FDRBench is a tool for FDR control evaluation in proteomics. It provides two main functions: (1) build entrapment databases using randomly shuffled target sequences or using sequences from foreign species, and (2) estimate the FDP using the lower bound, combined, and paired methods.
FDRBench is written using Java. Only Java is required to be installed to run FDRBench.
$ java -jar fdrbench-0.0.2.jar
usage: Options
-db <arg> Protein database file
-crux <arg> The peptide list generated by Crux
-target_pep <arg> The target peptide list to consider
-decoy Add decoy or not
-clip_n_m When digesting a protein starting with amino acid M, two copies of the
leading peptides (with and without the N-terminal M) are considered or
not. Default is false.
-fix_nc <arg> Fix N/C terminal amino acid. n/N: only N terminal, c/C: only C terminal
and nc/NC/cn/CN: both (default)
-o <arg> Output file
-I2L Convert I to L
-diann For DIA-NN
-uniprot For Uniprot
-prosit Generate Prosit input file
-charge <arg> For prosit input: charge range, 2,3,4
-nce <arg> For prosit input: NCE
-enzyme <arg> Enzyme used for protein digestion. 0:Non enzyme, 1:Trypsin, 2:Trypsin (no
P rule) (default), 3:Arg-C, 4:Arg-C (no P rule), 5:Arg-N, 6:Glu-C,
7:Lys-C
-miss_c <arg> The max missed cleavages, default is 1
-minLength <arg> The minimum length of peptide to consider, default is 7
-maxLength <arg> The maximum length of peptide to consider, default is 35
-export_db Export protein database or not
-seed <arg> Random seed for generating decoy peptides
-fix_seed Use a fixed random seed for all decoy peptides generation
-fold <arg> The number of folds for generating entrapment proteins/peptides
-r <arg> For FDP calculation: #entrapment/#target
-use_v1 <arg> Use the first version of FDP calculation for 1-fold
-pick <arg> If a group has multiple proteins, how to peak one protein: first
(default),last,random
-a Generate entrapment protein(s) for each target protein independently
-check Checking for duplicates and random shuffling
-method <arg> The method to generate a random peptide: 0:shuffle (default), 1:swap
-ms <arg> Multiple species entrapment: Fasta files of foreign species
-ns no shared peptides between entrapment and target protein
-swap Reverse the order of generated random peptide sequences
-i <arg> PSM/peptide/precursor/protein file
-score <arg> The score name for ranking precursor/peptide/protein for FDP calculation.
The format could be "score", "score:0" or "score:1". The second part is 0
or 1, 0: lower is better, 1: higher is better
-level <arg> PSM, peptide, precursor or protein
-pep <arg> peptide/protein pair file
-decoy_label <arg> Label for decoy: rev_ in default
-decoy_pos <arg> Position of decoy label: 0 (start, in default); 1 (end)
-entrapment_label <arg> Label for entrapment: _p_target in default
-entrapment_pos <arg> Position of entrapment label: 0 (start); 1 (end, in default)
-debug Print detailed information for debugging
-h Print this usage information
Generate a peptide level entrapment database using the target database UP000005640_9606.fasta:
java -jar fdrbench-0.0.1.jar -I2L -level peptide -db UP000005640_9606.fasta -o UP000005640_9606_entrapment_pep.txt -uniprot -diann -fix_nc cUsing the above command line, a peptide level entrapment database in FASTA format "UP000005640_9606_entrapment_pep.fasta" and a peptide tsv format file "UP000005640_9606_entrapment_pep.txt" will be generated:
The format of the Fasta file "UP000005640_9606_entrapment_pep.fasta" looks like below:
>sp|PSLDQLAAHPWMLGADGGVPESCDLR_target|PSLDQLAAHPWMLGADGGVPESCDLR_target
PSLDQLAAHPWMLGADGGVPESCDLR
>sp|PALLAVGGADSLLEDGHQPCSWDMPR_p_target|PALLAVGGADSLLEDGHQPCSWDMPR_p_target
PALLAVGGADSLLEDGHQPCSWDMPR
>sp|QLQGASWELQSLR_target|QLQGASWELQSLR_target
QLQGASWELQSLR
>sp|QQSWLSLQGLEAR_p_target|QQSWLSLQGLEAR_p_target
QQSWLSLQGLEAR
>sp|YPERDNR_target|YPERDNR_target
YPERDNR
The format of the tsv file "UP000005640_9606_entrapment_pep.txt" looks like below. This file is used in FDP calculation.
