Workflow for isobaric analysis (untested) #166

oliveralka · 2019-08-01T15:48:22Z

Reduced rt setting in IDMapper
TMT6-plex (K,N-term) in MSGFPlus
The correction Matrix for the TMT experiment has to be set by the user!

Reduced rt setting in IDMapper TMT6-plex (K,N-term) in MSGFPlus The correction Matrix for the TMT experiment has to be set by the user!

jpfeuffer · 2019-08-02T10:36:50Z

Very nice. I didn't have a look yet (will today).
But:

Did you make the input files accept relative paths inside the workspace?
Did you add a description of the workflow in the workflow settings for easy upload to the KNIME Hub?

jpfeuffer · 2019-08-02T10:37:01Z

I will write a TODO list for PRs here.

jpfeuffer · 2019-08-02T10:38:43Z

Ah this was just a change in one of the WFs but still, maybe you could add the abovementioned points. We should do it for every WF here anyway soon.

oliveralka · 2019-08-02T10:49:18Z

Did you make the input files accept relative paths inside the workspace?

Should we ship the input data with the workflow?

Did you add a description of the workflow in the workflow settings for easy upload to the KNIME Hub?

Can you please provide a reference / or example on how to do that?

jpfeuffer · 2019-08-02T11:01:31Z

No, otherwise it gets too big. Data goes here (assume the workspace root is this folder, i.e. specify paths relative to that root):
https://abibuilder.informatik.uni-tuebingen.de/archive/openms/Tutorials/Data/

Instructions for description is here:
https://docs.google.com/document/d/1uwVCz-4LH7VUzhcsE5FVyrBbRmzDnGQvdFF2ZsA8BYU/edit#heading=h.3hm698qmdoi3

jpfeuffer · 2019-08-02T11:03:54Z

The workflows have to run from the commandline. You can check by running the shell script executeBatch with arguments:

knime executable
workspace root

oliveralka · 2019-08-05T07:02:27Z

There are some errors in the workflow which need to be fixed first - I will update the PR, when it is ready.

oliveralka · 2019-09-10T07:36:56Z

We have to check if the IDMapper is switched to "precursor" see OpenMS/OpenMS#4216

pavel-shliaha · 2019-10-24T17:00:24Z

will the pipeline work for MS3 SPS?

oliveralka · 2019-10-24T18:44:21Z

The IsobaricAnalyzer should use MS3 automatically if possible (@cbielow).
Please use the current nightly build of OpenMS in KNIME to have full support.

oliveralka · 2020-02-24T17:05:17Z

@timosachsenberg @jpfeuffer
Added a rought draft for integration into the tutorial. Updated the workflow using epifany - runable - but I still have to check the results.
It would be great if you could take a look and make some comments and suggestions, also in terms of explanation of the experimental design for TMT.

Julianus, could you have a peek at the parameters used for epifany?

pavel-shliaha · 2020-02-25T16:51:52Z

@oliveralka could you please point to where the tutorial for MS3 TMT is and the actual workflow that you recommend?

into oliveralka-patch-2

pavel-shliaha · 2020-03-06T03:49:54Z

@oliveralka. I have found the workflow, but I cannot find the example files (mzMLs and also the MSstatsTMT table). Could you perhaps give a direct link to it?

oliveralka · 2020-03-06T08:23:17Z

@pavel-shliaha The small example can be found here:
https://abibuilder.informatik.uni-tuebingen.de/archive/openms/Tutorials/Data/latest/Example_Data/IsobaricLabellingTMT/

pavel-shliaha · 2020-03-07T01:51:21Z

@oliveralka. I downloaded the newest KNIME and OpenMS, the dataset and the database and set file locations on my computer. MSGFPlusAdapter and IsobaricAnalyzer nodes failed. Do they work in your hands?

oliveralka · 2020-03-08T18:01:12Z

@pavel-shliaha
Everything works in my case:
KNIME 4.1.1
OpenMS 2.5.0.20200201806

What are the error messages? Can you run the tools in debug mode?

pavel-shliaha · 2020-03-09T23:53:23Z

@oliveralka. I tried to execute the workflow on two separate computers. On both of them:

KNIME is 4.1.2
OpenMS is 2.5.0.202002241222

How can I run the software in debug mode (googled the question but could not find an answer)? Also if you are interested I can provide access to my computer so you can have a look.

jpfeuffer · 2020-03-09T23:55:18Z

What does the Console log in the lower right of KNIME say?

pavel-shliaha · 2020-03-10T00:05:13Z

@jpfeuffer it says I might be missing some of the requirements (trying to isntall them now). I appologise for this but I used the website https://www.openms.de/download/knime-plugin/ and it does not mention the prerequisites. I will now install proteowizard as well

pavel-shliaha · 2020-03-10T00:32:59Z

When I load the workflow I get the following message:

I installed all the prerequisites from the suggested link but it did not help

jpfeuffer · 2020-03-10T00:34:37Z

that can usually be ignored.

