MetaPathways v1.0 Installation

MetaPathways v1.0 Installation Niels W. Hanson, Kishori M. Konwar, Antoine P. Pagé, and Steven J. Hallam This document explains the basic installation and setup of the MetaPathways v1.0 pipeline on a typical unix-based machine including downloading and installing required software, Pathway Tools installation, reference sequence database installation, basic configuration, and Sun Grid engine setup. Further details of the MetaPathways v1.0 pipeline are described in the companion June 2013 BMC Bioinformatics article. 1 Downloading MetaPathways Download the zip file MetaPathways v1.0.zip from http://hallam.microbiology.ubc. ca/metapathways/ or the GitHub releases page. After you have downloaded the file, unzip and inspect the contents of the MetaPathways/ folder (Figure 1). A Tour of the MetaPathways/ folder: blastdb/ place where BLAST databases are stored along with name-mapping and taxonomic support files for specific databases like KEGG and COG daemon.py a script that carries out external operations on super-computing grids using the Sun Grid engine executables/ contains various analytical and data handling programs that process the inputs and outputs of different steps of the pipeline e.g. BLAST, Prodigal, trna-scan, etc. libs/ the code library folder contains different Perl and Python functions and code that coordinate different steps of the pipeline MetaPathways.py the starter script/program that runs the pipeline with specific configuration and parameter settings for each of the steps 1

Figure 1: An example of the MetaPathways/ folder from the MetaPathways v1.0.zip file. Notice that the folder has a number of different files and folders inside it. The template configuration (template config.txt) and parameter configuration (template param.txt) files are used to configure and set parameter settings of each of the analytical steps of the pipeline. Additionally, the Python script, MetaPathways.py, is used to start the pipeline. MetaPathwaysrc a source file that must be run to ensure that the computer system knows where the MetaPathways/ folder, sets the local python and perl paths, and compiles some executable code template config.txt a parameter file that specifies the analytical settings for all pipeline steps. e.g. BLAST cut-offs, steps to include in a run of the analysis, what order to annotate databases in, etc. template header.txt a template header for output GenBank (.gbk) files template param.txt a parameter file that specifies the analytical settings for all pipeline steps. e.g. BLAST cut-offs, steps to include in a run of the analysis, what order to annotate databases in, etc. testdata/ contains some simple.fasta files to do a dry-run to ensure that everything in the pipeline is working properly For simplicity we are going to perform this installation out of the user home folder /User/[username]/ by default. In unix commands the tilde character is equivalent to 2

your home directory. In OSX systems the home folder can be found through any of the following: Double-click the Macintosh HD on the Desktop Right-click (control-click) the Finder icon in the Dock and select New Finder Window Left-click the Finder icon and press (command + n) Go to home from any finder folder by pressing (shift + command + h) Drag-and-drop the newly extracted MetaPathways v1.0/ folder into the home directory. It should sit as /MetaPathways/ when accessing it through the terminal. MetaPathways requires the use of the unix command-line terminal to run. On OSX systems this is done through the Terminal program located in: Applications > Utilities > Terminal You may want to place this program on your OSX Dock for future convenience. 2 Installing programming languages Python, Perl, and GCC. Install the required Python 2.x, Perl 5.x, and GCC compiler. For OSX users, these are all contained within the current release of Xcode4 which can be obtained for free from https://developer.apple.com/xcode/ or on the Apple App Sore within modern releases of OSX. Alternatively, Perl, and Python installation files and documentation can be obtained from their respective websites: Python 2.x http://docs.python.org/using/unix.html Perl 5.x http://www.perl.org/get.html GCC http://gcc.gnu.org These also can be obtained through a package management system like Synaptic. Though in the case of many Unix distributions, like the popular Ubuntu, versions of Python, Perl, and GCC are included by default, but you will want to ensure that they are the proper versions. 3

In many instances, installing new programming languages is quite low-level from an OS perspective, and may require some discussion with your local system administrator. A restart of the computer might also be required. It is also a good idea to open the terminal after installation to check if these installations made it to your system s $PATH variable using the which command: # tests to see if a perl, python, or gcc are included in your $PATH variable $ which perl /usr/bin/perl $ which python /usr/bin/python $ which gcc /Developer/usr/bin/gcc 3 Install Pathway Tools One of the final steps of the MetaPathways pipeline uses the software Pathway Tools to build a Pathway/Genome Database (PGDB) from environmental nucleotide sequences. The Pathway Tools software can be obtained directly from SRI International and will require obtaining an academic licence for the software (http://biocyc.org/download.shtml). This is free for academic users and usually takes approximately 1-2 business days to approve. Problems with licensing can be emailed to ptools-info@ai.sri.com. SRI International provides installation instructions for OSX and Unix, and is extensively documented at its homepage: http://bioinformatics.ai.sri.com/ptools/. Eventually you receive an email from the Pathway Tools group that will allow you to download the Pathway Tools software (Figure 2) In short, you will obtain an install file like pathway-tools-17.0-macosx-tier1-install.dmg and upon mounting this folder to the desktop a folder with a file that starts an installation wizard (Figure 3). For ease of instruction we encourage the use of the default installation locations of Pathway Tools directories in the standard home folder locations: /pathway-tools and /ptools-local. pathway-tools/ contains the actual Pathway Tools software ptools-local/ pipeline contains the PGDBs once they have been built via the MetaPathways 4

