Replies: 11 comments 19 replies
-
|
How does the log look like? |
Beta Was this translation helpful? Give feedback.
-
|
Hi Vadim,
Thanks for getting back to me. I’m new to DIA analysis and new to DIA-NN, so perhaps I have set-up the task incorrectly.
This is part of the log:
Rod
2 files will be processed
diann.exe --f "C:\Users\rchalk\Desktop\#WORK IN PROGRESS\LC01_CTRL_Slot1-31_1_1285.d
" --f "C:\Users\rchalk\Desktop\#WORK IN PROGRESS\LC02_PD_Slot1-31_1_1287.d
" --lib "" --threads 4 --verbose 1 --out "C:\DIA-NN\1.8\report.tsv" --qvalue 0.01 --matrices --out-lib "C:\DIA-NN\1.8\report-lib.tsv" --gen-spec-lib --predictor --fasta "C:\Users\rchalk\Desktop\#WORK IN PROGRESS\UP000005640_9606.fasta" --fasta-search --min-fr-mz 200 --max-fr-mz 1800 --met-excision --cut K*,R* --missed-cleavages 1 --min-pep-len 7 --max-pep-len 30 --min-pr-mz 300 --max-pr-mz 1800 --min-pr-charge 1 --max-pr-charge 4 --unimod4 --reanalyse --smart-profiling
DIA-NN 1.8 (Data-Independent Acquisition by Neural Networks)
Compiled on Jun 28 2021 14:55:31
Current date and time: Fri Jul 29 09:50:22 2022
CPU: GenuineIntel Intel(R) Core(TM) i7-4790 CPU @ 3.60GHz
SIMD instructions: AVX AVX2 FMA SSE4.1 SSE4.2
Logical CPU cores: 8
Thread number set to 4
Output will be filtered at 0.01 FDR
Precursor/protein x samples expression level matrices will be saved along with the main report
A spectral library will be generated
Deep learning will be used to generate a new in silico spectral library from peptides provided
Library-free search enabled
Min fragment m/z set to 200
Max fragment m/z set to 1800
N-terminal methionine excision enabled
In silico digest will involve cuts at K*,R*
Maximum number of missed cleavages set to 1
Min peptide length set to 7
Max peptide length set to 30
Min precursor m/z set to 300
Max precursor m/z set to 1800
Min precursor charge set to 1
Max precursor charge set to 4
Cysteine carbamidomethylation enabled as a fixed modification
A spectral library will be created from the DIA runs and used to reanalyse them; .quant files will only be saved to disk during the first step
When generating a spectral library, in silico predicted spectra will be retained if deemed more reliable than experimental ones
DIA-NN will optimise the mass accuracy automatically using the first run in the experiment. This is useful primarily for quick initial analyses, when it is not yet known which mass accuracy setting works best for a particular acquisition scheme.
2 files will be processed
[0:00] Loading FASTA C:\Users\rchalk\Desktop\#WORK IN PROGRESS\UP000005640_9606.fasta
[0:03] Processing FASTA
[0:11] Assembling elution groups
[0:17] 4292512 precursors generated
[0:17] Gene names missing for some isoforms
[0:17] Library contains 20569 proteins, and 20322 genes
[0:17] Encoding peptides for spectra and RTs prediction
[0:26] Predicting spectra and IMs
[40:39] Predicting RTs
[45:04] Decoding predicted spectra and IMs
[45:27] Decoding RTs
[45:29] Saving the library to C:\DIA-NN\1.8\report-lib.predicted.speclib
[45:37] Initialising library
[45:39] First pass: generating a spectral library from DIA data
[45:39] File #1/2
[45:39] Loading run C:\Users\rchalk\Desktop\#WORK IN PROGRESS\LC01_CTRL_Slot1-31_1_1285.d
For most diaPASEF datasets it is better to manually fix both the MS1 and MS2 mass accuracies to 10 ppm.
