Nature Methods
SignalP 4.0: discriminating signal peptides from transmembrane regions
Thomas Nordahl Petersen, Søren Brunak, Gunnar von Heijne & Henrik Nielsen

Supplementary Methods
Supplementary Results

Nature Methods: doi:10.1038/nmeth.1701

Supplementary Methods
Sequence data
We extracted datasets for eukaryotes, Gram-positive bacteria and Gram-negative bacteria
from the Uniprot Knowledgebase release 2010_051. Only reviewed entries (i.e. from
UniProtKB/SwissProt) were used, and hypothetical proteins were not included. We
discarded sequences shorter than 30 amino acids. Fragments were allowed as long as
their N-terminal 70 amino acids were complete (i.e. they had no “FT NON_TER” line
pointing to position 1 and no “FT NON_CONS” line affecting the first 70 positions).
Gram-positive bacteria were defined as Firmicutes plus Actinobacteria. We did notinclude Tenericutes (Mycoplasma and related genera), since they do not seem to have atype I signal peptidase at all.2 Gram-negative bacteria were defined as all other bacteria.
We only considered positive data (signal peptide sequences) which had experimental
evidence for the cleavage site. Lipoproteins (cleaved by Lipoprotein signal peptidase)3
and signal peptides annotated as being Tat-dependent4 were excluded from the bacterial
sets. Signal peptides from eukaryote and Gram-negative organisms were restricted tohave a cleavage site between positions 15 and 45. For Gram-positive bacteria, which tend
to have longer signal peptides, the upper limit was extended to allow a cleavage site at
position 50.
We extracted two different negative sets. The first consisted of proteins with a subcellularlocation annotated as cytosolic (cytosolic and/or nuclear in eukaryotes) with experimental
evidence (note that UniProt uses the term “cytoplasm” for cytosol). Proteins with known
multiple locations (except for cytosolic and nuclear) were not included. The second
negative set consisted of transmembrane proteins with an annotated transmembraneregion within the first 70 positions. These had experimental evidence for being single- or
multi-pass membrane proteins (according to the subcellular location comment), but did
not necessarily have experimental evidence for the exact position of the transmembraneregion, as this criterion would have left too few sequences in the set.
All sequences were shortened to the 70 N-terminal amino acids. We then carried out
homology reduction using algorithm 2 of Hobohm et al.5 and cut-off criteria as described
previously6, i.e. a local alignment could include up to 17 identical amino acids foreukaryotic sequences, while for bacteria up to 21 identities were allowed (Table A).
In order to benchmark SignalP 4.0 against the previous version of SignalP, we assigned asubset of each of the positive and negative datasets as “comparison dataset” byperforming a homology reduction to sequences that were used to train SignalP 3.0 (Table
A). This means that no sequence in the comparison sets is homologous (according to theaforementioned cut-off criteria) to any sequence used to train SignalP 3.0. The
comparison datasets are also used for calculating performances of other methods. For
those that are newer than SignalP 3.0 (Philius, SPOCTOPUS, the two MEMSAT
versions, Signal-CF and Signal-3L), this can lead to slight overestimations of
performance, as these predictors to some extent have been trained on sequences identical

Nature Methods: doi:10.1038/nmeth.1701

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Signal peptides (SP)Cytosolic or Nuclear
Transmembrane (TM)
Total
Train
Comp. Total Train Comp. Total Train Comp.
Euk3,595(4) 1,640(2) 606(0) 8,956 5,131 1,000* 2,329
989
939
Gram+
277(1)
208(1) 48(1) 2,914
359
212
186
118
118
Gram–
612(6)
423(4) 104(1) 7,243
908
559 1,054
527
524
Table A. Sequences included in datasets for eukaryotes (Euk), Gram-positive bacteria (Gram+) and Gramnegative bacteria (Gram–). “Total” is the number before homology reduction, “Train” is the homology
reduced dataset used for training, “Comp.” is the dataset homology reduced against the SignalP 3.0 data,
used for comparison to SignalP 3.0 and other methods. The numbers in parentheses are sequences that
include both a signal peptide and a transmembrane region within the first 70 positions. The number marked
with (*) was artificially reduced to make a more balanced comparison set.

