spectral-counts fails to process percolator output

[Daryl W-M]

Hello. Thank you for the new 4.0 update. I'm using the latest build and trying to use spectral-counts to process percolator output, but it seems that spectral-counts is finding in the percolator target psms file a float where it expects to find the sequence string.

./crux-4.0.Linux.x86_64/bin/crux spectral-counts --overwrite T --output-dir /media/big-ssd/experiments/P3830/spectral-counts-output --protein-database ~/otf-peak-detect/fasta/ups1-ups2-yeast.fasta --verbosity 30 --fileroot P3830 /media/big-ssd/experiments/P3830/percolator-output-pasef-recalibrated/P3830.percolator.target.psms.txt

WARNING: The output directory '/media/big-ssd/experiments/P3830/spectral-counts-output' already exists.

Existing files will be overwritten.

WARNING: The file '/media/big-ssd/experiments/P3830/spectral-counts-output/P3830.spectral-counts.log.txt' already exists and will be overwritten.

INFO: CPU: deep-thought-home

INFO: Crux version: 4.0-fbfabf9-2021-04-06

INFO: Fri 7 May 09:43:48 AEST 2021

INFO: Beginning spectral-counts.

INFO: Reached protein 1000

INFO: Reached protein 2000

INFO: Reached protein 3000

INFO: Reached protein 4000

INFO: Reached protein 5000

INFO: Reached protein 6000

INFO: Total proteins found: 6097

FATAL: An exception occurred: Could not convert string '611.078'

The number seems to be the total matches/spectrum column of the psms file.

Is there a command I'm missing?

Regards,

Daryl.

[Rita Chupalov]

Hi Daryl,

sorry about the problem. Could you share your input files: the FASTA and the Percolator output so that I can reproduce the problem?

Thank you,

Rita

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Rita Chupalov

Software Developer

at UW Genome Sciences Department

[Daryl W-M]

Hello Rita; thanks for your reply. The FASTA and Percolator target psms file are attached. This is the first time I've tried spectral-counts so it could well be something I've done wrong. Thanks for looking into it.

Regards,

Daryl.

[Rita Chupalov]

  Daryl,

it looks like "matches per spectrum" column in your matches file contains a single float number: 611.078. It doesn't look right. It should be integer and it probably shouldn't be the same for all peptides. Could you provide the command line you use to generate this file? 

Sorry for the late reply.

Rita

On Fri, May 7, 2021 at 4:35 PM Daryl W-M <daryl...@gmail.com> wrote:

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[Rita Chupalov]

Thank you, Daryl,

I would also need the .fasta and the .params files to reproduce the issue.

Rita

On Wed, May 19, 2021 at 8:11 PM Daryl Wilding-McBride <daryl...@gmail.com> wrote:

Hi Rita,

Here's the percolator command I use to process comet output (actually for a different experiment but the issue is the same):

crux percolator --overwrite T --subset-max-train 1000000 --klammer F --maxiter 10 --output-dir ./P3856/percolator-output-pasef-recalibrated --picked-protein ./fasta/Human_Yeast_Ecoli.fasta --protein T --protein-enzyme trypsin --search-input auto --verbosity 30 --fileroot P3856 ./comet-output-pasef-recalibrated/P3856_YHE211_1_Slot1-1_1_5104.comet.pin

The comet command I use is this:

crux comet --parameter-file ./comet/TimsTOF-recalibration.params --output-dir ./comet-output-pasef-recalibrated --fileroot "P3856_YHE211_1_Slot1-1_1_5104" ./exp-P3856-run-P3856_YHE211_1_Slot1-1_1_5104-features-pasef-recalibrated.mgf ./fasta/Human_Yeast_Ecoli.fasta

Attached is the MGF.

 exp-P3856-run-P3856_YHE211_1_Slot1-1_1_5104-fea...

Thanks,

Daryl.

[Rita Chupalov]

Hi Daryl,

I experimented with your data a bit and it looks like the culprit here is percolator's --subset-max-training option you are using. If removed, the spectral matches per protein column becomes correct (integer-valued) and spectral-counts runs normally. Though you probably need to use Human_Yeast_Ecoli.fasta because the other fasta file doesn't have all the proteins.

The comet output is not too big and percolator runs reasonably fast without limiting the training set size even on my modest development machine. 

I will dig into it a bit more to find why it is happening, but at least you have a workaround for now.

Rita

[Daryl Wilding-McBride]

Hi Rita. Thank you for the workaround; much appreciated.