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Da Qi

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Sep 3, 2020, 2:56:33 AM9/3/20
to MS Amanda
Hi Viktoria,

I got the following output message when I try to search the iTRAQ data. There is no more message I could trace the cause. I here attach my setting.xml for your investigation.

Regards,
Da

----------------------
Using output fileformat .mzid
Using output folder '../RAW/mgf'
Reading settings file...
Settings file successfully read.
Perform Search
Input files contain 446604 ms2 spectra
Searching file 'Orbi - 091229_A549_8plex_IPG100_45min_fr_62.mgf'...
  Searching spectra 1 - 4000 of 446604 spectra
     Starting search against fwd database...
     Search against fwd database finished.
     Starting search against decoy database...
     Search against decoy database finished.
Search failed.
-------------------------
settings-hcd.xml

Viktoria Dorfer

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Sep 8, 2020, 8:09:50 AM9/8/20
to MS Amanda
Hi Da,

could you maybe provide me with the .mgf file you searched, such that I can trace down the error? And maybe also the fasta file?
Which version of MS Amanda are you using?
In addition, there should be a log file, I would be interested in. Under windows, it should be in "C:/ProgramData/MSAmanda2.0/Logs/" (attention: hidden folder), under linux there should also be a folder ".MSAmanda" in your home directory. Can you send that file to me as well?

Best regards,
Viktoria

Da Qi

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Sep 15, 2020, 12:11:44 AM9/15/20
to MS Amanda
Hi Viktoria,

I put all the files you requested in the following link. Let me know if you can access to them.


Regards,
Da

Viktoria Dorfer

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Sep 17, 2020, 11:27:31 AM9/17/20
to MS Amanda
Hi Da,

thanks for the files! We checked the logs and we think something went wrong with the digestion of the fasta file. We ran the provided mgf file with our latest MS AManda version - which worked - so we would like to ask you to do the following:
  • Download the latest MS Amanda standalone version 2.0.0.14828 here. (I just put it online today)
  • Delete the hidden MSAmanda2.0 folder
  • Re-run the mgf file you provided
  • Let us know if that worked ;)
Thanks!
Best regards,
Viktoria

Da Qi

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Sep 21, 2020, 4:23:39 AM9/21/20
to MS Amanda
Hi Viktoria,

I've done your steps. But no luck. I got the similar messages this time using your new version.
I have saved the relevant files/folders in the same link. 
I also noticed that as you mentioned the fasta file was wrongly generated in ./MSAmanda2.0 folder.
Then I tried another smaller test fasta file and it worked. It must be something went wrong when transforming fasta file.
I was running msamanda using mono in a cluster.  

Mono JIT compiler version 5.10.0.49 (tarball Wed Feb 14 20:23:54 HKT 2018)
Copyright (C) 2002-2014 Novell, Inc, Xamarin Inc and Contributors. www.mono-project.com
        TLS:           __thread
        SIGSEGV:       altstack
        Notifications: epoll
        Architecture:  amd64
        Disabled:      none
        Misc:          softdebug
        Interpreter:   yes
        LLVM:          supported, not enabled.
        GC:            sgen (concurrent by default)

===========  MSAmanda message ==============

Using output fileformat .mzid
Using output folder '../RAW/mgf'
Reading settings file...
Settings file successfully read.
Perform Search
Input files contain 446604 ms2 spectra
Searching file 'Orbi - 091229_A549_8plex_IPG100_45min_fr_62.mgf'...
  Searching spectra 1 - 7906 of 446604 spectra
     Starting search against fwd database...
     Search against fwd database finished.
     Starting search against decoy database...
     Search against decoy database finished.
Search failed.

