Sorcerer Proteomics Edition 4.3 software introduces support for TPP’s GUI interface which is called Petunia. By using Petunia, you have easier access to all the tools and options which TPP provides. This tutorial will explain how to use Petunia with your Sorcerer search results.
Background
When TPP is enabled as a post search analysis, Sorcerer uses a default set of options to run TPP as specified with the xinteract options.
For the xinteract options “-Op”, Sorcerer does the following:
Run PeptideProphet
Run ProteinProphet afterwards
In Sorcerer versions 4.3 and later, our default xinteract options have changed to “-OplA”. See your search log file to verify the options used.
The option “l” tells PeptideProphets not to downweight peptide hits with the asterisked DeltaCn scores. Top peptide hits for each spectrum are marked with asterisked DeltaCn score when runner up peptide(s) share at least 75% sequence identity with the top peptide hit. Normally TPP penalizes such peptides but with this option enabled, they are left alone. Enabling this option is useful for phospho analysis.
The option “A” tells PeptideProphet to use accurate mass binning which we’ve found improves both sensitivity and error rates.
By running TPP with these options, you automatically get the links to the pepXML viewer and protXML viewer on the Sorcerer TPP results page. The pepXML viewer is actually a link to the file interact.pep.xml. The protXML is a link to interact.prot.xml.
With the addition of Petunia, you now have the ability to specify your own options to TPP or to run TPP yourself if it wasn’t selected when you submitted the search initially. For an example of additional options possible, you can enable iProphet or use the quantitation tools XPress and ASAPRatio. While specifying your own options to TPP has always been possible on the Sorcerer through the use of the xinteract options and re-run analysis option, Petunia makes the process much easier and understandable.
Intended Usage
Petunia was originally designed as a start to finish pipeline analysis tool, from submitting the search to analyzing the results. However Petunia’s integration on Sorcerer is only intended as a post search analysis tool. For example, it is not possible to submit Sorcerer searches to the system using Petunia. While you can browse to such a database search page in Petunia, it’s not compatible with submitting Sorcerer searches.
The intended usage scenario with Petunia on the Sorcerer is you want to analyze your Sorcerer search results with TPP and specify you own options to TPP. Under the Petunia menu, the tested and approved of functions so far are under Analysis Pipeline (Sequest), they are Analyze Peptides and Analyze Proteins. The tutorial below will cover those options.
Using Petunia on the Sorcerer
1. Under the TPP Analysis tab of a completed search on your Sorcerer’s web interface, you should see a link to Petunia Viewer. Click on this link.
2. The first time you access Petunia, you will be prompted for a username and password. The default login/password is guest/guest. Once you login, you will not need to login again unless you clear your browser’s cookies.
3. After logging in, you will be at the Home page of Petunia. For a shortcut to your search results, close this Petunia page and click on the Petunia Viewer link on the Sorcerer interface again. Since you logged in already this time, you will now be at the filechooser step of Analyze Peptides in the pipeline.
4. The Analyze Peptides step is designed to take in your search result files in pepXML format and run PeptideProphet on them. At the filechooser step, any file which ends in .xml is displayed. If you ran TPP previously, you will see the files it generated here since they end in .xml (ex. interact.pep.xml, interact.prot.xml). However, at this step you only want to select the pepXML files associated with each sample you searched, do not just select every xml file you see. So for example, if you searched sample1.raw and sample2.raw, you will see sample1.pep.xml and sample2.pep.xml in this list, check the boxes next to those two files only and press the Select button.
5. You will now be at the main Analyze Peptide page. Take a moment to look at the file(s) to be analyzed, they should be the pepXML files you just selected. If you see any old files from previous analyses you performed in this list, remove them. The files to be analyzed should all be located in the same folder. While you can add files from different folders, TPP will not work correctly if the pepXML files are not from the same folder. Notice the file path set should be the location of your current search (/home/sorcerer/output/###/original/) which contains the pepXML file(s) you selected.
6. You may now select the TPP options you want to run. If you want to recreate what Sorcerer normally does, you would need to verify the following are checked: RUN PeptideProphet, Use accurate mass binning, Leave alone all entries with asterisked score values, and Run ProteinProphet afterwards. Press the Run Xinteract button at the bottom when you are done.
7. Once your analysis is submitted, you will be at TPP’s Jobs status page. This page keeps a record of all jobs submitted through Petunia. It will auto refresh as your analysis progresses. Click the View link under the Output column once your analysis status is finished.
8. You will be at the Job page again, scroll down until you see the Output Files section. You will see the links to the output files you generated such as interact.pep.xml, interact.prot.xml if ProteinProphet was enabled, or interact.iproph.pep.xml if iProphet was enabled. Click on the links to view the output.
Tips
To access your TPP result links, you are not limited to the the Jobs page of Petunia as that list is difficult to trace to specific jobs. Given the output links are just file links, if Sorcerer initially ran TPP automatically and your Petunia analysis later created new interact.pep.xml and interact.prot.xml files, the links to those files from the Sorcerer’s TPP Analysis page can be used as they will reflect your new analysis since the old files would be overwritten. If you created a new file which isn’t linked, for example interact.iproph.pep.xml, use the Results Path link to browse to that file under the original folder.
The Jobs page in Petunia is just a record of jobs submitted to it. It is safe to click on the Delete Log link to clean up this page, doing so does not delete the TPP data. Since it’s easier to access the results through the Sorcerer’s queue and result links as described above, it’s not necessary to keep the Jobs list around. It does serve as a record of the TPP options you used to generate the results from Petunia, so you may want to keep it around for that purpose if you wish.
If you enabled Sorcerer’s Automatic decoy peptide generation, remember to enable the PeptideProphet option “Use decoy hits to pin down the negative distribution.” For the “Decoy Protein names begin with” option, specify “decoy” (no quotes when you enter it the box). If you searched your own fasta which contains decoys, enter the appropriate decoy identifier here instead.
You are free to change the guest password, remove the guest account, or create additional Petunia accounts. Please note that while multiple Petunia accounts can be created, there’s no separation between users or data. Once logged in, there’s nothing to separate users from one another as for example everyone will have access to the same Jobs status page.
If you want to access more advanced ProteinProphet options, then under Analysis Pipeline (Sequest), choose the Analyze Proteins link. For the files to analyze, this step takes in PeptideProphet results so choose the interact.pep.xml file only. Then choose your options and run ProteinProphet. A new interact.prot.xml file will be generated.
Petunia is a powerful feature in that it gives the user the freedom to browse and write to any output folder. Users should be aware of what folders they are writing to.
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