or similar to the sequences in our comparison datasets. The data used to train and test
SignalP 4.0 are available at http://www.cbs.dtu.dk/services/SignalP/ under the heading“Data.”
Neural Networks
We trained standard feed forward neural networks by the use of a back-propagation
procedure7 to recognize the signal peptide and the cleavage site in protein sequences. A
sliding window of up to 41 amino acids was fed into the first layer of the neural networkand predictions were made for the central position in the window. Amino acids weresparsely encoded as an orthogonal vector of length 21, where the last entry represented aposition outside the protein sequence. In general we trained neural networks for two
different tasks, one to predict positions within the signal peptide (SP) and another topredict the cleavage site in each sequence (CS). The output representing ‘signal peptide’in the SP networks is called the S-score, while the output representing ‘cleavage site’ in
the CS networks is called C-score (Figure A).
In contrast to the earlier versions of SignalP, where target values of 0 and 1 were used,
SignalP 4.0 uses target values of 0.1 and 0.9 for incorrect and correct outputs,
respectively. This creates a visual difference in the output compared to that of SignalP 3.0(Figure A).
A number of different architectures were tried. For the SP networks, the symmetricalinput window was varied from 29 to 41. For the CS networks, the input window wasasymmetrical with 16 to 21 positions before the putative cleavage site and 1 to 6
positions after. The size of the hidden layer was varied from 2 to 20 units (Table B). Not
all intermediate values have been used. In addition to the sequence window inputs, twoother input types were used: the relative position of the input window in the sequence,
encoded as a number between 0 and 1, and the composition of the entire input sequence(up to 70 residues), expressed as 20 numbers between 0 and 1. For the SP networks,
networks both with and without these additional inputs were tried, while for the CS
networks, they were always included, since preliminary tests showed them to beadvantageous (results not shown).

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Figure A. Output from the SignalP 4.0 web-server for the sequence EGFR_HUMAN. ‘C-score’ is thepredicted cleavage site value, ‘S-score’ is the predicted signal peptide value and ‘Y-score’ is a combinationof C- and S-scores as described in Methods. The predicted cleavage site is between position 24 and 25 withthe local sequence context “SRA-LE.” In a summary line below the plot we show the calculated D-score,
the associated cutoff value and which networks were used to make the prediction, in this case: “D=0.868 Dcutoff=0.450 Networks=SignalP-noTM”

Each network was trained for a maximum of 200 training epochs. Optimal architecture
and training cycle were selected by the optimization set (see next subsection) according
to the MCC (Matthews Correlation Coefficient)8, where the true and false positives andnegatives were counted on the single position level (i.e. each single position was counted
as a true or false positive or negative).
Nested cross-validation
When prediction methods based on machine learning algorithms are trained and tested, it
is customary to use a cross-validation procedure, where the data set is divided into kpartitions and for each partition a version of the method is trained on k-1 partitions, while
one partition is left out as a test set for measuring the performance. This is adequate aslong as the test set data are not used in any way to optimize the parameters of the method,
but if the test set performance is used for “stopping” the network training (i.e. selecting
the best training cycle) or for selecting the optimal network architecture, the test set
performance is no longer an unbiased estimate of how the method will perform on newdata, i.e. data that have not been used in its development. This may lead to an

Nature Methods: doi:10.1038/nmeth.1701

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EukWindow size
Hidden units
Relative position
Composition
Gram+
Window size
Hidden units
Relative position
Composition
Gram–
Window size
Hidden units
Relative position
Composition

SignalP-TM
SP
41
16 - 2095%
100%
SignalP-TM
SP
29 - 37
8 - 2080%
90%
SignalP-TM
SP
33 - 41
8 - 2095%
100%

CS
17+1 - 20+5
2-8
Always on
Always onCS
17+2 - 21+6
2-8
Always on
Always onCS
16+1 - 21+6
2-8
Always on
Always on

SignalP-noTM
SPCS
41
20+3 - 20+5
8 - 202-855%
Always on
95%
Always on
SignalP-noTM
SPCS
29 - 33
18+1 - 20+5
18 - 202-860%
Always on
90%
Always on
SignalP-noTM
SPCS
31 - 41
17+1 - 21+6
8 - 202-860%
Always on
90%
Always on

Table B. Parameters selected for training of the neural networks for the eukaryote (Euk), Gram-positive(Gram+) and Gram-negative (Gram–) datasets. The columns SP and CS show the optimal parameters forneural network training of signal peptide and cleavage site prediction, respectively. ‘Window size’ is thenumber of input positions used in the first layer of the neural networks for sparse encoding of the aminoacids. For the CS networks, the input window is given as number of positions before the cleavage site plus
number of positions after the cleavage site. ‘Hidden units’ is the number of neurons used in the second
layer. For ‘Window size’ and ‘Hidden units’, the numbers show the range in the 20 networks trained during
nested 5-fold cross-validation. ‘Relative position’ is central position in the sliding window, relative to thelength of the protein sequence. ‘Composition’ is the amino acid composition of the entire sequence. For‘Relative position’ and ‘Composition’, the numbers show the percentage of the 20 networks that wereselected to have the corresponding input option on.

overestimation of performance and is a problem with many published methods, including
our own SignalP version 3.0.9
To overcome this problem, we used a nested cross-validation procedure, where the data
were divided into homology-screened training, optimization and evaluation sets. Data
were divided into five partitions that are all used in each of the three roles. Whenpartition 1 was used as evaluation set, it was kept out of the process while a full four-foldcross-validation was performed on partitions 2 through 5. In each of these runs, threepartitions were used for training the networks, while the optimization set was used forselecting the optimal training cycle and network architecture, and the evaluation set could
then be used to obtain an unbiased estimate of the performance. The whole process wasthen repeated with partition 2 as evaluation set, and so on until all partitions had beenused. This gave in total 20 versions of each network.

