What does this warning mean?

[sunyusui]

Warning: scores is null for index 0 in scan 1448

Warning: scores is null for index 0 in scan 1448

Warning: scores is null for index 0 in scan 1479

Warning: scores is null for index 0 in scan 1479

[alchemistmatt]

That means that the spectrum did not have a good match. Examine the data for scans 1448 and 1479. I suspect they are sparse spectra with few ions above the noise background. As long as you're not seeing 1000's of warnings like that, just ignore them.

[sunyusui]

There are 231’s such warnings. Two other warnings were also reported today. What caused it? … Warning: scores is null for index 0 in scan 1547

Warning: scores is null for index 0 in scan 1547

Warning: scores is null for index 0 in scan 1560

Warning: scores is null for index 1 in scan 1560

Warning: scores is null for index 0 in scan 2174

Warning: scores is null for index 0 in scan 2453

Warning: scores is null for index 0 in scan 2522

Warning: scores is null for index 0 in scan 2557

Warning: scores is null for index 0 in scan 2953 Estimated matched sequences: 0 Decoy-spectrum match E-value calculation elapsed Time: 0.3 sec

_Warning: Error computing FDR: Cannot compute FDR Scores; decoy results file is empty Error processing 2DLCH4.pbf: Cannot compute FDR Scores; decoy results file is empty

The following are the parameters used in my experiment.

MSPathFinderT version 1.0.6619 (February 14, 2018) MaxThreads: 4 SpectrumFilePath: 2DLC_H4.pbf DatabaseFilePath: uniprot-histone.fasta FeatureFilePath: 2DLC_H4.ms1ft OutputDir: .\ InternalCleavageMode: SingleInternalCleavage Tag-based search: True Tda: Target+Decoy PrecursorIonTolerancePpm: 10 ProductIonTolerancePpm: 10 MinSequenceLength: 21 MaxSequenceLength: 500 MinPrecursorIonCharge: 2 MaxPrecursorIonCharge: 50 MinProductIonCharge: 1 MaxProductIonCharge: 20 MinSequenceMass: 3000 MaxSequenceMass: 50000 MaxDynamicModificationsPerSequence: 4 Modifications: C(2) H(2) N(0) O(1) S(0),R,opt,Everywhere,Acetyl C(2) H(2) N(0) O(1) S(0),K,opt,Everywhere,Acetyl C(1) H(2) N(0) O(0) S(0),R,opt,Everywhere,Methyl C(1) H(2) N(0) O(0) S(0),K,opt,Everywhere,Methyl C(2) H(4) N(0) O(0) S(0),R,opt,Everywhere,Dimethyl C(2) H(4) N(0) O(0) S(0),K,opt,Everywhere,Dimethyl C(3) H(6) N(0) O(0) S(0),R,opt,Everywhere,Trimethyl C(0) H(1) N(0) O(3) S(0) P(1),S,opt,Everywhere,Phospho C(0) H(1) N(0) O(3) S(0) P(1),T,opt,Everywhere,Phospho C(0) H(1) N(0) O(3) S(0) P(1),Y,opt,Everywhere,Phospho

[FarmGeek4Life]

You specified a target+decoy search, but there were no results from the decoy search; either something failed with running the decoy search, or there simply were no matches with the decoy database. I can't say for certain without more of the command line output.

[sunyusui]

