DIA-NN 1.9.2 (Data-Independent Acquisition by Neural Networks)
Compiled on Oct 20 2024 02:59:53
Current date and time: Thu Jun 19 14:49:31 2025
Logical CPU cores: 256
/diann-1.9.2/diann-linux --cfg ./ProteoBench/Search3/Search3.txt-- 

Thread number set to 100
Output will be filtered at 0.01 FDR
Min fragment m/z set to 50
Max fragment m/z set to 2000
Min precursor m/z set to 400
Max precursor m/z set to 1000
In silico digest will involve cuts at K*,R*
Maximum number of missed cleavages set to 1
Min peptide length set to 6
Max peptide length set to 30
Min precursor charge set to 1
Max precursor charge set to 4
Modification UniMod:4 with mass delta 57.0215 at C will be considered as fixed
Maximum number of variable modifications set to 2
Modification UniMod:35 with mass delta 15.9949 at M will be considered as variable
Modification UniMod:1 with mass delta 42.0106 at *n will be considered as variable
A spectral library will be generated
N-terminal methionine excision enabled
The spectral library (if generated) will retain the original spectra but will include empirically-aligned RTs
DIA-NN will carry out FASTA digest for in silico lib generation
High accuracy quantification mode enabled
Deep learning will be used to generate a new in silico spectral library from peptides provided
N-terminal methionine excision enabled
Precursor/protein x samples expression level matrices will be saved along with the main report
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
Peptidoform scoring enabled
The spectral library (if generated) will include empirical spectra and empirically-aligned RTs
WARNING: unrecognised option [--]
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.
WARNING: it is strongly recommended to first generate an in silico-predicted library in a separate pipeline step and then use it to process the raw data, now without activating FASTA digest
The following variable modifications will be scored: UniMod:35 UniMod:1 

6 files will be processed
[0:00] Loading FASTA /nas/longleaf/home/slyon/DIANN/ProteoBench/ProteoBenchFASTA_MixedSpecies_HYE.fasta
[0:05] Processing FASTA
[0:12] Assembling elution groups
[0:19] 5441048 precursors generated
[0:19] Protein names missing for some isoforms
[0:19] Gene names missing for some isoforms
[0:19] Library contains 31685 proteins, and 0 genes
[0:27] [0:42] [5:51] [6:37] [6:50] [6:55] Saving the library to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-lib.predicted.speclib
[7:07] Initialising library
[7:24] Loading spectral library /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-lib.predicted.speclib
[7:32] Library annotated with sequence database(s): /nas/longleaf/home/slyon/DIANN/ProteoBench/ProteoBenchFASTA_MixedSpecies_HYE.fasta
[7:33] Spectral library loaded: 31837 protein isoforms, 51765 protein groups and 5441048 precursors in 2890025 elution groups.
[7:33] Loading protein annotations from FASTA /nas/longleaf/home/slyon/DIANN/ProteoBench/ProteoBenchFASTA_MixedSpecies_HYE.fasta
[7:34] Annotating library proteins with information from the FASTA database
[7:34] Protein names missing for some isoforms
[7:34] Gene names missing for some isoforms
[7:34] Library contains 31685 proteins, and 0 genes
[7:39] Initialising library

First pass: generating a spectral library from DIA data

[7:58] File #1/6
[7:58] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_A_REP1.mzML
[10:24] 5337568 library precursors are potentially detectable
[10:25] Calibrating with mass accuracies 30 (MS1), 20 (MS2)
[11:05] RT window set to 0.83699
[11:05] Peak width: 2.744
[11:05] Scan window radius set to 6
[11:06] Recommended MS1 mass accuracy setting: 2.31639 ppm
[11:59] Optimised mass accuracy: 8.45298 ppm
[13:22] Removing low confidence identifications
[14:07] Precursors at 1% peptidoform FDR: 61622
[14:08] Removing interfering precursors
[14:17] Training neural networks on 309558 PSMs
[14:29] Number of IDs at 0.01 FDR: 96825
[14:33] Precursors at 1% peptidoform FDR: 83436
[14:34] Calculating protein q-values
[14:34] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[14:34] Quantification
[14:35] Precursors with monitored PTMs at 1% FDR: 2578 out of 21337 considered
[14:35] Unmodified precursors with monitored PTM sites at 1% FDR: 16138
[14:35] Precursors with PTMs localised (when required) with > 90% confidence: 2512 out of 2578
[14:36] Quantification information saved to /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_A_REP1.mzML.quant