pavel-shliaha · 2020-03-10T00:37:05Z

I installed the prerequisites and the prerequisites for visual studio under this link

https://support.microsoft.com/en-us/help/2977003/the-latest-supported-visual-c-downloads

but it did not help

jpfeuffer · 2020-03-10T00:39:43Z

As long as it just shows "... FileConverter requirements" this can also be ignored. You just might not be able to use Raw file conversion inside KNIME. We just check registry keys here. Sometimes it does not work correctly.

pavel-shliaha · 2020-03-10T00:49:20Z

it still fails to execute the workflow

jpfeuffer · 2020-03-10T01:15:05Z

I thought so. Without the requirements no OpenMS node would work. That is why you need to check the Console and the Standard Output/Error of the nodes for info now.

jpfeuffer · 2020-03-10T01:18:20Z

Just a random guess: did you configure the Input database in the ID Metanode?

pavel-shliaha · 2020-03-10T01:20:10Z

I have installed:

KNIME
OpenMS-2.5.0-Win64.exe from https://www.openms.de/download/openms-binaries
OpenMS extension through KNIME
OpenMS-2.5-prerequisites-installer.exe

the workflow still does not run. Should I install anything else? This is the error message.

(or a complete workflow) generated with an older version of the tool.
If you do not reconfigure the node (marked with an exclamation mark),
the current defaults will be loaded instead.
- Entry for parameter MzTabExporter.1.opt_columns not found in settings.xml.

ERROR LoadWorkflowRunnable Errors during load: Status: Error: Identification_quantification_isobaric_MSstatsTMT 0 loaded with errors
ERROR LoadWorkflowRunnable Status: Error: Identification_quantification_isobaric_MSstatsTMT 0
ERROR LoadWorkflowRunnable Status: Error: MzTabExporter 0:227
ERROR LoadWorkflowRunnable Status: Error: Loading model settings failed:
ERROR LoadWorkflowRunnable GenericKNIMENodes:
ERROR LoadWorkflowRunnable Maybe you are loading node settings (or a complete workflow) generated with an older version of the tool.
ERROR LoadWorkflowRunnable If you do not reconfigure the node (marked with an exclamation mark),
ERROR LoadWorkflowRunnable the current defaults will be loaded instead.
ERROR LoadWorkflowRunnable - Entry for parameter MzTabExporter.1.opt_columns not found in settings.xml.
WARN IsobaricAnalyzer 0:235 Can't continue loop as the workflow was restored with the loop being partially executed. Reset loop start and execute entire loop again.
WARN MSGFPlusAdapter 0:237:3 Can't continue loop as the workflow was restored with the loop being partially executed. Reset loop start and execute entire loop again.
WARN IsobaricAnalyzer 0:235 Can't continue loop as the workflow was restored with the loop being partially executed. Reset loop start and execute entire loop again.

pavel-shliaha · 2020-03-10T01:32:42Z

I have reimported the workflow and it seems to be working

jpfeuffer · 2020-03-10T22:32:15Z

@oliveralka did you check: #166 (comment)

oliveralka · 2020-03-11T07:29:14Z

@jpfeuffer Yes, checked some time ago - is set to precursor.

Note: The current workflow does not have the protein level anymore, since we have to fix the inference first and input it to the MSstatsConverter.

jpfeuffer

Looks good. Minor changes.
Then we can go over the workflow together via Skype.

Handout/isobaric.tex

jpfeuffer · 2020-06-22T11:16:23Z

Handout/isobaric.tex

+The workflow has three input nodes, the first for the experimental design to allow for MSstatsTMT compatible export (.tsv). The second for the .mzML files with the centroided spectra data from the isobaric labeling experiment and the last one for the .fasta database used for identification. The quantification (A) is performed using the \KNIMENODE{IsobaricAnalzyer}. The tool is able to extract and normalize quantiative information from TMT and iTRAQ data. The values can be assessed from centroided MS2 or MS3 spectra and isotopte correction is performed based on the specified correction matrix (as provided by the manufacturer). The identification (C) is performed as known from the previous chapters by using database search and a target-decoy database.
+
+The workflow is performed peptide level (B, D, F, H), were the posterior error probability (PEP) estimation and FDR filtering is performed on PSM level for each file individually (B). Afterwards the identification (PSM) and quantiative information is combined using the \KNIMENODE{IDMapper}. After the processing of all available files, the intermediate results are aggregated (D) and can be exported via \KNIMENODE{MzTabExporter} (F) or further processed to obtain a MSstatsTMT
+compatible version. Here, the R package MSstatsTMT can be used for further processing. \\


Add a TODO that protein inference will be added in a later version of the tutorial.

pavel-shliaha · 2020-06-22T18:15:56Z

Looks good. Minor changes.
Then we can go over the workflow together via Skype.