Nature Methods: doi:10.1038/nmeth.1701

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Range (d)Weight (w)Dcut

SignalP-TM
Euk
Gram+
Gram–
13-20
13
12
0.58-0.60 0.61
0.51-0.63
0.50-0.51 0.45
0.51-0.53

SignalP-noTM
Euk
Gram+
Gram–
20-24
12-15
12
0.41-0.54 0.45
0.45-0.53
0.36-0.51 0.44-0.57 0.56-0.59

Table C. Parameters optimized on test datasets for the integration of predictions from signal peptide andcleavage site neural networks. ‘Range’ is the number of amino acid positions before and after a potential
cleavage site from which the mean signal peptide likeliness is calculated. ‘Weight’ is the term used in thelinear combination of Smean and Ymax. ‘Dcut’ is the optimized threshold for the prediction of a potential
signal peptide.

When measuring the performance on the evaluation set, the output values of the four
networks in the inner loop of the nested cross-validation were arithmetically averaged togive a final score. This tended to give a better result than each of the networks alone(Table D in Supplementary Results). In the final version of SignalP 4.0 made available
on the website, the score is an average of the output values of all twenty versions in thenested cross-validation.
The division into training, optimization and evaluation sets is independent of theassignment of part of the data as comparison set. When calculating the comparison
performances for SignalP 4.0 (Fig. 1 in the main text and Table E in Supplementary
Results), we basically used the evaluation performances, but reduced each evaluation set
to those sequences belonging to the comparison sets.
Calculating results at sequence level
After training, the C-score will typically be high at the cleavage site, while the S-score
drops from a high value before the cleavage site to a low value after the cleavage site. If
the C-score shows several peaks, the correct cleavage site can often be inferred bychoosing the peak that occurs where the slope of the S-score is highest. This is formalized
by the Y-score introduced in the first SignalP version, which is defined as the geometricaverage between the C-score and a smoothed derivative of the S-score:i + d −1⎞
1 ⎛ i −1
Yi = Ci Δ d S i where Δ d S i = ⎜⎜ ∑ S j − ∑ S j ⎟⎟d ⎝ j =i −dj =i⎠The cleavage site is then predicted to be at the position where the Y-score is maximal(see also Fig. A).
The mean S-score is calculated as the arithmetic average of the S-score from position 1 tothe position of the maximal Y-score. The prediction of whether the entire sequence
contains a signal peptide or not is then done by the D-score introduced in the third
SignalP version,9 which is a weighted average of the mean S-score and the maximal Yscore:D = wYmax + (1 − w)S mean
A signal peptide is predicted if the D-score is larger than a cut-off value, Dcut.

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The values of the parameters d, w and Dcut were optimized to yield the highest MCC,
where true and false positives and negatives are calculated per sequence instead of per
position. This optimization was done in a standard five-fold cross-validation (Table C).
Methods for comparison
In addition to the previous version of SignalP (3.0), ten methods were selected forcomparison of predictive performances on novel data. The methods Philius,10
MEMSAT311 and MEMSAT-SVM12 were downloaded and run locally on our computers.
Phobius,13 Signal-BLAST,14 SPOCTOPUS15 and PrediSi16 were used directly on theirrespective websites. For the methods SPEPlip,17 Signal-CF18 and Signal-3L19 thesituation was more complicated, since their websites only allow one sequence to besubmitted; in these three cases, we wrote Perl scripts to automate the process ofsubmitting a sequence and collecting the results.

Supplementary Results
As mentioned in the main text, SignalP 4.0 uses two different kinds of negative data: thefirst, corresponding to the negative data in earlier versions of SignalP, consists ofcytosolic and, for the eukaryotes, nuclear proteins; the second consists of sequences
without signal peptides but with one or more transmembrane helices within the first 70
positions of the sequence.
The Neural Networks have been trained on two types of input data; one method, SignalPnoTM, has been trained with only the first negative set (i.e. cytosolic and nuclear
sequences) while the other method, SignalP-TM, is trained with both negative sets. The
SignalP-noTM method is similar to the previous versions of SignalP where two output
neurons are used both for the SP (signal peptide or not) and the CS networks (cleavage
site or not). The SignalP-TM method is different as the SP networks have three output
states, representing signal peptide, transmembrane region, or anything else.
We found that the two methods SignalP-TM and SignalP-noTM were to some extent
complementary, i.e. SignalP-TM did not yield as good results as SignalP-noTM when
there were no transmembrane sequences involved (Table E). Since the overall goal is tomake one combined predictor that can be used to discriminate between signal peptide andtransmembrane regions while still having a high performance on any kind of input
sequence, the best generic predictor is the one that can decide which of the two methods
should be used for any possible sequence.
The solution was to use the ability of SignalP-TM to predict TM-regions, such that if acertain number of positions or more were predicted as part of a TM-region, then the
SignalP-TM predictor should be used, with the SignalP-noTM predictor as the default.
The number 4 was found to give the best performance for the combined predictor for the
eukaryotic and Gram-negative bacterial data (this means that if SignalP-TM predicts a