[14:36] File #2/6
[14:36] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_A_REP2.mzML
[16:23] 5337568 library precursors are potentially detectable
[16:24] Calibrating with mass accuracies 30 (MS1), 18.8884 (MS2)
[17:05] RT window set to 0.849475
[17:05] Recommended MS1 mass accuracy setting: 2.15375 ppm
[18:17] Removing low confidence identifications
[19:02] Precursors at 1% peptidoform FDR: 63793
[19:03] Removing interfering precursors
[19:13] Training neural networks on 330676 PSMs
[19:27] Number of IDs at 0.01 FDR: 99245
[19:31] Precursors at 1% peptidoform FDR: 84824
[19:33] Calculating protein q-values
[19:33] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[19:33] Quantification
[19:34] Precursors with monitored PTMs at 1% FDR: 2626 out of 21833 considered
[19:34] Unmodified precursors with monitored PTM sites at 1% FDR: 15999
[19:34] Precursors with PTMs localised (when required) with > 90% confidence: 2562 out of 2626
[19:35] Quantification information saved to /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_A_REP2.mzML.quant

[19:35] File #3/6
[19:35] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_A_REP3.mzML
[21:41] 5337568 library precursors are potentially detectable
[21:42] Calibrating with mass accuracies 30 (MS1), 18.2184 (MS2)
[22:04] RT window set to 0.911574
[22:04] Recommended MS1 mass accuracy setting: 2.18489 ppm
[23:03] Removing low confidence identifications
[23:36] Precursors at 1% peptidoform FDR: 62390
[23:37] Removing interfering precursors
[23:47] Training neural networks on 324844 PSMs
[24:01] Number of IDs at 0.01 FDR: 98161
[24:05] Precursors at 1% peptidoform FDR: 83051
[24:06] Calculating protein q-values
[24:06] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[24:07] Quantification
[24:07] Precursors with monitored PTMs at 1% FDR: 2503 out of 21578 considered
[24:07] Unmodified precursors with monitored PTM sites at 1% FDR: 15940
[24:07] Precursors with PTMs localised (when required) with > 90% confidence: 2438 out of 2503
[24:08] Quantification information saved to /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_A_REP3.mzML.quant

[24:08] File #4/6
[24:08] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_B_REP1.mzML
[26:10] 5337568 library precursors are potentially detectable
[26:11] Calibrating with mass accuracies 30 (MS1), 18.8708 (MS2)
[26:53] RT window set to 0.899967
[26:53] Recommended MS1 mass accuracy setting: 2.30006 ppm
[28:06] Removing low confidence identifications
[28:52] Precursors at 1% peptidoform FDR: 64941
[28:53] Removing interfering precursors
[29:02] Training neural networks on 336128 PSMs
[29:15] Number of IDs at 0.01 FDR: 99538
[29:19] Precursors at 1% peptidoform FDR: 85845
[29:20] Calculating protein q-values
[29:21] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[29:21] Quantification
[29:22] Precursors with monitored PTMs at 1% FDR: 3106 out of 22700 considered
[29:22] Unmodified precursors with monitored PTM sites at 1% FDR: 16332
[29:22] Precursors with PTMs localised (when required) with > 90% confidence: 3027 out of 3106
[29:23] Quantification information saved to /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_B_REP1.mzML.quant

