OpenMS parsing

[enryH]

First of all thanks for the great tool. I use it within the nextflow pipelinebigbio/quantms and try to analyze the ProteoBench DDA ion quant module. Therefore I subselected the relevant rows from the SDRF file provide along side PXD028735. The file is the following.

dda_lfq_proteobench_SDRF.sdrf.txt

If I parse this for openms as describe in the latest nfcore workflow step:

https://github.com/bigbio/quantms/blob/655b159251206d862039d8e3990558607355c62b/modules/local/sdrfparsing/main.nf

so translated for the example this would be

parse_sdrf convert-openms -t2 -l  --extension_convert raw:mzML -s dda_lfq_proteobench_SDRF.sdrf.txt

However for OpenMS ProteomicsLFQ this produces an invalid experimental design file. The concentration duplicates for the mixture of samples are not reduced leading to a an error saying ((Fraction Group, Fraction, Label) combination can only appear once), whos combination are indeed not unique.

Fraction_Group	Fraction	Spectra_Filepath	Label	Sample
1	1	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_01.mzML	1	1
1	1	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_01.mzML	1	1
1	1	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_01.mzML	1	1
2	1	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_02.mzML	1	2
2	1	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_02.mzML	1	2
2	1	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_02.mzML	1	2
3	1	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_03.mzML	1	3
3	1	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_03.mzML	1	3
3	1	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_03.mzML	1	3
4	1	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_01.mzML	1	4
4	1	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_01.mzML	1	4
4	1	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_01.mzML	1	4
5	1	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_02.mzML	1	5
5	1	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_02.mzML	1	5
5	1	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_02.mzML	1	5
6	1	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_03.mzML	1	6
6	1	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_03.mzML	1	6
6	1	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_03.mzML	1	6

Sample	MSstats_Condition	MSstats_BioReplicate
1	cond_A	1
2	cond_A	2
3	cond_A	3
4	cond_B	4
5	cond_B	5
6	cond_B	6

instead of

Fraction_Group	Fraction	Spectra_Filepath	Label	Sample
1	3	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_01.mzML	1	1
2	3	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_02.mzML	1	2
3	3	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_03.mzML	1	3
4	3	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_01.mzML	1	4
5	3	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_02.mzML	1	5
6	3	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_03.mzML	1	6

Sample	MSstats_Condition	MSstats_BioReplicate
1	cond_A	1
2	cond_A	2
3	cond_A	3
4	cond_B	4
5	cond_B	5
6	cond_B	6

I can fix it manueally by removing duplicated raw file entries from the SDRF file, but I wonder if this should be handled better?

[fabianegli]

This seems to work now on the dev branch. Do you still experience the issue?

% wget https://github.com/user-attachments/files/18442149/dda_lfq_proteobench_SDRF.sdrf.txt
--2025-04-29 21:05:57--  https://github.com/user-attachments/files/18442149/dda_lfq_proteobench_SDRF.sdrf.txt
Resolving github.com (github.com)... 140.82.121.3
Connecting to github.com (github.com)|140.82.121.3|:443... connected.
HTTP request sent, awaiting response... 302 Found
Location: https://objects.githubusercontent.com/github-production-repository-file-5c1aeb/251399011/18442149?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAVCODYLSA53PQK4ZA%2F20250429%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20250429T190558Z&X-Amz-Expires=300&X-Amz-Signature=e25932403e5f4d90806c31cb42188fa010f35dfae89437ace59423a139196e71&X-Amz-SignedHeaders=host&response-content-disposition=attachment%3Bfilename%3Ddda_lfq_proteobench_SDRF.sdrf.txt&response-content-type=text%2Fplain [following]
--2025-04-29 21:05:58--  https://objects.githubusercontent.com/github-production-repository-file-5c1aeb/251399011/18442149?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAVCODYLSA53PQK4ZA%2F20250429%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20250429T190558Z&X-Amz-Expires=300&X-Amz-Signature=e25932403e5f4d90806c31cb42188fa010f35dfae89437ace59423a139196e71&X-Amz-SignedHeaders=host&response-content-disposition=attachment%3Bfilename%3Ddda_lfq_proteobench_SDRF.sdrf.txt&response-content-type=text%2Fplain
Resolving objects.githubusercontent.com (objects.githubusercontent.com)... 185.199.111.133, 185.199.109.133, 185.199.110.133, ...
Connecting to objects.githubusercontent.com (objects.githubusercontent.com)|185.199.111.133|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 10805 (11K) [text/plain]
Saving to: ‘dda_lfq_proteobench_SDRF.sdrf.txt’

dda_lfq_proteobench_SDRF.sdrf.txt                    100%[=====================================================================================================================>]  10.55K  --.-KB/s    in 0.01s   

2025-04-29 21:05:58 (787 KB/s) - ‘dda_lfq_proteobench_SDRF.sdrf.txt’ saved [10805/10805]

