How to export TMT11plex quantification?

[sorenwacker]

I wonder whether it is possible to export the quantification results from shaker. I saw the reports that can be exported, but they seem to be identical to the once that PeptideShaker creates? Ideally, I am looking for a way to do export the quantification data from the commandline in linux.

[hbarsnes]

Reporter does have its own command line support:

• run java -cp Reporter-0.7.16.jar eu.isas.reporter.cli.ReporterCLI to see the options for quantifiying the data
• and run java -cp Reporter-0.7.16.jar eu.isas.reporter.cli.ReportCLI to see the report options

It should also be possible to attach the -reports options from ReportCLI directly to the ReporterCLI command line.

And while the names of the reports may be the same as for PeptideShaker, they should contain extra columns with the quantification data.

Note however that the command line support for Reporter it is still in beta mode and yet not fully tested.

[sorenwacker]

When I run

java -cp ~/Software/Reporter/Reporter-0.7.16/Reporter-0.7.16.jar eu.isas.reporter.cli.ReportCLI -in "/home/.../workspace/uofc/lsarp/proteomics/0002-D003_MS2-TMT/run/S8926_A_TMT_T_MS2_181116/TMT11a/proteinshaker.cpsx" -reports "1,2,3,4,5,6,7,8,9,10,11" -out_reports "/home/../workspace/uofc/lsarp/proteomics/0002-D003_MS2-TMT/run/S8926_A_TMT_T_MS2_181116/TMT11a/reporter/"

I get :

Thu Feb 14 13:25:24 MST 2019 An error occurred while reading: .../peptideshaker.cpsx Please verify that the Reporter version used to create the file is compatible with your version of Reporter.

I did not use Reporter to create that file, I used PeptideShaker 1.16.38.

[sorenwacker]

I am trying to convert the file with Reporter using java -cp ~/Software/Reporter/Reporter-0.7.16/Reporter-0.7.16.jar eu.isas.reporter.cli.ReporterCLI -in ".../peptideshaker.cpsx" -zip ".../reporter.zip"

The log file shows that these keywords -zip or -out are not known.

org.apache.commons.cli.UnrecognizedOptionException: Unrecognized option: -out at org.apache.commons.cli.Parser.processOption(Parser.java:363) at org.apache.commons.cli.Parser.parse(Parser.java:199) at org.apache.commons.cli.Parser.parse(Parser.java:85) at eu.isas.reporter.cli.ReporterCLI.main(ReporterCLI.java:815)

Thu Feb 14 13:32:46 MST 2019: Reporter version 0.7.16. Memory given to the Java virtual machine: 16869490688 b. Total amount of memory in the Java virtual machine: 1056964608 b. Free memory: 1048576000 b. Java version: 11.0.2.

Thu Feb 14 13:33:16 MST 2019: Reporter version 0.7.16. Memory given to the Java virtual machine: 16869490688 b. Total amount of memory in the Java virtual machine: 1056964608 b. Free memory: 1048576000 b. Java version: 11.0.2. org.apache.commons.cli.UnrecognizedOptionException: Unrecognized option: -zip at org.apache.commons.cli.Parser.processOption(Parser.java:363) at org.apache.commons.cli.Parser.parse(Parser.java:199) at org.apache.commons.cli.Parser.parse(Parser.java:85) at eu.isas.reporter.cli.ReporterCLI.main(ReporterCLI.java:815)

[sorenwacker]

I opened the peptideshaker.cpsx with reporter and safed the project as reporter.cpsx. Then tried it to process it, but I get the same error message about file incompatibility.

[hbarsnes]

Seems like I introduced a bug in recent code clean up were I mixed the parameters for ReportCLI and ReporterCLI. I will correct this and release a new version of Reporter as soon as possible.