The following is the command line output; some lines are replaced by "......". …… Getting MS1 features from 2DLC_H4.ms1ft Reading pbf file... Total Progress: 0.00%, 0d 0h 0.00m elapsed, Current Task: Reading spectra file, estimated remaining: 0d 0h 0.00m Elapsed Time: 4.2 sec Reading Fasta File Generating uniprot-histone.icseq and Generating uniprot-histone.icanno ... Done Reading ProMex results... 0/0 features loaded...Elapsed Time: 0.2 sec Generating deconvoluted spectra for MS/MS spectra... Elapsed Time: 44.7 sec Generating sequence tags for MS/MS spectra... Number of spectra: 2698 Processing, 0 spectra done, 0.0% complete, 0.1 sec elapsed Processing, 1090 spectra done, 40.4% complete, 15.1 sec elapsed Processing, 1945 spectra done, 72.1% complete, 30.1 sec elapsed Processing, 2424 spectra done, 89.8% complete, 45.2 sec elapsed Generated sequence tags: 34372 Elapsed Time: 51.2 sec Caching peaks in MS1 spectra: 270 scans Sorting MS1 peaks: 4,973,249 peaks Reading the target database... Elapsed Time: 0.0 sec Tag-based searching the target database Number of spectra containing sequence tags: 2698 Processing, 0 spectra done, 0.0% complete, 0.0 sec elapsed Processing, 115 spectra done, 4.3% complete, 25.5 sec elapsed Processing, 190 spectra done, 7.0% complete, 41.3 sec elapsed Processing, 232 spectra done, 8.6% complete, 57.6 sec elapsed Processing, 351 spectra done, 13.0% complete, 74.4 sec elapsed Processing, 383 spectra done, 14.2% complete, 90.8 sec elapsed Processing, 424 spectra done, 15.7% complete, 107.0 sec elapsed Processing, 494 spectra done, 18.3% complete, 141.1 sec elapsed Total Progress: 6.28%, 0d 0h 5.00m elapsed, Current Task: Tag-based searching the target database, estimated remaining: 0d 1h 14.60m …… Processing, 2145 spectra done, 79.5% complete, 815.1 sec elapsed Processing, 2199 spectra done, 81.5% complete, 875.5 sec elapsed Processing, 2273 spectra done, 84.2% complete, 935.9 sec elapsed Processing, 2323 spectra done, 86.1% complete, 996.2 sec elapsed Processing, 2377 spectra done, 88.1% complete, 1057.5 sec elapsed Total Progress: 7.22%, 0d 0h 20.00m elapsed, Current Task: Tag-based searching the target database, estimated remaining: 0d 4h 16.93m Processing, 2426 spectra done, 89.9% complete, 1121.9 sec elapsed Processing, 2547 spectra done, 94.4% complete, 1194.4 sec elapsed Collected candidate matches: 1078 Target database tag-based search elapsed Time: 1296.4 sec Searching the target database Generating uniprot-histone.icplcp ... Done Estimated Sequences: 2,387,364 Processing, 0 proteins done, 0.0% complete, 0.0 sec elapsed Processing, 1133 proteins done, 0.0% complete, 15.1 sec elapsed Processing, 1626 proteins done, 0.1% complete, 30.1 sec elapsed Processing, 2365 proteins done, 0.1% complete, 45.1 sec elapsed Processing, 2941 proteins done, 0.1% complete, 60.2 sec elapsed Total Progress: 7.43%, 0d 0h 25.00m elapsed, Current Task: Searching the target database, estimated remaining: 0d 5h 11.35m …… Processing, 2378741 proteins done, 99.6% complete, 66906.4 sec elapsed Total Progress: 46.37%, 0d 19h 0.07m elapsed, Current Task: Searching the target database, estimated remaining: 0d 21h 58.57m Collected candidate matches: 1078 Target database search elapsed Time: 67135.0 sec Calculating spectral E-values for target-spectrum matches