Figure 2: Pathways tools software download table outlining the different versions. For most metagenomic purposes the basic configuration with just the EcoCyc and MetaCyc databases will be fine (outlined in red). Figure 3: The Pathway Tools install wizard for OSX. We recommend that installation defaults are followed, placing the pathway-tools/ and ptool-local directories in their default location of the user root folder. On typical installations these are placed in the user s home directory by default. OSX installations may prompt the installation of XQuartz please allow the installation of XQuartz to finish before continuing on with the Pathway Tools installation. 5

On OSX systems the a window during the Pathway Tools installation will prompt installation of xquartz. This will download an additional.dmg file to install xquartz. Allow the installation of xquartz to finish before continuing with the Pathway Tools installation. On some systems, installation of xquartz may require a manual restart. Please restart your system prior to running Pathway Tools for the first time. After installing Pathway Tools you can launch it from the terminal by executing the following from the command line: $ cd $./pathway-tools/pathway-tools Or from the shortcut icons that it placed on your desktop during installation. 4 BLAST Databases The Basic Local Alignment Search Tool (BLAST) is used for a number of pipeline steps; specifically the Open Reading Frame (ORF) functional annotation and the taxonomic identification of sequences through RNA homology. In order to perform this step locally you need a copy of some sequence reference databases to search. We have provided a few databases to get started: MetaCyc (metacyc-v5-2011-10-21) a sub-set of Uniprot corresponding with the sequences in the MetaCyc database. This is included with the Pathway Tools software (uniprot-seq-ids.seq) just reformatted into the common.fasta format Cluster of Orthologous Groups of proteins (COG 2013-02-05) A protein database containing taxonomically specific clusters of functional proteins Silva LSU (LSURef 111 tax silva) LSU rrna nucleotide sequences for taxonomic identification However, the choice of database often depends on the specific scientific question you are asking. As such, many databases are freely maintained for download from public ftp servers. However, these databases are large and they grow in size every day. Downloads add up to many gigabytes (GBs), so a high-speed internet connection will be required. Also many of these are hosted on file transfer protocol (ftp) servers, we recommend Cyberduck http://cyberduck.ch as a free, simple, and user-friendly ftp client. By default, MetaPathways is configured to detect databases in the blastdb/ folder. Below we outline some basic instructions for obtaining other popular databases for metagenomic analysis. 6

Protein Databases RefSeq RefSeq is a major protein reference database maintained by the National Center of Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov/refseq/. Refseq provides formatted BLAST databases on its ftp server: connect to the BLAST database ftp server ftp://ftp.ncbi.nlm.nih.gov/blast/db download the set of files named refseq protein.xx.tar.gz, where XX are numbers extract the.tar.gz archives (usually by simply double-clicking on them) MetaPathways actually requires the original fasta sequences of the RefSeq database to start. Extract the sequences from the refseq protein BLAST database using the blastdbcmd or the older fastacmd: $ blastdbcmd -db refseq protein -dbtype prot -outfmt %f -out Refseq 2013 $ fastacmd -D 1 -d refseq protein -o Refseq 2013 Both the blastdccmd and the legacy fastacmd can be found from the BLAST Software and Databases webiste provided by the NCBI. KEGG The Kyoto Encyclopedia of Genes and Genomes http://www.genome.jp/kegg/ and http://www.bioinformatics.jp/en/keggftp.html. MetaPathways is configured to handle KEGG annotations and provide summary tables. Unfortunately, KEGG now requires a subscription fee to access its databases. However, once sequences are obtained they can be simply placed in the blastdb/ folder. Nucleotide Taxonomic Databases Silva Silva is a comprehensive ribosomal database project. Visit the Silva website http://www.arb-silva.de/download/ navigate links: Download Archive Current Exports download the current SSU database (SSURef 111 NR tax silva.fasta.tgz) and the current LSU database (LSURef 111 tax silva.fasta.tgz) 7

