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Nesvilab / FragPipe

A cross-platform proteomics data analysis suite
http://fragpipe.nesvilab.org
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Nonspecific-peptidome workflow #403

Closed BenSamy2020 closed 3 years ago

BenSamy2020 commented 3 years ago

Greetings,

Currently, I am trying to perform non-enzyme searches using fragpipe with the default workflow (with reviewed UniProt Proteome Database) of non-specific-peptidome with the following changes ----->

Peptide length: 3 - 50 AA Peptide mass range: 350 - 6000 Variable modification: None

I am consistently experience failed searches using my high performance desktop. I am providing the log , .params and config files here for your reference. Lastly, I would like to thank you for your assistance with this issue.

Regards, Ben log_2021-07-19_17-15-05.txt fragger_Params.txt fragpipe_Config.txt

fcyu commented 3 years ago

The error was from the split script. @guoci Can you take a look when you have time?

Thanks,

Fengchao

guoci commented 3 years ago

@BenSamy2020 that has been fixed in that latest version, download link: https://drive.google.com/file/d/1qZ--_6dKFxIfBlh0nNLhqxVjAp54YkPA/view?usp=sharing

BenSamy2020 commented 3 years ago

Greetings @guoci,

Thank you for your quick reply. After using the latest version of FragPipe, I am able to progress further with the search. But now I am facing errors with ProteinProphet as per error log attatched below for your reference.

Regards Ben log_2021-07-20_14-35-34.txt

fcyu commented 3 years ago

Hi Ben,

That is because we changed the command of running ProteinProphet. You need the new version of Philosopher.

We are going to release new versions of our tools soon. But if you don't want to wait, we can send you a pre-released version.

Best,

Fengchao

BenSamy2020 commented 3 years ago

Greetings @fcyu,

Please do send me the pre-released version. Would require to complete the search asap. Sorry for any trouble caused.

Regards, Ben

fcyu commented 3 years ago

Hi Ben,

No worries!

Please find the pre-released versions of all tools from https://www.dropbox.com/sh/qhg4npww6wwe3tw/AADjJFaUUilx20F7Clzp7J6xa?dl=0

Best,

Fengchao

On Tue, Jul 20, 2021 at 9:43 AM BenSamy2020 @.***> wrote:

Greetings @fcyu https://github.com/fcyu,

Please do send me the pre-released version. Would require to complete the search asap. Sorry for any trouble caused.

Regards, Ben

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/Nesvilab/FragPipe/issues/403#issuecomment-883404395, or unsubscribe https://github.com/notifications/unsubscribe-auth/ABU27WZPUA366QLFXU2P3HTTYV4QRANCNFSM5ATDHIIQ .

-- Dr. Fengchao Yu Research Investigator University of Michigan

BenSamy2020 commented 3 years ago

Thank you brother

BenSamy2020 commented 3 years ago

Greetings @fcyu,

I tested the run with a small file and the search happened successfully. Thank you.

Regards, Ben

BenSamy2020 commented 3 years ago

Greetings @fcyu,

Can the Nonspecific-peptidome workflow be extended to add label-free quantification? (Based on your experience, is the work suitable for such add on?)

Regards, Ben

fcyu commented 3 years ago

Yes, it can. Just checking IonQuant and clicking "load quant default".

I think there is no big difference between nonspecific and tryptic searches from the LFQ point-of-view since both them are closed searches.

Best,

Fengchao

On Tue, Jul 20, 2021 at 7:09 PM BenSamy2020 @.***> wrote:

Greetings @fcyu https://github.com/fcyu,

Can the Nonspecific-peptidome workflow be extended to add label-free quantification? (Based on your experience, is the work suitable for such add on?)

Regards, Ben

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/Nesvilab/FragPipe/issues/403#issuecomment-883763393, or unsubscribe https://github.com/notifications/unsubscribe-auth/ABU27W66GACAK37PL75ILV3TYX6Z5ANCNFSM5ATDHIIQ .

