Installation¶
TTT Proteotyping Pipeline consists of several steps:
- raw to mzXML conversion with ReAdW.
- peptide identification with X!Tandem.
- X!Tandem xml output to FASTA conversion with convert_tandem_xml_2_fasta.py.
- Peptide to reference database alignment with BLAT.
- Antibiotic resistance detection with Proteotyping.
- Taxonomic composition estimation with Proteotyping.
- Listing distinct/unique proteins in X!Tandem output with create_unique_protein_list.py.
ReAdW, X!Tandem, and BLAT are external programs. This documentation will give
brief instructions on how to install and use them together with TTT
Proteotyping Pipeline. The Python programs are a part of this Python package,
available in the TTT_proteotyping_pipeline
folder
in the repository.
The steps can be run manually, or preferably via the included Snakemake script.
Download the code¶
To download the code, clone the repository:
$ hg clone https://bitbucket.org/chalmersmathbioinformatics/TTT_proteotyping_pipeline
This will clone the entire repository to a folder called TTT_proteotyping_pipeline in your current directory.
ReAdW¶
ReAdW is meant to be run under Windows, but can be run under Linux using Wine, see instructions below. Running ReAdW in Wine requires a Linux system with a working 32-bit Wine installation.
Note
It is important that you use 32-bit Wine, as ReAdW cannot be run under 64-bit Wine. As of this writing, 32-bit Wine is only available for RedHat Enterprise Linux 6 and below. Support for 32-bit Wine was removed in RHEL 7.
Get ReAdW¶
ReAdW can be downloaded from the ReAdW Github repository. Either clone the
entire repository or download the binary suitable for your system. Note the
information about the dependencies on three Windows DLL files:
XRawfile2.dll
, fileio.dll
, fregistry.dll
. These files are NOT
supplied with this pipeline.
Create a 32-bit Wine prefix¶
Install Wine. It is important that a 32-bit version of Wine is installed, this normally means packages named
<package>.i686
instead of<package>.x86_64
. In RHEL/CentOS it can be installed like this:yum install wine
Create a Win32 prefix from which to run ReAdW. Make sure to set and export
WINEARCH=win32
during the creation of the wine prefix. Modify the command below to a path of your choice. Note that this step likely requires working X11-forwarding:export WINEARCH=win32 export WINEPREFIX=/path/to/your/desired/wineprefix winecfg
Click OK in any configuration windows that pop up.
Download winetricks to install the required Visual Studio C++ runtimes.
vcrun2010
is required for ReAdW andvcrun2008
is required for the Thermo DLL’s. Again, note that this step requires X11-forwarding to be enabled:wget https://raw.githubusercontent.com/Winetricks/winetricks/master/src/winetricks sh winetricks vcrun2010 vcrun2008
Click through any installation prompts that pop up, and after they complete, finish by registering XRawfile2.dll in your wine prefix:
wine regsvr32 XRawfile2.dll
Running ReAdW¶
Make sure to set the WINEPREFIX
environment variable to the correct path
(same directory you specified when creating the 32 bit wine prefix), then run ReAdW from
your Linux command prompt via Wine:
export WINEPREFIX=/path/to/your/desired/wineprefix
wine /path/to/ReAdW.201510.xcalibur.exe [options] /path/to/sample.raw
Now it should run.
X!Tandem¶
Download and install X!Tandem according to the instructions on the X!Tandem homepage.
To run a sample in X!Tandem, several xml-files must be prepared. There is a Python program
in TTT Proteotyping Pipeline called run_xtandem.py
that will automatically create the
required input files and run X!Tandem for you. It makes it very easy to use X!Tandem:
run_xtandem.py --output OUTFILE --db /PATH/TO/FASTA --threads N --xtandem /PATH/TO/TANDEM.EXE