MetaPathways 0.8 can be downloaded

MetaPathways 0.8 Requirements:
  • Linux/Unix, Mac OSX 10.6.x or later
  • Python 2.x
  • Perl 5.x
  • GCC
  • Pathway Tools v15.5 or later

  • Academic licenses are required for BLAST and Pathway Tools


A downloadable version of this installation page can be found here.
1. Downloading MetaPathways.

Download the zip file After you have downloaded the file, unzip and inspect the contents of the MetaPathways/ folder (Figure 1).
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Figure 1 - An example of the MetaPathways/ folder from the file. Notice that the folder has a number of additional 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,, used to start the pipeline.
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 your home directory. In OSX systems the home folder can be found by any of the following methods:
  • Double-click the "Macintosh HD" on the Desktop
  • Right-click (control-click) the "Finder" icon in the Dock finder_icon and select "New Finder Window"
  • Left-click the "Finder" icon and press: (command + n)
Click home symbol in the left-hand panel of the new window if the home directory does not appear by default. This should be named after your username.
Drag and drop the newly extracted MetaPathways/ folder into the home directory. It should sit as ~/MetaPathways/ when accessing it through the terminal.

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
  • - a script that carries out external operations on supercomputing grids using the Sun Grid engine, if available
  • 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
  • — the starter script/program that runs the pipeline with specific configuration and parameter settings for each of the steps
  • MetaPathwaysrc — a unix source file that ensures 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 configuration file that specifies the location of different programming resources on the computer. e.g. the Location of BLAST databases, Perl, Python, etc.
  • 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
Note: MetaPathways requires the use of the unix terminal to issue commands. On OSX systems this is done through the Terminal program located in:
  • Applications > Utilities > Terminal
  • Double-click to open
  • Drag the Terminal program to your Dock for convenience

2. Installing programming languages Python, Perl, and C.

Install the required Python 2.x, Perl 5.x, and C. For OSX users these are all contained within the current release of Xcode4 which can be obtained for free at or on the Apple App Sore directly within modern releases of OSX.
Note: Apple Xcode installs a large number of programming languages and related packages which makes it a large download (approx. 2GB).
Alternatively, Perl and Python installation files and documentation can be obtained from their respective websites:
These also can be obtained through a package management system like Synaptic.

Note: In many instances this may require some discussions with your local system administrator in an academic setting. A restart of the computer might also be required after installation of any new programming language. 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 perl is included in your Unix $PATH variable
$ which perl
$ which python
$ which gcc
3. Pathway Tools.

One of the final steps of the MetaPathways pipeline uses the software Pathway Tools to build a Pathway/Genome Database (PGDB) from your metagenomic sample. The Pathway Tools 16.0 software can be obtained directly from SRI International and will require obtaining an academic licence for the software ( This is free for academic users and usually takes approximately 1-2 business days to approve. Problems with licensing can be emailed to SRI International provides installation instructions for OSX and Unix, and is extensively documented at its homepage:

In short, you will obtain an install file akin to pathway-tools-16.0-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 — contains the PGDBs once they have been built
After installing Pathway Tools you can test to see if it runs by launching it from the Terminal:
$ cd ~
$ ./pathway-tools/pathway-tools
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Figure 3 - The Pathway Tools 16.0 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 Mac OSX installations these are ~pathway-tools and ~/ptools-local, respectively.
Note: There is a possibility that Pathway Tools will not launch because X11 is not installed. This is included on your original OSX install CD. On recent versions of OSX (Snow Lion 10.8.x) X11 has been dropped in favor of XQuatrz
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. Essentially we are searching for similarity between our query sequence and a set of known sequences contained in public databases. In order to perform this step locally you need a copy of the databases on your computer. We only provide the MetaCyc database (metacyc-v5-2011-10-21) which is the same as a file coupled with the Pathway Tools software (uniprot-seq-ids.seq), just reformatted into the common .fasta format.

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.
Note: These databases are large and they grow in size every day. Downloads add into the 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 as a free, simple, and user-friendly ftp client.
We will use a directory to store and compile these databases:
• create a folder in the metapathways/ directory named blastDB/

We will discuss a number of these here and go through how to obtain them:

Protein Databases

RefSeq - a major protein reference database maintained by the National Center of Biotechnology Information (NCBI)
  • connect to the BLAST database ftp server
  • 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)
Note: RefSeq comes as a pre-compiled BLAST database in a number of separate folders after extraction from their tar.gz archives You may find the following helpful to quickly transfer these to the MetaPathways/blastDB folder:

cd Downloads/
mv refseq_protein.0*/* ~/MetaPathways/blastDB/

COG - Clusters of Orthologous Groups of proteins (COGs) which is also maintained by the NCBI
  • connect to the COG ftp server
  • download the file myva which is a .fasta file containing the sequences

KEGG - The Kyoto Encyclopedia of Genes and Genomes,
Note: As of writing KEGG has requires a subscription fee.

Nucleotide Taxonomic Databases

Silva — comprehensive ribosomal database project
  • 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)

GreeneGenes — 16S rRNA gene database and workbench compatible with ARB
  • navigate links: Download > Sequence Data > Fasta_data_files
  • download current_GREENGENES_gg16S_unaligned.fasta.gz

Note: one need only download the databases in .fasta format in place them in the blastDB/ folder. MetaPathways is programed 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:
$ 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
follow the on-screen instructions and update the PERL_EXECUTABLE, PYTHON_EXECUTABLE, PATHOLOGIC_EXECUTABLE, and METAPATHWAYS_PATH variables in template_config.txt (Figure 3):
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Figure 3 — An example of the template_config.txt file containing all the configuration settings that tells MetaPathways where all its resources reside on a specific computer. Run source MetaPathwaysrc in the MetaPathways directory to find the correct entries for PYTHON_EXECUTABLE, PERL_EXECUTABLE, and METAPATHWAYS_PATH. Type in the full path of the location where you installed Pathway Tools under the PATHOLOGIC_EXECUTABLE variable.
6. Configuring template_param.txt.

The template_param.txt file needs to be updated with the exact names of your protein and nucleotide databases in the blastDB folder (Figure 4).

  • 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:
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Figure 4 — The template_param.txt folder. The exact names of the BLAST databases need to be listed in the above highlighted lines. These must be the exact names of the files in the blastDB/ folder. This will replace the generic metacyc, kegg, cog, etc. above. For instance metacyc needs to be replaced with metacyc-v5-2011-10-21, which should have been included in the original download of MetaPathways in the blastDB/ directory .
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 access and sufficient user permissions to set up password-less on a super computing server. This might be a good time to check with your local system administrator and ask if this kind of setup is permissible.
  • test to see if you can connect to your account via ssh:
    $ ssh
  • You should be asked for your password.
  • check to see there is a .ssh/ folder in your home directory
    $ ls ~/.ssh/
    authorized_keys known_hosts

  • if not you should create it:
    $ mkdir ~/.ssh/
  • press control + d to return to your local computer
  • navigate to the ~/.ssh/ directory
    $ cd ~/.ssh/
  • 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
    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

  • Copy your public key to your grid .ssh/ folder with scp
    $ scp
  • Log back in to your external server account using ssh
    $ ssh
  • Navigate to the ~/.ssh/ directory again
    $ cd ~/.ssh
  • append the public key to a file called authorized_keys
    $ cat >> authorized_keys
  • 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/

  • logout to your local computer pressing control + d
  • again try to login using ssh, you should not need to type in your password this time
    $ ssh username@server.address

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 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 detailed examples and use cases.