% parse_sdrf convert-openms -t2 -l  --extension_convert raw:mzML -s dda_lfq_proteobench_SDRF.sdrf.txt 
PROCESSING: dda_lfq_proteobench_SDRF.sdrf.txt"
Factor columns: ['factor value[phenotype]']
Characteristics columns (those covered by factor columns removed): ['characteristics[organism]', 'characteristics[organism part]', 'characteristics[biological replicate]', 'characteristics[cell line]', 'characteristics[cell type]', 'characteristics[concentration of]', 'characteristics[disease]', 'characteristics[spiked compound]']
Conditions (2): dict_keys(['cond_A', 'cond_B'])
Files per condition: dict_values([3, 3])
WARNING: "No precursor mass tolerance set. Assuming 10 ppm." occurred 18 times.
WARNING: "No fragment mass tolerance set. Assuming 20 ppm." occurred 18 times.
WARNING: "No dissociation method provided. Assuming HCD." occurred 18 times.
WARNING: "The comment[proteomics data acquisition method] column is missing, default Data-Dependent Acquisition" occurred 18 times.
WARNING: "No sample identifier" occurred 36 times.
SUCCESS (WARNINGS=5): dda_lfq_proteobench_SDRF.sdrf.txt
% cat openms.tsv 
URI	Filename	FixedModifications	VariableModifications	Proteomics Data Acquisition Method	Label	PrecursorMassTolerance	PrecursorMassToleranceUnit	FragmentMassTolerance	FragmentMassToleranceUnit	DissociationMethod	Enzyme
ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/10/PXD010000/LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_01.raw	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_01.raw	Carbamidomethyl (C)		Data-Dependent Acquisition	label free sample	10	ppm	20	ppm	HCD	Trypsin
ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/10/PXD010000/LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_02.raw	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_02.raw	Carbamidomethyl (C)		Data-Dependent Acquisition	label free sample	10	ppm	20	ppm	HCD	Trypsin
ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/10/PXD010000/LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_03.raw	LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_03.raw	Carbamidomethyl (C)		Data-Dependent Acquisition	label free sample	10	ppm	20	ppm	HCD	Trypsin
ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/10/PXD010000/LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_01.raw	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_01.raw	Carbamidomethyl (C)		Data-Dependent Acquisition	label free sample	10	ppm	20	ppm	HCD	Trypsin
ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/10/PXD010000/LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_02.raw	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_02.raw	Carbamidomethyl (C)		Data-Dependent Acquisition	label free sample	10	ppm	20	ppm	HCD	Trypsin
ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/10/PXD010000/LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_03.raw	LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_03.raw	Carbamidomethyl (C)		Data-Dependent Acquisition	label free sample	10	ppm	20	ppm	HCD	Trypsin

[enryH]

This looks promising. I can re-run the workflow with using the dev branch and the full SDRF file.

[enryH]

So I finally came about to test it. Unfortunately although openms.tsv looks fine, ProteomicsLFQ from OpenMS uses the experimental_design.tsv which still is not correctly parsed for the current design. I upload the file here (but it looks the same as before in my first post). I had to add .txt to be able to upload it.

experimental_design.tsv.txt

[enryH]

Just for completeness the error as for quantms 1.5.0:

Command executed:

  ProteomicsLFQ \
      -threads 12 \
      -in LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_01.mzML LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_02.mzML LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_03.mzML LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_01.mzML LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_02.mzML LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_03.mzML \
      -ids LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_01_comet_perc_filter.idXML LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_02_comet_perc_filter.idXML LFQ_Orbitrap_DDA_Condition_A_Sample_Alpha_03_comet_perc_filter.idXML LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_01_comet_perc_filter.idXML LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_02_comet_perc_filter.idXML LFQ_Orbitrap_DDA_Condition_B_Sample_Alpha_03_comet_perc_filter.idXML \
      -design dda_lfq_proteobench_SDRF.sdrf_openms_design.tsv \
      -fasta ProteoBenchFASTA_MixedSpecies_HYE_decoy.fasta \
      -protein_inference aggregation \
      -quantification_method feature_intensity \
      -targeted_only false \
      -feature_with_id_min_score 0.10 \
      -feature_without_id_min_score 0.75 \
      -mass_recalibration false \
      -Seeding:intThreshold 1000 \
      -protein_quantification unique_peptides \
      -alignment_order star \
       \
      -psmFDR 0.01 \
      -proteinFDR 0.01 \
      -picked_proteinFDR true \
      -out_cxml dda_lfq_proteobench_SDRF.sdrf_openms_design_openms.consensusXML \
      -out dda_lfq_proteobench_SDRF.sdrf_openms_design_openms.mzTab \
      -out_msstats dda_lfq_proteobench_SDRF.sdrf_openms_design_msstats_in.csv \
       \
      -debug 0 \
      2>&1 | tee proteomicslfq.log
  
  cat <<-END_VERSIONS > versions.yml
  "BIGBIO_QUANTMS:QUANTMS:LFQ:PROTEOMICSLFQ":
      ProteomicsLFQ: $(ProteomicsLFQ 2>&1 | grep -E '^Version(.*)' | sed 's/Version: //g' | cut -d ' ' -f 1)
  END_VERSIONS

Command exit status:
  8

Command output:
  Error: Unexpected internal error ((Fraction Group, Fraction, Label) combination can only appear once)

[jpfeuffer]

OpenMS is right. There are clearly triplicates for each entry.

[jpfeuffer]

Not sure if the sdrf parser supports annotating the same file with multiple samples in lfq.
Was Sdrf supposed to model that?

[jpfeuffer]

Although technically correct for a mixture of proteins from different organisms, one would need to philosophize if that is even valid sdrf.
Besides reference database generation this information is actually never needed in any processing tools.

[jpfeuffer]

For now I would definitely suggest to only use one sample per file and just enter a list of organisms for that sample

[enryH]

The sdrf file parses as valid also with the last release of sdrf-pipelines.