sequence decoy proteins peptide_type peptide_pair_index
PSLDQLAAHPWMLGADGGVPESCDLR No sp|Q86V86|PIM3_HUMAN target 0
PALLAVGGADSLLEDGHQPCSWDMPR No sp|Q86V86_p_target|PIM3_HUMAN_p_target p_target 0
QLQGASWELQSLR No sp|Q96N95|ZN396_HUMAN target 1
QQSWLSLQGLEAR No sp|Q96N95_p_target|ZN396_HUMAN_p_target p_target 1
YPERDNR No sp|Q5T200|ZC3HD_HUMAN target 2
YDRPENR No sp|Q5T200_p_target|ZC3HD_HUMAN_p_target p_target 2
SYKALADQMNLLLSK No sp|Q9UBJ2|ABCD2_HUMAN target 3
YSALSNMDLQKLALK No sp|Q9UBJ2_p_target|ABCD2_HUMAN_p_target p_target 3
Generate a peptide level entrapment database using the target database UP000005640_9606.fasta:
java -jar fdrbench-0.0.1.jar -level protein -db UP000005640_9606.fasta -o UP000005640_9606_I2L_entrapment.fasta -I2L -diann -uniprot -fix_nc c -checkUsing the above command line, a protein level entrapment database in FASTA format "UP000005640_9606_I2L_entrapment.fasta" will be generated:
The format of the Fasta file "UP000005640_9606_I2L_entrapment.fasta" looks like below:
>sp|A0A0G2JMI3|HV692_HUMAN Immunoglobulin heavy variable 1-69-2 OS=Homo sapiens OX=9606 GN=IGHV1-69-2 PE=3 SV=2
MDCTWRLLLLVAAATGTHAEVQLVQSGAEVKKPGATVKLSCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETLYAEKFQGRVTLTADTSTDTAYMELSSLRSEDTAVYYCAT
>sp|A0A0G2JMI3_p_target|HV692_HUMAN_p_target Immunoglobulin heavy variable 1-69-2 OS=Homo sapiens OX=9606 GN=IGHV1-69-2_p_target PE=3 SV=2
CWTMDRTGAVEAVQVQAESAGLALTVLHLLKKTAGPVKCSLKYYMATPVDWTQQFSYVGHGKDAPGEEELDVMEGLGYLWTKQFGREAMLTLADYVLDSTTSTSTRDVTACESAYYT
For protein level FDP calculation, an example input is shown below. Only the three columns protein, q_value and score are required.
Protein.Group PG.Q.Value q_value protein score
P62857 1.64447e-4 1.64447e-4 P62857 1
P29590 1.64447e-4 1.64447e-4 P29590 2
O95989 1.64447e-4 1.64447e-4 O95989 3
P61006 1.64447e-4 1.64447e-4 P61006 4
Q70J99 1.64447e-4 1.64447e-4 Q70J99 5
P36404 1.64447e-4 1.64447e-4 P36404 6
Q71SY5 1.64447e-4 1.64447e-4 Q71SY5 7
Q9BPW8 1.64447e-4 1.64447e-4 Q9BPW8 8
Q8WUP2 1.64447e-4 1.64447e-4 Q8WUP2 9
Below is an example command line to run protein FDP calculation. The database used to generate the identification result must be generated using FDRBench. The command line parameter -score is set to use the column "score" as a secondary ranking score in FDP calculation. The number 0 indicates lower score is better (more confident).
java -jar fdrbench-0.0.1.jar -i protein-fdp_protein_input.tsv -level protein -o protein-diann_fdp_protein.csv -score 'score:0' -fold 1 -pick firstThe output file "protein-diann_fdp_protein.csv" contains estimated FDP values using different methods.
Wen, Bo, et al. "Assessment of false discovery rate control in tandem mass spectrometry analysis using entrapment." bioRxiv (2024): 2024-06.