Regards,
Da

Da Qi

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Sep 21, 2020, 5:44:33 AM9/21/20
to MS Amanda
Hi Viktoria,

I have encounter another error message as follows

Using output fileformat .mzid
Using output folder '../RAW/mgf'
Reading settings file...
Settings file successfully read.
Perform Search
Input files contain 446604 ms2 spectra
Searching file 'Orbi - 091229_A549_8plex_IPG100_45min_fr_62.mgf'...
  Searching spectra 1 - 7906 of 446604 spectra
     Starting search against fwd database...
     Search against fwd database finished.
     Starting search against decoy database...
     Search against decoy database finished.
  Search finished for 7906/446604 spectra
File 'Orbi - 091229_A549_8plex_IPG100_45min_fr_62.mgf' finished.

Generating .mzid file...

Searching file 'Orbi - 091229_A549_8plex_IPG100_45min_fr_63.mgf'...
  Searching spectra 7907 - 14996 of 446604 spectra
     Starting search against fwd database...
     Search against fwd database finished.
     Starting search against decoy database...
     Search against decoy database finished.
  Search finished for 14996/446604 spectra
File 'Orbi - 091229_A549_8plex_IPG100_45min_fr_63.mgf' finished.

Generating .mzid file...

w32error-unix.c: unknown error (122) "Disk quota exceeded"
Search failed.

In our cluster, user home directory only gets 150M quota. Could this be the possible reason for the error?

Regards,
Da

Viktoria Dorfer

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Sep 22, 2020, 7:10:51 AM9/22/20
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Hi Da,

that definitely might be the reason for the error. MS Amanda is first generating a digested target and decoy database (that depending on the minimum and maximum peptide length will probably be double the size of your fasta file, for semi specific enzymes of course even bigger). Then it writes intermediate results to a temporary file (in the hidden folder) and in the end combines all results to one mzid file (which might also get pretty large). You can change the directory of the digested fasta and the intermediate results by changing the parameter "data_folder" in the settings file. Is there a folder in your cluster you can use where there is more space?

Best,
Viktoria 

Da Qi

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Sep 24, 2020, 12:24:52 AM9/24/20
to MS Amanda
Hi Viktoria,

Since I changed the setting of "data_folder", search failed message was not there any more. However, the search never end. I have one file being searched for over 24 hours and still didn't get any result.
I put the two output files "run-msamanda.sh.e5263351" and "run-msamanda.sh.o5263351" together with .MSAmanda folder to the share link.
Thanks for your help.

Regards,
Da

Viktoria Dorfer

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Sep 24, 2020, 7:37:19 AM9/24/20
to MS Amanda
Hi Da,

according to the stack trace of your output file we assume you run into another memory issue, an out of RAM error in mono itself. We are currently working on a mono-free linux version of MS Amanda however this is still development in progress. 
There are several things you can try:
* change the available heap size for mono to a high number depending on what is available on your cluster: setenv MONO_GC_PARAMS max-heap-size=64G
* reduce the values in the parameters "LoadedProteinsAtOnce" (especially this one) and "LoadedSpectraAtOnce"
* if that all still does not help: run each mgf file separately

Best regards,
Viktoria 

Da Qi

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Sep 25, 2020, 6:20:57 AM9/25/20
to MS Amanda
Hi Viktoria,

Many thanks. I finally got MSAmanda running successfully by setting both "LoadedProteinsAtOnce" and "LoadedSpectraAtOnce" to the minimum value.
I also noticed that the first file in the folder was searched for very long time and the rest files were equally short.
Looking forward to testing your mono-free linux version.

Regards,
Da

Viktoria Dorfer

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Sep 28, 2020, 8:03:15 AM9/28/20
to MS Amanda
Hi Da!
great that it finally works!
Concerning the different durations of the files: this is expected, as the fasta file gets digested when searching the first file, which definitely takes some time, and is then re-used for all the other files - and for subsequent searches provided that you use the same fasta, same folder and the same fasta specific settings (enzyme, proteinsAtOnce, missedCleavages,...).

We will inform you as soon as the mono-free version is available!
Thanks :)
Regards,
Viktoria
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