 in the distribution folder; however, it is key that the dependencies are available from the 
 command line interface. 

 DETAILS
 The -f flag specifies the starting fileType. If -f 1, then the mandatory input is the name
 or path to the corresponding starting file, which will be run in the current working directory. 
 If -f 2, then mandatory input is the name or path to the working directory (type '.' for current 
 directory, or supply a relative or absolute path).
 
 The -e flag sets the name of the RAxML executable that will be called. The user may wish to
 change this to something specific to their install, or to something generic like 'raxml'.
 The default setting should work on local machine or supercomputing cluster installs.
 
 The -b flag sets the number of boostrap pseudoreplicates for RAxML to perform while estimating 
 the gene tree for each locus. The default is 100; remove bootstrapping by setting to 0.

 The -r flag sets the RAxML model for each locus. This uses the full default GTRGAMMA model,
 and at present it is not possible to vary the model across loci. If you want to use HKY
 or K80, you will need to use the -s flag (below).

 The -s flag sets a simple RAxML model for each locus/partition, which will override any
 model set using the -r flag above and apply to all partitions. In the current version of 
 RAxML, it is possible to specify the JC69, K80, and HKY85 models as overrides. By default,
 this option is turned off and the model set under the -r flag is used instead.

 The following two options are available **ONLY** if you are starting from a NEXUS input file:

	The -g flag supplies a 'gap threshold' to an R script, which deletes all column sites in 
	the DNA alignment with a proportion of gap characters '-' at or above the threshold value. 
	If no gap threshold is specified, all sites with gaps are removed by default. If end goal
	is to produce a file for G-PhoCS, you  will want to leave gapThreshold at the default. 
	However, if the next step in your pipeline involves converting from .gphocs to other data 
	formats, you will likely want to set gapThreshold=1 (e.g. before converting to PHYLIP 
	format for RAxML). 

	The -m flag allows users to choose their level of tolerance for individuals with missing
	data. The default is indivMissingData=1, allowing individuals with runs of 10 or more 
	missing nucleotide characters ('N') to be kept in the alignment. Alternatively, setting
	indivMissingData=0 removes all such individuals from each locus; thus, while the input
	file would have had the same number of individuals across loci, the resulting file could
	have varying numbers of individuals for different loci.

 The -o flag sets the outgroup exactly the same way as that described in the RAxML v8 user's
 manual, as a single name or as a comma-separated list with no spaces between taxon names. 
 The first name in the list is prioritized, e.g. when members of the list are not monophyletic.

 CITATION
 Bagley, J.C. 2017. MAGNET v0.1.6. GitHub package, Available at: 
	<http://github.com/justincbagley/MAGNET>.
 or
 Bagley, J.C. 2017. MAGNET v0.1.6. GitHub package, Available at: 
	<http://doi.org/10.5281/zenodo.166024>.

 REFERENCES
 Gronau I, Hubisz MJ, Gulko B, Danko CG, Siepel A (2011) Bayesian inference of ancient human 
	demography from individual genome sequences. Nature Genetics, 43, 1031-1034.
 Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of 
	large phylogenies. Bioinformatics, 30, 1312-1313.

NOTES ON NEXUS2gphocs USAGE

  • You may use NEXUS2gphocs.sh as a standalone script for converting prior to running G-PhoCS on your data.
  • However, in its current form, you must move NEXUS2gphocs.sh (out of the shell folder) and rmGapSites.r (out of the R folder) into the MAGNET directory in order to run NEXUS2gphocs as a standalone script (this assumes the target inputFile is also located in the MAGNET dir). You could also move both scripts into another working directory containing your target inputFile.
  • You can get the usage info for NEXUS2gphocs.sh, in similar fashion to that above, by typing ./NEXUS2gphocs.sh, ./NEXUS2gphocs.sh -h ., or ./NEXUS2gphocs.sh -help into the command line, and then pressing enter. The NEXUS2gphocs usage text is sufficiently similar to the latter part of the MAGNET usage printed above that it doesn't bear repeating here.

USAGE EXAMPLES

Below I give some examples of how to use the software under the two most common scenarios:

SCENARIO 1. If your data contain very little missing data and, in particular, they contain no individuals with all missing data for a locus, then it should be fine to run MAGNET using the default options on either a single input file (-f 1) or multiple PHYLIP input files (-f 2), as follows:

##--Scenario 1, generic usage:
./MAGNET.sh -f1 inputFile
./MAGNET.sh -f2 workingDir     ## multiple PHYLIP input files case.

##--Examples:
cd ~/Downloads/MAGNET-master/
./MAGNET.sh -f1 example.nex
./MAGNET.sh -f2 .     ## multiple PHYLIP input files case.