-- Dr. Fengchao Yu Research Investigator University of Michigan

BenSamy2020 commented 3 years ago

Thanks!

Regards, Ben

anesvi commented 3 years ago

Do we have ‘generate peptide report’ checked for nonspecific ? Need that for peptide-level table with quant

Sent from my iPhone

On Jul 20, 2021, at 7:12 PM, Fengchao @.***> wrote:

 External Email - Use Caution

Yes, it can. Just checking IonQuant and clicking "load quant default".

I think there is no big difference between nonspecific and tryptic searches from the LFQ point-of-view since both them are closed searches.

Best,

Fengchao

On Tue, Jul 20, 2021 at 7:09 PM BenSamy2020 @.***> wrote:

Greetings @fcyu https://github.com/fcyu,

Can the Nonspecific-peptidome workflow be extended to add label-free quantification? (Based on your experience, is the work suitable for such add on?)

Regards, Ben

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/Nesvilab/FragPipe/issues/403#issuecomment-883763393, or unsubscribe https://github.com/notifications/unsubscribe-auth/ABU27W66GACAK37PL75ILV3TYX6Z5ANCNFSM5ATDHIIQ .

-- Dr. Fengchao Yu Research Investigator University of Michigan

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fcyu commented 3 years ago

No, we don't have that checked.

Best,

Fengchao

On Tue, Jul 20, 2021 at 7:19 PM Alexey Nesvizhskii @.***> wrote:

Do we have ‘generate peptide report’ checked for nonspecific ? Need that for peptide-level table with quant

Sent from my iPhone

On Jul 20, 2021, at 7:12 PM, Fengchao @.***> wrote:

 External Email - Use Caution

Yes, it can. Just checking IonQuant and clicking "load quant default".

I think there is no big difference between nonspecific and tryptic searches from the LFQ point-of-view since both them are closed searches.

Best,

Fengchao

On Tue, Jul 20, 2021 at 7:09 PM BenSamy2020 @.***> wrote:

Greetings @fcyu https://github.com/fcyu,

Can the Nonspecific-peptidome workflow be extended to add label-free quantification? (Based on your experience, is the work suitable for such add on?)

Regards, Ben

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/Nesvilab/FragPipe/issues/403#issuecomment-883763393,

or unsubscribe < https://github.com/notifications/unsubscribe-auth/ABU27W66GACAK37PL75ILV3TYX6Z5ANCNFSM5ATDHIIQ>

.

-- Dr. Fengchao Yu Research Investigator University of Michigan

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-- Dr. Fengchao Yu Research Investigator University of Michigan

BenSamy2020 commented 3 years ago

Greetings @fcyu,

I just completed the search. Using this workflow, I am obtaining 2654 proteins in the protein.tsv file with 0 total peptide count. I am providing the list here for your reference.

Is this a bug?

Regards, Ben Protein.xlsx

BenSamy2020 commented 3 years ago

The log file for your reference. log_2021-07-21_10-29-00.txt

fcyu commented 3 years ago

Hi Ben,

This is a feature in Philosopher, not a bug ;)

Because of "--prot 1" in the filter command, the protein FDR threshold is 100%. But the PSM FDR threshold is still 1%, which makes some proteins having no PSMs mapped after filtered by PSM FDR. Ideally, Philosopher could remove the proteins with 0 PSMs mapped.

Best,

Fengchao

On Tue, Jul 20, 2021 at 8:34 PM BenSamy2020 @.***> wrote:

Greetings @fcyu https://github.com/fcyu,

I just completed the search. Using this workflow, I am obtaining 2654 proteins in the protein.tsv file with 0 total peptide count. I am providing the list here for your reference.

Is this a bug?

Regards, Ben Protein.xlsx https://github.com/Nesvilab/FragPipe/files/6851946/Protein.xlsx

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/Nesvilab/FragPipe/issues/403#issuecomment-883796281, or unsubscribe https://github.com/notifications/unsubscribe-auth/ABU27W53XYK4B4ZNH5LXLK3TYYIZVANCNFSM5ATDHIIQ .