DIA-NN exited
DIA-NN-plotter.exe "C:\DIA-NN\1.8\report.stats.tsv" "C:\DIA-NN\1.8\report.tsv" "C:\DIA-NN\1.8\report.pdf"
PDF report will be generated in the background
From: Vadim Demichev ***@***.***>
Sent: 29 July 2022 12:18
To: vdemichev/DiaNN ***@***.***>
Cc: Rod Chalk ***@***.***>; Author ***@***.***>
Subject: Re: [vdemichev/DiaNN] Program is not generating output (Discussion #457)
How does the log look like?
—
Reply to this email directly, view it on GitHub<#457 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/A2JASB564EAPZDBLN3QIL2DVWO4YLANCNFSM55AMX7AQ>.
You are receiving this because you authored the thread.Message ID: ***@***.******@***.***>>
|
Beta Was this translation helpful? Give feedback.
-
|
Hi Rod, DIA-NN crashed when loading the run. Maybe not enough RAM in the system? Best, |
Beta Was this translation helpful? Give feedback.
-
|
Hi Vadim,
This PC has 3.6 GHz processor with 16 GB RAM
My TIMS-TOF PC has 3.7 GHz processor with 64 GB RAM and does the same. How much RAM do you recommend?
Best wishes,
Rod
From: Vadim Demichev ***@***.***>
Sent: 29 July 2022 12:34
To: vdemichev/DiaNN ***@***.***>
Cc: Rod Chalk ***@***.***>; Author ***@***.***>
Subject: Re: [vdemichev/DiaNN] Program is not generating output (Discussion #457)
Hi Rod,
DIA-NN crashed when loading the run. Maybe not enough RAM in the system?
Best,
Vadim
—
Reply to this email directly, view it on GitHub<#457 (reply in thread)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/A2JASB4TLWFL4ODOVA7MXQTVWO6SBANCNFSM55AMX7AQ>.
You are receiving this because you authored the thread.Message ID: ***@***.******@***.***>>
|
Beta Was this translation helpful? Give feedback.
-
|
The memory consumption required to load a Bruker .d folder might be up to 3x the size of the folder. So 64Gb should be fine, unless something is also sonsuming lots of RAM on that system. Also, please generate a .predicted.speclib in silico predicted spectral library in a separate pipeline step. |
Beta Was this translation helpful? Give feedback.
-
|
Hi Vadim,
I managed to quire 64Gb RAM and DIA-NN is working well and I’m hoping to use it a lot more.
Thanks for your help,
Rod
From: Vadim Demichev ***@***.***>
Sent: 06 August 2022 12:54
To: vdemichev/DiaNN ***@***.***>
Cc: Rod Chalk ***@***.***>; Author ***@***.***>
Subject: Re: [vdemichev/DiaNN] Program is not generating output (Discussion #457)
The memory consumption required to load a Bruker .d folder might be up to 3x the size of the folder. So 64Gb should be fine, unless something is also sonsuming lots of RAM on that system. Also, please generate a .predicted.speclib in silico predicted spectral library in a separate pipeline step.
—
Reply to this email directly, view it on GitHub<#457 (reply in thread)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/A2JASB7CN47VIB6XZTPTFTTVXZG6RANCNFSM55AMX7AQ>.
You are receiving this because you authored the thread.Message ID: ***@***.******@***.***>>
|
Beta Was this translation helpful? Give feedback.
-
|
Hello,
I have tried to run this both, with 16 or 64 GB RAM, but both failed. With the information, that I might need 3x the folder size of RAM, in this example I exceeded the RAM I have, but this also keeps happening, if I run single/smaller files that should use less. Thank you in advance |
Beta Was this translation helpful? Give feedback.
-
|
Hi Stef, Things to try:
Best, |
Beta Was this translation helpful? Give feedback.
-
|
hi I have the same problem |
Beta Was this translation helpful? Give feedback.
-
|
Hi Vadim, we are running dia-PASEF files with methylthio(C) (UniMod:39) modification instead of carbamidomethyl(C) modification. We use either external library from Spectronaut, or use Fasta-based library. We add the following into Additional options: |
Beta Was this translation helpful? Give feedback.