After this fix a command line of the following type should work: java -cp Reporter-0.7.17.jar eu.isas.reporter.cli.ReporterCLI -id C:\...\PeptideShaker_output.cpsx -isotopes C:\...\Reporter-0.7.17\resources\conf\defaultMethods.xml -method "TMT 10-plex" -out C:\...\tmt.cpsx

[hbarsnes]

Just released Reporter v0.7.17 which should solve the issues with the command line.

Here's the kind of command line that now works fine for me: java -cp Reporter-0.7.17.jar eu.isas.reporter.cli.ReporterCLI -id C:\...\PeptideShaker_output.cpsx -isotopes C:\...\Reporter-0.7.17\resources\conf\defaultMethods.xml -method "TMT 10-plex" -out C:\...\tmt.cpsx -ref_samples 1 -reports 9

[sorenwacker]

The new version runs on commandline. Awesome!

[sorenwacker]

I can create a bunch of reports from the commandline now, but I don't find the quantification data. This is the list of files that was generated:

Default_Hierarchical_Report.txt Default_Peptide_Phosphorylation_Report.txt Default_Peptide_Report.txt Default_Peptide_Report_with_non-validated_matches.txt Default_Protein_Phosphorylation_Report.txt Default_Protein_Report.txt Default_Protein_Report_with_non-validated_matches.txt Default_PSM_Phosphorylation_Report.txt Default_PSM_Report.txt Default_PSM_Report_with_non-validated_matches.txt Extended_PSM_Report.txt

Which of these files should contain the quantification data and which columns would that be? Is it possible to get the quantification data exported? If not could you recommend a different workflow? I am quite new to this. Any help is greatly appreciated.

[hbarsnes]

All of these should contain the quantification data. There should be columns with this data to the far right in the reports. Are you saying that these columns are not there? If so, maybe you can share your Default Protein Report?

[sorenwacker]

The column names would be helpful. I have these columns:

- Unnamed: 0
- Main Accession
- Description
- Gene Name
- Chromosome
- Protein Inference
- Secondary Accessions
- Protein Group
- #Peptides
- #Validated Peptides
- #Unique
- #Validated Unique
- #Unique to Group
- #Validated Unique to Group
- #PSMs
- #Validated PSMs
- Coverage [%]
- Possible Coverage [%]
- MW [kDa]
- Spectrum Counting
- Confidently Localized Modification Sites
- # Confidently Localized Modification Sites
- Ambiguously Localized Modification Sites
- #Ambiguously Localized Modification Sites
- Confidence [%]
- Validation
- Raw Unique Ratios
-  
-  .1
-  .2
-  .3
-  .4
-  .5
-  .6
-  .7
-  .8
-  .9
- Raw Shared Ratios
-  .10
-  .11
-  .12
-  .13
-  .14
-  .15
-  .16
-  .17
-  .18
-  .19
- Raw Ratios
-  .20
-  .21
-  .22
-  .23
-  .24
-  .25
-  .26
-  .27
-  .28
-  .29
- Unique Ratios
-  .30
-  .31
-  .32
-  .33
-  .34
-  .35
-  .36
-  .37
-  .38
-  .39
- Shared Ratios
-  .40
-  .41
-  .42
-  .43
-  .44
-  .45
-  .46
-  .47
-  .48
-  .49
- Ratios
-  .50
-  .51
-  .52
-  .53
-  .54
-  .55
-  .56
-  .57
-  .58
-  .59

How can I make this appear as raw text? The column names have symbols in it that are interpreted as markdown. I have 11plex data. So I expect 11 columns for quantification?

Download

[hbarsnes]

I have 11plex data. So I expect 11 columns for quantification?

TMT is generally used as relative quantification, i.e. you retain one TMT tag for the reference sample that you divide the values from the other TMT tags on to make them comparable. Hence you will have ten ratios for 11plex, nine ratios for 10plex etc.

The reference sample is set by using the -ref_samples option.

[sorenwacker]

Alright. Thanks!

If I set -ref_sample 1, is the first or the second sample that is used as a reference? Is counting starting with 1 or 0?