-- Dr. Fengchao Yu Research Investigator University of Michigan

BenSamy2020 commented 3 years ago

Greetings @fcyu,

My apologies for presuming it as a bug. Based on your above response, what you mean is:

Since the workflow is using protein level FDR at 100% (and PSM level FDR 1%), proteins that do not have any corresponding PSM are still listed in protein.tsv list?

Is it possible that you elaborate more about the algorithm or workflow in the context of this setting? (I am really sorry, I am extremely new to this)

Regards, Ben

fcyu commented 3 years ago

Hi Ben,

No worries!

I think Felipe @prvst and Alexey @anesvi are better persons than me explaining the procedure of PSM and protein filtering.

Best,

Fengchao

anesvi commented 3 years ago

I think it would be better if philosopher did not print any entries that have 0 counts. But it does now.

So your options are : 1) Just filter them out yourself. 2)Since you do peptidome, you do not even need to look at protein.tsv. Just use peptide files 3) If you do want to look at that protein file, change protein FDR to 1 percent

Sent from my iPhone

On Jul 20, 2021, at 8:54 PM, BenSamy2020 @.***> wrote:

 External Email - Use Caution

Greetings @fcyuhttps://github.com/fcyu,

My apologies for presuming it as a bug. Based on your above response, what you mean is:

Since the workflow is using protein level FDR at 100% (and PSM level FDR 1%), proteins that do not have any corresponding PSM are still listed in protein.tsv list?

Is it possible that you elaborate more about the algorithm or workflow in the context of this setting? (I am really sorry, I am extremely new to this)

Regards, Ben

— You are receiving this because you commented. Reply to this email directly, view it on GitHubhttps://github.com/Nesvilab/FragPipe/issues/403#issuecomment-883802993, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AIIMM66MFRQESG6GU4BUSKLTYYLFJANCNFSM5ATDHIIQ.

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BenSamy2020 commented 3 years ago

Greetings @fcyu,

I tried performing MS1 quant (IonQuant + MBR). I had hit a error message of:

**"2021-07-22 18:08:37 [INFO] - Updating Philosopher's tables... java.lang.NullPointerException: Cannot find certain columns from D:\Parthiban\RAW_MS_Files\BRCA_Panel\Biological_Experiment_Test\TNBC_VS_Non-TNBC_Peptidomics\Non-TNBC/protein.tsv. at q.(Unknown Source) at ionquant.IonQuant.main(Unknown Source) 2021-07-22 18:08:38 [ERROR] - Cannot find certain columns from D:\Parthiban\RAW_MS_Files\BRCA_Panel\Biological_Experiment_Test\TNBC_VS_Non-TNBC_Peptidomics\Non-TNBC/protein.tsv. Process 'IonQuant' finished, exit code: 1 Process returned non-zero exit code, stopping


Cancelling 4 remaining tasks"**

I am also attaching the log here for your reference. I am using the latest version of FragPipe, MSFragger and Philosopher. Please do advise me how to troubleshoot.

Regards,
Ben
[log_2021-07-22_18-08-38.txt](https://github.com/Nesvilab/FragPipe/files/6866007/log_2021-07-22_18-08-38.txt)
BenSamy2020 commented 3 years ago

log_2021-07-22_18-08-38.txt

fcyu commented 3 years ago

Hi Ben,

I cannot reproduce the error. We just released the new versions. Can you download it from https://github.com/Nesvilab/FragPipe/releases/tag/16.0 and try again? You also need the new MSFragger which can be updated by clicking "download/update" in FragPipe.

If the error is still there, can you send me your files?

Best,

Fengchao

BenSamy2020 commented 3 years ago

Greetings @fcyu,

Will try the new version with the laterest MSFragger JAR file too. Will update you soon.

Regards, Ben

BenSamy2020 commented 3 years ago

Greetings @fcyu,

With the latest of everything, everything worked.

Regards, Ben

fcyu commented 3 years ago

Hi Ben,

Glad to hear that. Please feel free to contact us if there is any questions in the future.

Best,

Fengchao