-
|
"We use either external library from Spectronaut" - no need to declare anything in DIA-NN. If using MBR, just switch Library generation to 'IDs, RT & IM profiling'. This will fix the error you are getting. FASTA: need to declare it at the library generation stage and also the analysis stage - the same way. To avoid conversion to the UniMod format can use --original-mods. |
Beta Was this translation helpful? Give feedback.
-
|
Specifically the error you get pops ub because --fixed-mod declares it as 'UniMod:39' whereas in the library it's 'Methylthio (C)' |
Beta Was this translation helpful? Give feedback.
-
|
Hi Vadim, I am trying to generate a in silico library with DIA-NN and I am having some problems. I tried a few different fasta files that contain different number of proteins (file size varied from 35 MB, to 1 KB). I got output for .predicted.spclib files but no report.tsv output files. I am using a computer with 32G ram and 16 CPU cores. Here is the screen shot and log files that I got.
Can you please let me know what is the reason for no tsv report files? Thanks, |
Beta Was this translation helpful? Give feedback.
-
|
Hi Leah, .predicted.speclib is the library being generated, you can now specify it in the 'Spectral library' field and analyse the raw files of interest, can also convert it to .tsv (but this is very rarely needed). Best, |
Beta Was this translation helpful? Give feedback.
-
|
Hi Vadim,
Thanks for your fast response. I tried to use the .predicted.speclib to
generate a .tsv file. Here are the columns being generated in the tsv file:
[1] "FileName" "PrecursorMz" "ProductMz"
"Tr_recalibrated" "IonMobility"
[6] "transition_name" "LibraryIntensity" "transition_group_id"
"decoy" "PeptideSequence"
[11] "Proteotypic" "QValue" "PGQValue"
"Ms1ProfileCorr" "ProteinGroup"
[16] "ProteinName" "Genes"
"FullUniModPeptideName" "ModifiedPeptide" "PrecursorCharge"
[21] "PeptideGroupLabel" "UniprotID" "NTerm"
"CTerm" "FragmentType"
[26] "FragmentCharge" "FragmentSeriesNumber" "FragmentLossType"
"ExcludeFromAssay"
When I looked at the tsv files that people generated are the following:
[1] "File.Name" "Run" "Protein.Group"
"Protein.Ids"
[5] "Protein.Names" "Genes" "PG.Quantity"
"PG.Normalised"
[9] "PG.MaxLFQ" "Genes.Quantity"
"Genes.Normalised" "Genes.MaxLFQ"
[13] "Genes.MaxLFQ.Unique" "Modified.Sequence"
"Stripped.Sequence" "Precursor.Id"
[17] "Precursor.Charge" "Q.Value" "PEP"
"Global.Q.Value"
[21] "Protein.Q.Value" "PG.Q.Value"
"Global.PG.Q.Value" "GG.Q.Value"
[25] "Translated.Q.Value" "Proteotypic"
"Precursor.Quantity" "Precursor.Normalised"
[29] "Precursor.Translated" "Translated.Quality"
"Ms1.Translated" "Quantity.Quality"
[33] "RT" "RT.Start" "RT.Stop"
"iRT"
[37] "Predicted.RT" "Predicted.iRT"
"First.Protein.Description" "Lib.Q.Value"
[41] "Lib.PG.Q.Value" "Ms1.Profile.Corr" "Ms1.Area"
"Evidence"
[45] "Spectrum.Similarity" "Averagine"
"Mass.Evidence" "CScore"
[49] "Decoy.Evidence" "Decoy.CScore"
"Fragment.Quant.Raw" "Fragment.Quant.Corrected"
[53] "Fragment.Correlations" "MS2.Scan"
"PTM.Informative" "PTM.Specific"
[57] "PTM.Localising" "PTM.Q.Value"
"PTM.Site.Confidence" "Lib.PTM.Site.Confidence"
[61] "IM"
So I feel like in the report file that I have it is missing a lot of
columns. I am thinking about using the in silico library to analyze data in
DIA-NN and also use the library generated in DIA-NN to look at data in
Skyline. When Skyline generates a library, it requires "Precursor.Id", "
Q.Value" and "RT" in the tsv file, which are all missing in the generated
tsv file.