Dear Jennifer and Oliver, could you please add me to the skype discussion as well? I use a lot of MS3 SPS and I think I could provide some valuable input to the discussion, as well as clarify some questions for myself. I wanted to ask about whether certain functionalities are implemented in openMS. In particular:

Could yo clarify why IDConflictResolver is used with isobaric mass tagging?
I understand that OpenMS workflow suggests using MSStats node for protein inference, but I actually wanted to export the ids with quant values and then look through the table myself. Is there a node that will convert IDConflictResolver output consensusXML to a csv output where the quantitation values are one line for PSM (wide format) or one line per channel (long format). I tried TextExporter, but it does not seem to generate this output
is there a node to estimate precursor contamination?
is there a node to estimate which of the fragments selected for MS3 originate from MS2 fragments which have been identified as true fragments from the identified peptide?

jpfeuffer · 2020-06-23T11:21:41Z

Hi Pavel,

sorry, the meeting is right now. But you will be in the data clinic tomorrow right?

It only makes sure that the top hit for the corresponding MS2 spectrum is retained (and not more). You could probably achieve the same with an IDFilter after the search engine.
The MSstats node just rearranges the tables to be compatible with MSstats. It does not perform any inference.
TextExporter should export one line per PSM in the "FEATURE" section. The intensities should be in the columns intensity_0 - intensity_9 (or similar). An alternative would be the MzTabExporter (but I did not double-check the completeness of the output for TMT yet).
Yes, if you export meta values in the TextExporter, there should be a column with a precursor_purity score.
No, but that is a good idea.

jpfeuffer · 2020-06-23T22:05:55Z

@pavel-shliaha Regarding 4): How do you get the information about the isolated ions in the MS2. I have an SPS mzML here and all the MS3 spectra only report a single selected precursor ion:

              <selectedIonList count="1">
                <selectedIon>
                  ...

Not sure if this is a bug in the mzML converters (e.g. proteowizard) or if this information is just not available in the raw file.

pavel-shliaha · 2020-06-25T14:51:26Z

I am looking at the MSGFPlusAdapter and it does not have the TMTPro modification implemented. Could you please have a look?

jpfeuffer · 2020-06-25T14:56:05Z

Hi, which Unimod accession is this?

pavel-shliaha · 2020-06-25T17:12:06Z

accession number is 2016

into oliveralka-patch-2

oliveralka · 2020-07-29T10:15:59Z

I added the data here: https://abibuilder.informatik.uni-tuebingen.de/archive/openms/Tutorials/Data/isobaric_MSV000084264/

@jpfeuffer The path to the data has still to be added to the document, but please let me know what you think of the current version.

handout_isobaric_20200729.pdf

Workflow for isobaric analysis (untested)

c664da6

Reduced rt setting in IDMapper TMT6-plex (K,N-term) in MSGFPlus The correction Matrix for the TMT experiment has to be set by the user!

timosachsenberg requested a review from jpfeuffer August 1, 2019 15:50

oliveralka mentioned this pull request Aug 1, 2019

Identification / Quantification #165

Closed

oliveralka added 2 commits August 5, 2019 09:30

[FIX] metanode, FileMerger

575049d

[UPDATE] example data path, fixes mime tpye

8fa97d6

oliveralka mentioned this pull request Oct 24, 2019

Support for TMT 16-plex (IsobaricAnalyzer) OpenMS/OpenMS#4242

Closed

[ADD] In_design; MSstatsConverter; Check mapping by precursor

82a1056

[ADD] Draft for Tutorials, [UPDATE] workflow use epifany for inference

4be53fb

Update isobaric.tex

e760ec0

oliveralka added 2 commits February 26, 2020 16:38

[RM] inference, [ADD] Edited WF, Draft explanation experimental design

2e7ef24

Merge branch 'oliveralka-patch-2' of https://github.com/OpenMS/Tutorials

9f91839

into oliveralka-patch-2

jpfeuffer requested changes Jun 22, 2020

View reviewed changes

jpfeuffer added 2 commits June 22, 2020 18:18

Update isobaric.tex

c1a6cee

Describe special values.

10efe46

jpfeuffer mentioned this pull request Jun 23, 2020

Count number of matching MS2 fragments used for MS3 scan in MS3-SPS OpenMS/OpenMS#4792

Open

Check parameters of the workflow

1af9605

Add MSstatsTMT R script to workflow.

feea4b9

oliveralka and others added 4 commits July 28, 2020 16:56

rewrite - unfinished

c94c436

Merge branch 'master' into oliveralka-patch-2

ef3f2db

add MSstatsTMT and results

7faec92

Merge branch 'oliveralka-patch-2' of https://github.com/OpenMS/Tutorials

c9fc587

into oliveralka-patch-2

jpfeuffer merged commit c636f46 into master Sep 11, 2020

jpfeuffer deleted the oliveralka-patch-2 branch September 30, 2020 22:22

Workflow for isobaric analysis (untested) #166

Workflow for isobaric analysis (untested) #166

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