Warning: scores is null for index 0 in scan 1448 …… Warning: scores is null for index 1 in scan 2959 Estimated matched sequences: 855 Processing, 0 proteins done, 0.0% complete, 2.9 sec elapsed Processing, 23 proteins done, 2.7% complete, 18.9 sec elapsed Processing, 42 proteins done, 4.9% complete, 34.0 sec elapsed Processing, 61 proteins done, 7.1% complete, 49.4 sec elapsed Processing, 84 proteins done, 9.8% complete, 65.0 sec elapsed Processing, 104 proteins done, 12.2% complete, 80.3 sec elapsed Total Progress: 47.19%, 0d 19h 5.07m elapsed, Current Task: Calculating spectral E-values for target-spectrum matches, estimated remaining: 0d 21h 21.43m …… Processing, 542 proteins done, 63.4% complete, 416.6 sec elapsed Processing, 621 proteins done, 72.6% complete, 476.9 sec elapsed Processing, 703 proteins done, 82.2% complete, 537.0 sec elapsed Processing, 781 proteins done, 91.3% complete, 597.1 sec elapsed Processing, 847 proteins done, 99.1% complete, 657.5 sec elapsed Target-spectrum match E-value calculation elapsed Time: 739.5 sec Creating uniprot-histone.icsfldecoy.fasta Generating uniprot-histone.icsfldecoy.icseq and Generating uniprot-histone.icsfldecoy.icanno ... Done Reading the decoy database... Elapsed Time: 0.0 sec Tag-based searching the decoy database Number of spectra containing sequence tags: 2698 Processing, 0 spectra done, 0.0% complete, 0.0 sec elapsed Total Progress: 52.01%, 0d 19h 15.07m elapsed, Current Task: Tag-based searching the decoy database, estimated remaining: 0d 17h 45.70m Processing, 284 spectra done, 10.5% complete, 17.2 sec elapsed Processing, 404 spectra done, 15.0% complete, 40.0 sec elapsed Processing, 458 spectra done, 17.0% complete, 57.8 sec elapsed Processing, 533 spectra done, 19.8% complete, 77.1 sec elapsed Processing, 637 spectra done, 23.6% complete, 92.4 sec elapsed Processing, 691 spectra done, 25.6% complete, 108.0 sec elapsed Processing, 814 spectra done, 30.2% complete, 147.6 sec elapsed Processing, 1218 spectra done, 45.1% complete, 178.7 sec elapsed Processing, 1337 spectra done, 49.6% complete, 210.1 sec elapsed Processing, 1855 spectra done, 68.8% complete, 241.7 sec elapsed Processing, 2100 spectra done, 77.8% complete, 278.1 sec elapsed Total Progress: 53.11%, 0d 19h 20.08m elapsed, Current Task: Tag-based searching the decoy database, estimated remaining: 0d 17h 4.05m Processing, 2354 spectra done, 87.2% complete, 341.4 sec elapsed Processing, 2523 spectra done, 93.5% complete, 408.7 sec elapsed Collected candidate matches: 15 Decoy database tag-based search elapsed Time: 466.2 sec Searching the decoy database Generating uniprot-histone.icsfldecoy.icplcp ... Done Estimated Sequences: 2,387,364 Processing, 0 proteins done, 0.0% complete, 0.0 sec elapsed Processing, 1064 proteins done, 0.0% complete, 15.0 sec elapsed Processing, 1440 proteins done, 0.1% complete, 30.0 sec elapsed Processing, 1805 proteins done, 0.1% complete, 45.0 sec elapsed Processing, 2480 proteins done, 0.1% complete, 60.1 sec elapsed Processing, 2938 proteins done, 0.1% complete, 75.2 sec elapsed Processing, 3430 proteins done, 0.1% complete, 90.2 sec elapsed Processing, 4098 proteins done, 0.2% complete, 105.2 sec elapsed Total Progress: 53.47%, 0d 19h 25.08m elapsed, Current Task: Searching the decoy database, estimated remaining: 0d 16h 54.00m …… Processing, 2368541 proteins done, 99.2% complete, 68095.2 sec elapsed Total Progress: 92.27%, 1d 14h 20.15m elapsed, Current Task: Searching the decoy database, estimated remaining: 0d 3h 12.82m Processing, 2380297 proteins done, 99.7% complete, 68395.3 sec elapsed Total Progress: 92.45%, 1d 14h 25.15m elapsed, Current Task: Searching the decoy database, estimated remaining: 0d 3h 8.18m Collected candidate matches: 15 Decoy database search elapsed Time: 68569.7 sec Calculating spectral E-values for decoy-spectrum matches

Warning: scores is null for index 0 in scan 1547 …… Warning: scores is null for index 0 in scan 2953 Estimated matched sequences: 0 Decoy-spectrum match E-value calculation elapsed Time: 0.3 sec

Warning: Error computing FDR: Cannot compute FDR Scores; decoy results file is empty Error processing 2DLC_H4.pbf: Cannot compute FDR Scores; decoy results file is empty

[FarmGeek4Life]

So, it did perform the decoy search with a shuffled database, and in this case did not find any matches with high enough scores to pass internal thresholds. If you want to try the decoy search again with a different decoy database, you can delete the uniprot-histone.icsfldecoy.* files and run the decoy search again (which will create a new shuffled decoy database).