SCENARIO 2. If your data are relatively lower quality data (e.g. from NGS runs) and you have lots of missing data, including individuals with all missing data for a locus (as is common for RAD tag/SNP data), then RAxML will not run properly under the default MAGNET options. You will likely get up to ~10 messages like "ERROR: Sequence XXXXX consists entirely of undetermined values which will be treated as missing data", follwed by a summary like this: "ERROR: Found 10 sequences that consist entirely of undetermined values, exiting...", and RAxML will quit. The rest of the pipeline will be affected, for example the final summary gene tree file will make no sense because it will simply include a concatenation of all files in the working directory.

To avoid the above issues caused by large amounts of missing data, you should run MAGNET while setting the -m flag to 0 (indivMissingData=0) to specify that individuals with missing data are NOT allowed:

##--Scenario 2, all params except indivMissingData set to default options:
./MAGNET.sh -f1 -m0 inputFile
./MAGNET.sh -f1 -m0 workingDir     ## multiple PHYLIP input files case.

##--Example:
cd ~/Downloads/MAGNET-master/
./MAGNET.sh -f1 -m0 example.nex
./MAGNET.sh -f2 -m0 .     ## multiple PHYLIP input files case.

In addition to the above, here are illustrations of varying the RAxML options:

##--Scenario 1, GTRCAT model, instead of the default GTRGAMMA model:
./MAGNET.sh -f1 -rGTRCAT inputFile
./MAGNET.sh -f2 -rGTRCAT .     ## multiple PHYLIP input files case.

##--Scenario 1, adding name of an outgroup taxon:
./MAGNET.sh -f1 -rGTRCAT -o outgroup inputFile
./MAGNET.sh -f2 -rGTRCAT -o outgroup .     ## multiple PHYLIP input files case.

##--Scenario 1, overriding -r model with HKY85 and adding an outgroup:
./MAGNET.sh -f1 -rGTRCAT -sHKY85 -o outgroup inputFile
./MAGNET.sh -f2 -rGTRCAT -sHKY85 -o outgroup .     ## multiple PHYLIP input files case.

##--Scenario 2, 500 bootstrap reps per locus, instead of the default 100:
./MAGNET.sh -f1 -b500 -m0 inputFile
./MAGNET.sh -f2 -b500 -m0 .     ## multiple PHYLIP input files case.

##--Scenario 2, *zero* bootstrap reps per locus:
./MAGNET.sh -f1 -b0 -m0 inputFile
./MAGNET.sh -f2 -b0 -m0 .     ## multiple PHYLIP input files case.

ACKNOWLEDGEMENTS

I gratefully acknowledge Nayoki Takebayashi, who wrote and freely provided some Perl scripts I have used in PIrANHA and MAGNET. I also thank the Brigham Young University Fulton Supercomputing Lab (FSL) for providing computational resources used during the development of this software. J.C.B. received stipend support from a Ciência Sem Fronteiras (Science Without Borders) postdoctoral fellowship from the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Processo 314724/2014-1). Lab and computer space was also supplied by The University of Alabama, during an internship in the Lozier Lab in the UA Department of Biological Sciences.

REFERENCES

  • Chifman J, Kubatko L (2014) Quartet inference from SNP data under the coalescent model. Bioinformatics, 30, pages 3317–3324.
  • Eaton DAR (2014) PyRAD: assembly of de novo RADseq loci for phyloge-netic analyses. Bioinformatics, 30, 1844–1849.
  • Gronau I, Hubisz MJ, Gulko B, Danko CG, Siepel A (2011) Bayesian inference of ancient human demography from individual genome sequences. Nature Genetics, 43, 1031-1034.
  • Larget BR, Kotha SK, Dewey CN, Ané C (2010) BUCKy: gene tree/species tree reconciliation with Bayesian concordance analysis. Bioinformatics, 26(22):2910-2911.
  • Liu L, Yu L, Edwards SV (2010) A maximum pseudo-likelihood approach for estimating species trees under the coalescent model. BMC Evol Biol, 10(1):302.
  • Liu L, Yu L (2011) Estimating species trees from unrooted gene trees. Syst Biol, 60(5):661-667.
  • Mirarab S, Warnow T (2015) ASTRAL-II: coalescent-based species tree estimation with many hundreds of taxa and thousands of genes. Bioinformatics, 30:44-52.
  • Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. PLoS One, 7, e37135.
  • Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30.9, 1312-1313.
  • Vachaspati P, Warnow T (2015) ASTRID: Accurate Species TRees from Internode Distances. BMC Genomics, 16(Suppl 10):S3.

September 27, 2017 Justin C. Bagley, Richmond, VA, USA