GreeneGenes 16S rrna gene database and workbench compatible with ARB. Visit the GreeneGenes website http://greengenes.lbl.gov/cgi-bin/nph-index.cgi navigate links: Download Sequence Data Fasta data files download current GREENGENES gg16s unaligned.fasta.gz Once again, one need only download the databases in.fasta format in place them in the blastdb/ folder. MetaPathways is programmed to do automatic formatting of them on-the-fly. 5 Configuring the template config.txt The template config.txt file configures the pipeline to find the resources it needs to run. Paths will have to be set for the PERL EXECUTABLE, PYTHON EXECUTABLE, PATHOLOGIC EXECUTABLE, REFDBS, and METAPATHS PATH. Direct the Terminal to the MetaPathways/ folder and source the MetaPathwaysrc file compiling the Perl and Python code and locating Perl, Python and the MetaPathways directory for the config file: $ cd MetaPathways/ $ source MetaPathwaysrc Checking for Python and Perl: Python found in /usr/bin/python Please set variable PYTHON_EXECUTABLE in file template_config.txt as: PYTHON_EXECUTABLE /usr/bin/python Perl found in /usr/bin/perl Please set variable PERL_EXECUTABLE in file template_config.txt as: PERL_EXECUTABLE /usr/bin/perl Adding installation folder of MetaPathways to PYTHONPATH Your MetaPathways is installed in : Please set variable METAPATHWAYS_PATH in file template_config.txt as: METAPATHWAYS_PATH /Users/username/MetaPathways 8

Follow the printed instructions and update the PYTHON EXECUTABLE, PERL EXECUTABLE, METAPATHWAYS PATH, PATHOLOGIC EXECUTABLE, and SYSTEM keyword in template config.txt (Figure 4). The METAPATHWAYS PATH and PATHOLOGIC EXECUTABLE represent the absolute paths to MetaPathways and Pathways Tools, respectively. Figure 4: An example of how to edit the template config.txt file for MetaPathways setup. In most cases, one only needs to edit the PYTHON EXECUTABLE, PERL EXECUTABLE, METAPATHWAYS PATH, the PATHOLOGIC EXECUTABLE, and then replace the SYSTEM keyword with ether mac, linux, or win depending on the operating system. These are highlighted in the red boxes on the left, and in blue boxes on the right during an example setup on the for a Mac OSX operating system. 6 Configuring the template param.txt The template param.txt file defines the parameter settings of all the analytical steps in a MetaPathways run. It needs to be updated with the exact names of your protein and nucleotide databases in the blastdb/ folder (Figure 5). 7 Connecting with the Grid (optional) MetaPathways has capability to externalize computationally heavy tasks like protein BLAST searches to super computing facilities, provided they use the Sun Grid Engine. This is an optional, but highly recommended step. However, this requires having ssh 9

Figure 5: The template param.txt file. The exact names of the BLAST databases need to be listed in the above highlighted lines. These must be the exact names of the database sequence files in the blastdb/ folder. access and sufficient user permissions to set up password-less access on a compute server. This might be a good time to check with your local system administrator and ask if this kind of setup is permissible. We ve outlined some basic steps of this process: 1. test to see if you can connect to your account via ssh: $ ssh username@server.address.com 2. You should be asked for your password 3. check to see there is a.ssh/ folder in your remote home directory $ ls /.ssh/ $ authorized keys known hosts 4. if not you should create it $ mkdir /.ssh/ 5. return to your local computer (control + d) 6. navigate to the local /.ssh/ directory $ cd /.ssh/ 10

7. run ssh-key to create a RSA public and private key $ ssh-keygen -t rsa Generating public/private rsa key pair. Enter passphrase (empty for no passphrase): Enter same passphrase again: Your identification has been saved in id_rsa. Your public key has been saved in id_rsa.pub. Enter file in which to save the key (/Users/username/.ssh/id_rsa): Enter passphrase (empty for no passphrase): Enter same passphrase again: Your identification has been saved in id_rsa. Your public key has been saved in id_rsa.pub. 8. Copy your public key to your grid.ssh/ folder with scp $ scp id rsa.pub user@user.server.com: /.ssh/ 9. Log back in to your external server account using ssh $ ssh username@server.address.com 10. Navigate to the /.ssh/ directory again $ cd /.ssh/ 11. append the public key to a file called authorized keys $ cat id rsa.pub >> authorized keys 12. change the permissions of the authorized keys file and.ssh/ directory such that only your username can read/write it $ chmod 600 /.ssh/authorized keys $ chmod 700 /.ssh/ 13. logout to your local computer pressing (control + d) 14. again try to login using ssh, you should not need to type in your password this time $ ssh username@server.address.com If this above procedure did not help then you likely have a more complicated setup on your hands. At this point it would be good to speak with a local system administrator to help you setup keyless login. If this is not possible, a Google term would be ssh keyless login Congratulations! You have completed what is in some cases an convoluted and unintuitive setup, but with some luck the MetaPathways pipeline ready for action. Now that you 11