[29:23] File #5/6
[29:23] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_B_REP2.mzML
[31:24] 5337568 library precursors are potentially detectable
[31:25] Calibrating with mass accuracies 30 (MS1), 19.0406 (MS2)
[31:45] RT window set to 0.830138
[31:45] Recommended MS1 mass accuracy setting: 2.44179 ppm
[32:39] Removing low confidence identifications
[33:09] Precursors at 1% peptidoform FDR: 64952
[33:10] Removing interfering precursors
[33:19] Training neural networks on 331546 PSMs
[33:33] Number of IDs at 0.01 FDR: 99036
[33:37] Precursors at 1% peptidoform FDR: 85567
[33:38] Calculating protein q-values
[33:38] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[33:39] Quantification
[33:39] Precursors with monitored PTMs at 1% FDR: 3229 out of 22668 considered
[33:39] Unmodified precursors with monitored PTM sites at 1% FDR: 16357
[33:39] Precursors with PTMs localised (when required) with > 90% confidence: 3160 out of 3229
[33:40] Quantification information saved to /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_B_REP2.mzML.quant

[33:40] File #6/6
[33:40] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_B_REP3.mzML
[35:51] 5337568 library precursors are potentially detectable
[35:52] Calibrating with mass accuracies 30 (MS1), 19.024 (MS2)
[36:30] RT window set to 0.792754
[36:30] Recommended MS1 mass accuracy setting: 2.07223 ppm
[37:33] Removing low confidence identifications
[38:10] Precursors at 1% peptidoform FDR: 64873
[38:11] Removing interfering precursors
[38:20] Training neural networks on 330702 PSMs
[38:31] Number of IDs at 0.01 FDR: 99169
[38:36] Precursors at 1% peptidoform FDR: 82988
[38:37] Calculating protein q-values
[38:38] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[38:38] Quantification
[38:38] Precursors with monitored PTMs at 1% FDR: 2765 out of 22774 considered
[38:38] Unmodified precursors with monitored PTM sites at 1% FDR: 15709
[38:38] Precursors with PTMs localised (when required) with > 90% confidence: 2693 out of 2765
[38:39] Quantification information saved to /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_B_REP3.mzML.quant

[38:39] Cross-run analysis
[38:39] Reading quantification information: 6 files
[38:49] Quantifying peptides
[39:27] Assembling protein groups
[39:30] Quantifying proteins
[39:30] Calculating q-values for protein and gene groups
[39:32] Calculating global q-values for protein and gene groups
[39:32] Protein groups with global q-value <= 0.01: 13145
[39:36] Compressed report saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-first-pass.parquet. Use R 'arrow' or Python 'PyArrow' package to process
[39:36] Writing report
[39:47] Report saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-first-pass.tsv.
[39:47] Saving precursor levels matrix
[39:47] Precursor levels matrix (1% precursor and protein group FDR) saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-first-pass.pr_matrix.tsv.
[39:47] Manifest saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-first-pass.manifest.txt
[39:47] Stats report saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-first-pass.stats.tsv
[39:47] Generating spectral library:
[39:49] 124844 target and 1181 decoy precursors saved
WARNING: 2335 precursors without any fragments annotated were skipped
[39:49] Spectral library saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-lib.parquet

[39:50] Loading spectral library /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-lib.parquet
[39:51] Spectral library loaded: 13762 protein isoforms, 14686 protein groups and 126025 precursors in 118001 elution groups.
[39:51] Loading protein annotations from FASTA /nas/longleaf/home/slyon/DIANN/ProteoBench/ProteoBenchFASTA_MixedSpecies_HYE.fasta
[39:51] Annotating library proteins with information from the FASTA database
[39:51] Gene names missing for some isoforms
[39:51] Library contains 13736 proteins, and 0 genes
[39:52] Initialising library
[39:52] Saving the library to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report-lib.parquet.skyline.speclib