So what am I missing here?
Thanks a lot,
Leah
…On Fri, Apr 21, 2023 at 2:55 PM Vadim Demichev ***@***.***> wrote:
Hi Leah,
.predicted.speclib is the library being generated, you can now specify it
in the 'Spectral library' field and analyse the raw files of interest, can
also convert it to .tsv (but this is very rarely needed).
Best,
Vadim
—
Reply to this email directly, view it on GitHub
<#457 (reply in thread)>,
or unsubscribe
<https://github.com/notifications/unsubscribe-auth/A7HWXBVK5EDRJPSJBGM2HYTXCLJZ3ANCNFSM55AMX7AQ>
.
You are receiving this because you commented.Message ID:
***@***.***>
|
Beta Was this translation helpful? Give feedback.
-
|
Yes, a library and a report have different formats. |
Beta Was this translation helpful? Give feedback.
-
|
Hi Vadim, I am running into a very similar problem. It is interesting because the first file will run completely, but the second one will exit every time. We have tried it on a different computer and it worked. I have also checked the RAM on my computer and it says we have more than 70 GB of RAM installed. During the run, the memory allocation never got above 2,000 MB. Here is the log for my run: diann.exe --f "Z:\2024\10\GPFlibrary\LL2076_SQp5_HYEA_10ng_blo1_GC10_rep3_ch2_DIAwet10ng45.raw 2 files will be processed First pass: generating a spectral library from DIA data [0:36] File #1/2 [6:11] File #2/2 DIA-NN exited Any suggestions? Thanks, |
Beta Was this translation helpful? Give feedback.
-
|
Hi Alex, Is the problem with this specific run, even if you have it first in the list of runs?
Best, |
Beta Was this translation helpful? Give feedback.
-
|
Vadim, Yes, here is the OneDrive link: Let me know if that works! Again, this one file is giving us trouble on about half of our computers. Other files do run on the computers, just not this one. Thanks, |
Beta Was this translation helpful? Give feedback.
-
|
Hi Alex, Did you check mzML? Best, |
Beta Was this translation helpful? Give feedback.
-
|
Vadim, I did just check the mzML converted files. It did run on the same computer. Is there a chance the problem is stemming from the warning code: "WARNING: cannot save the .quant file to the raw data folder; using the current working folder instead"? Best, |
Beta Was this translation helpful? Give feedback.
-
|
Strangely, the .raw file works fine on my PC with both 1.9 and 1.9.2. Try maybe moving it to a different folder, e.g. C:/Temp, and running DIA-NN quickly just on this file with a small lib? |
Beta Was this translation helpful? Give feedback.
-
|
I have tried putting them in a different folder. Here is what I did: I have 10 files, (file1, file2, ... , file10) that are all replicates of the same experiment. These 10 files all run on some other computers, but not on my computer. On my computer, files 1, 2, 3, 5, 7, and 9 run just fine on their own, but files 4, 6, 8, and 10 give the same exit log after loading the run. I converted these files to mzML and they ran! However, all of the data we want to run is in .raw, so we would love to figure out how to run it in .raw. I then did what you suggested. Using the spec lib generated from the mzML run, I ran these same 10 files on a local folder C:/. Here is the log of what happened (files 1-10 are given in the name as "rep#"): diann.exe --f "C:\DIA-NN\Files_share\LL2076_SQp5_HYEA_10ng_blo1_GC10_rep1_ch2_DIAwet10ng45.raw 10 files will be processed First pass: generating a spectral library from DIA data [0:08] File #1/10 [0:34] File #2/10 [1:02] File #3/10 [1:31] File #4/10 DIA-NN exited We cannot figure out why these files (4, 6, 8, and 10) exit on my computer and not on others. Even more strange is that their mzML versions still run. Any thoughts? Thanks, |
Beta Was this translation helpful? Give feedback.
-
Hi,
I have run a number of searches using the default Output file location or specifying a different location.
When the run is complete, Dia-NN will generate a spectral library but does not generate a report in any of the report formats.
Is there something basic I am not doing?
Beta Was this translation helpful? Give feedback.
All reactions