have come so far you will likely want to use it. You can now proceed to obtain some.fasta files full of sample sequences and let the analysis commence. Its use is simple if you are familiar with the Unix command line, however, we have provided some basic examples and use cases. 8 MetaPathways Use and Setup Running MetaPathways 1. Setting Parameters Preparing for your MetaPathways run Before we start our first run of the pipeline we will again take a look at the parameters contained in template param.txt. This file gives all the instructions and settings to be run for each step of the pipeline. Many of the default settings found in template param.txt are general and should be adequate for many metagenomic analyses. However, often one will have to remember to change these to reflect the questions and goals one has about their specific dataset. Settings in this file are in the form of parameter/value separated by spaces; multiple values are separated by commas: parameter value parameter value1,value2,... INPUT: format specifies the type of input file. Possible values include: fasta, gbk-annotated, and gbk-unannotated. Annotated and unannotated correspond to the existing gene annotations contained within the or GenBank (gbk) input files. QC parameters quality control:min length specifies the minimum number of nucleotides a sequence must have during the QC phase quality control:delete replicates removes duplicate sequences from input ORF prediction parameters orf prediction:algorithm specifies the ORF prediction algorithm that is used. Currently only Prodigal is available orf prediction:min length specifies the minimum number of amino acids in a predicted ORF Annotation parameters 12

annotation:algorithm specifies which homology search algorithm to use for ORF annotation. Current options are blast and last are more-efficient implementation of the seed-and-extend approximation algorithm annotation:dbs specifies which protein databases and in what order they will be used for annotation. Database names are separated by commas, and the names must exactly match the naming convention in the database folder blastdb/ annotation:min bsr specifies the minimum blast-score ratio threshold. Only hits greater than the threshold will be kept. annotation:max evalue specifies the maximum e-value threshold. Only e-values smaller (more statistically significant) than this threshold will be kept. annotation:min score specifies the minimum bit-score threshold. Only hits greater than this score will be kept. annotation:min length specifies the minimum length threshold. Only annotations with a greater length will be kept. annotation:max length specifies the maximum number of annotations to be kept for each search. Usually the top-5 or top-10 homology hits are sufficient for most pourposes. RNA parameters Analogous to the protein homology search settings above: rrna:refdbs specifies the databases to be searched against. These database names must match the names of the nucleotide BLAST databases found in the blastdb/ folder specified in pipeline configuration file rrna:max evalue sets the 16s rrna maximum expect value threshold. Only hits less than (more statistically significant) than this threshold will be kept rrna:min identity sets the minimum percent identity threshold. Only annotations with a greater percent identity with the query sequence will be kept rrna:min bitscore only annotations with bit-scores greater than this minimum threshold will be kept. Grid Settings Settings associated with running protein homology searches on the grid grid engine:batch size specifies the number of sequences to be included in each grid job. This should be set to respect the memory and cpu time requirements of the grid you are using grid engine:max concurrent batches sets the maximum number of jobs to be submitted to a grid at one time. MetaPathways will maintain a job queue of this size waiting to be scheduled grid engine:walltime sets the maximum amount of time an individual job can take. Setting this value too high affects your scheduling by the SunGrid 13

scheduler. Setting it too low allows you to be schedule but your job will be stopped before completion. grid engine:ram the maximum ram usage for the job. Also can affect the scheduling of your jobs. Becomes an issue for larger databases such as RefSeq grid engine:user username used to access the grid via ssh grid engine:server the address of the compute grid via ssh Pathway Tools parameters ptools settings:taxonomic pruning specifies if the epgdb in Pathway Tools should be built with taxonomic pruning enabled (yes) or disabled (no). Disabled is recommended for metagenomic samples. Single-cell analyses may want to consider enabling it. 2. Pipeline Execution Flags yes, skip, stop, redo, grid For each step of the pipeline one must specify one of the following actions: yes perform the operation with the above settings skip do not perform this operation (note that this could cause later dependent steps in the pipeline to fail) stop stop the pipeline run after completing the previous step redo recompute a specific step of the pipeline (after incomplete execution or error may have corrupted the output) grid compute this step on the grid. Currently only available for the BLAST/LAST homology search step 3. Starting a Run The MetaPathways pipeline is run using the MetaPathways.py script from the command line: $./MetaPathways.py -i [input file/folder] -o [output directory] -c [config file] -p [parameter file] -r [overwrite/overlay] For example, $./MetaPathways.py -i testdata/ -o /MetaPathways/output -c /MetaPathways/template_config.txt -p /MetaPathways/template_param.txt -r overlay -v 14

where, -i specifies the input file directory or specific.fasta file -o specifies the output directory -c the configuration file to be used for this run -p the parameter file to be used for this run -r the run-style to be use for this run: overlay check for existing run in place and uses existing files as it finds them except if the pipeline step is set to redo overwrite overwrites existing output -v verbose output displays the exact commands being run for each step The script testmetapathways.sh will do a simple run on sequences in the testdata/ folder: $ testmetapathways.sh 15