Second pass: using the newly created spectral library to reanalyse the data

[39:52] File #1/6
[39:52] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_A_REP1.mzML
[42:34] 124844 library precursors are potentially detectable
[42:34] Calibrating with mass accuracies 30 (MS1), 18.1143 (MS2)
[42:35] RT window set to 0.379584
[42:35] Recommended MS1 mass accuracy setting: 2.71874 ppm
[42:37] Removing low confidence identifications
[42:41] Precursors at 1% peptidoform FDR: 77967
[42:41] Removing interfering precursors
[42:42] Training neural networks on 163284 PSMs
[42:46] Number of IDs at 0.01 FDR: 107510
[42:50] Precursors at 1% peptidoform FDR: 93046
[42:50] Calculating protein q-values
[42:50] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[42:50] Quantification
[42:50] Precursors with monitored PTMs at 1% FDR: 3174 out of 23208 considered
[42:50] Unmodified precursors with monitored PTM sites at 1% FDR: 17598
[42:50] Precursors with PTMs localised (when required) with > 90% confidence: 3095 out of 3174

[42:51] File #2/6
[42:51] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_A_REP2.mzML
[44:32] 124844 library precursors are potentially detectable
[44:32] Calibrating with mass accuracies 30 (MS1), 18.4276 (MS2)
[44:33] RT window set to 0.39771
[44:33] Recommended MS1 mass accuracy setting: 2.61411 ppm
[44:35] Removing low confidence identifications
[44:38] Precursors at 1% peptidoform FDR: 77582
[44:38] Removing interfering precursors
[44:39] Training neural networks on 163674 PSMs
[44:45] Number of IDs at 0.01 FDR: 108091
[44:50] Precursors at 1% peptidoform FDR: 92796
[44:50] Calculating protein q-values
[44:50] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[44:50] Quantification
[44:50] Precursors with monitored PTMs at 1% FDR: 3163 out of 22992 considered
[44:50] Unmodified precursors with monitored PTM sites at 1% FDR: 17515
[44:50] Precursors with PTMs localised (when required) with > 90% confidence: 3097 out of 3163

[44:51] File #3/6
[44:51] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_A_REP3.mzML
[46:38] 124844 library precursors are potentially detectable
[46:38] Calibrating with mass accuracies 30 (MS1), 17.8295 (MS2)
[46:38] RT window set to 0.366739
[46:38] Recommended MS1 mass accuracy setting: 2.50649 ppm
[46:40] Removing low confidence identifications
[46:44] Precursors at 1% peptidoform FDR: 77072
[46:44] Removing interfering precursors
[46:45] Training neural networks on 163635 PSMs
[46:50] Number of IDs at 0.01 FDR: 106991
[46:55] Precursors at 1% peptidoform FDR: 92840
[46:55] Calculating protein q-values
[46:55] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[46:55] Quantification
[46:55] Precursors with monitored PTMs at 1% FDR: 3142 out of 22946 considered
[46:55] Unmodified precursors with monitored PTM sites at 1% FDR: 17487
[46:55] Precursors with PTMs localised (when required) with > 90% confidence: 3063 out of 3142

[46:55] File #4/6
[46:55] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_B_REP1.mzML
[49:03] 124844 library precursors are potentially detectable
[49:03] Calibrating with mass accuracies 30 (MS1), 18.4737 (MS2)
[49:04] RT window set to 0.42558
[49:04] Recommended MS1 mass accuracy setting: 2.79038 ppm
[49:06] Removing low confidence identifications
[49:10] Precursors at 1% peptidoform FDR: 79788
[49:10] Removing interfering precursors
[49:11] Training neural networks on 165093 PSMs
[49:16] Number of IDs at 0.01 FDR: 108881
[49:20] Precursors at 1% peptidoform FDR: 94580
[49:20] Calculating protein q-values
[49:20] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[49:20] Quantification
[49:20] Precursors with monitored PTMs at 1% FDR: 3449 out of 23854 considered
[49:20] Unmodified precursors with monitored PTM sites at 1% FDR: 17926
[49:20] Precursors with PTMs localised (when required) with > 90% confidence: 3381 out of 3449

[49:21] File #5/6
[49:21] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_B_REP2.mzML
[51:16] 124844 library precursors are potentially detectable
[51:16] Calibrating with mass accuracies 30 (MS1), 18.5947 (MS2)
[51:17] RT window set to 0.391225
[51:17] Recommended MS1 mass accuracy setting: 2.5857 ppm
[51:19] Removing low confidence identifications
[51:22] Precursors at 1% peptidoform FDR: 79463
[51:22] Removing interfering precursors
[51:23] Training neural networks on 164632 PSMs
[51:28] Number of IDs at 0.01 FDR: 109151
[51:32] Precursors at 1% peptidoform FDR: 94560
[51:32] Calculating protein q-values
[51:32] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[51:32] Quantification
[51:32] Precursors with monitored PTMs at 1% FDR: 3478 out of 23754 considered
[51:32] Unmodified precursors with monitored PTM sites at 1% FDR: 17871
[51:32] Precursors with PTMs localised (when required) with > 90% confidence: 3413 out of 3478

[51:33] File #6/6
[51:33] Loading run /work/users/s/l/slyon/ProteoBench_Astral_DIA/LFQ_Astral_DIA_15min_50ng_Condition_B_REP3.mzML
[53:21] 124844 library precursors are potentially detectable
[53:22] Calibrating with mass accuracies 30 (MS1), 18.7351 (MS2)
[53:22] RT window set to 0.417396
[53:22] Recommended MS1 mass accuracy setting: 2.49398 ppm
[53:25] Removing low confidence identifications
[53:28] Precursors at 1% peptidoform FDR: 78498
[53:28] Removing interfering precursors
[53:30] Training neural networks on 164724 PSMs
[53:35] Number of IDs at 0.01 FDR: 108902
[53:38] Precursors at 1% peptidoform FDR: 93674
[53:38] Calculating protein q-values
[53:38] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only)
[53:38] Quantification
[53:39] Precursors with monitored PTMs at 1% FDR: 3416 out of 23609 considered
[53:39] Unmodified precursors with monitored PTM sites at 1% FDR: 17710
[53:39] Precursors with PTMs localised (when required) with > 90% confidence: 3340 out of 3416

[53:39] Cross-run analysis
[53:39] Reading quantification information: 6 files
[53:41] Quantifying peptides
[55:11] Quantification parameters: 0.310316, 0.00147709, 0.0013423, 0.0164819, 0.0163377, 0.016407, 0.139633, 0.153693, 0.133123, 0.0177932, 0.0459829, 0.0364797, 0.240836, 0.0509868, 0.0613075, 0.106353
[55:28] Quantifying proteins
[55:29] Calculating q-values for protein and gene groups
[55:29] Calculating global q-values for protein and gene groups
[55:29] Protein groups with global q-value <= 0.01: 13305
[55:33] Compressed report saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report.parquet. Use R 'arrow' or Python 'PyArrow' package to process
[55:33] Writing report
[55:43] Report saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report.tsv.
[55:43] Saving precursor levels matrix
[55:44] Precursor levels matrix (1% precursor and protein group FDR) saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report.pr_matrix.tsv.
[55:44] Saving protein group levels matrix
[55:44] Protein group levels matrix (1% precursor FDR and protein group FDR) saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report.pg_matrix.tsv.
[55:44] Saving gene group levels matrix
[55:44] Gene groups levels matrix (1% precursor FDR and protein group FDR) saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report.gg_matrix.tsv.
[55:44] Saving unique genes levels matrix
[55:44] Unique genes levels matrix (1% precursor FDR and protein group FDR) saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report.unique_genes_matrix.tsv.
[55:44] Manifest saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report.manifest.txt
[55:44] Stats report saved to /nas/longleaf/home/slyon/DIANN/ProteoBench/Search3/Search3_report.stats.tsv