Nature Methods: doi:10.1038/nmeth.1701

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Euk.
S-corr.
Training
0.908
Optimization 0.872
Evaluation
0.870(individual)
Evaluation
0.881(averaged)

C-corr.
0.736
0.654
0.628

Gram+
S-corr.
0.935
0.825
0.806

0.648

0.833

C-corr.
0.903
0.631
0.543

Gram–
S-corr.
0.909
0.857
0.850

C-corr.
0.908
0.793
0.747

0.579

0.866

0.766

Table D. The effects of the nested cross-validation and of averaging on the SignalP-TM networks. ‘S-corr.’denotes Matthews Correlation Coefficient (MCC) for the S-score (signal peptide score in the SP networks),
while ‘C-corr.’ denotes MCC for the C-score (cleavage site score in the CS networks). Evaluation
performance is calculated in two different ways: for each optimization network individually or after
averaging the scores of the four optimization networks.

transmembrane helix that is shorter than 4 residues, SignalP-noTM is used). The final
predictor SignalP 4.0 uses this selection scheme for these two organism groups, while forGram-positive bacterial data no improvement was found after applying the selection
scheme, so in this case the output is always that of SignalP-TM.
During training, the data were divided into three parts (see Supplementary Methods fordetails regarding nested cross-validation): training (for adjusting the weights of the neuralnetworks), optimization (for selecting the optimal training cycle and neural networkarchitecture), and independent evaluation (for measuring the final performance). The
performances of the SignalP-TM networks on the three parts of the data sets are shown asMatthews Correlation Coefficients (MCC)8 with true and false positives and negatives
counted at single position level; the same values that were used for optimization (Table
D). The S-correlation is a measure of how good the SP networks are at distinguishingpositions within signal peptides from positions after the cleavage site and positions in
negative sequences. The C-correlation is a measure of how good the CS networks are atdistinguishing cleavage site positions from all other positions.
It is evident that the performance measured on the evaluation data were not much lower
than that measured on the optimization data (except for C-correlation for the Grampositive data). The difference would probably be larger if the data had not been subjectedto the strict homology reduction as described in Supplementary Methods.
The effect of averaging the output scores of the four different optimization networks
before calculating the performance on each evaluation set is also shown (Table D). This
gives a quite remarkable rise in performance – measured by S-correlation, the evaluation
performance using averaging even exceeds the optimization performance. This averaging
assumes that each network has the optimal architecture parameters for its corresponding
optimization set.
The optimal parameter settings for the neural networks are shown in Supplementary
Methods (Table B). For most of the data sets, there was quite a large variation in the

Nature Methods: doi:10.1038/nmeth.1701

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optimal parameters over the twenty versions of the nested cross-validation, both in input
window size and hidden layer size. The extra inputs (relative position in the sequence andamino acid composition) were selected to be included in most of the SP networks. Note,however, that relative position was more important for SignalP-TM than for SignalPnoTM, indicating that this information helps in discriminating between signal peptides
and transmembrane helices.
We benchmarked SignalP 4.0 against the previous version and ten other signal peptide
prediction methods (Fig. 1 in the main text and Table E i-iii). Performance values for thebest prediction methods have been highlighted in bold in the table. The first column(signal peptide correlation for the entire data set) corresponds to the values shown in the
figure. Benchmarking is done using the comparison datasets (Table A). For the methods
developed in this project (SignalP-noTM, SignalP-TM and SignalP 4.0), the results areevaluation performances, i.e. for each version of the network they are calculated on thecorresponding evaluation part of the comparison dataset. Signal peptide performance isgiven as the MCC,8 where true and false positives and negatives are counted at thesequence level (i.e. each sequence was counted as a true or false positive or negative).
Cleavage site performance is given as two measures: sensitivity, i.e. the percentage ofsignal peptides that are predicted as a signal peptide and have the correct cleavage site
assigned, and precision, i.e. the percentage of cleavage site predictions that are correct. In
addition, we show the false positive rate specifically for the negative set withtransmembrane proteins, and the signal peptide correlation for the case where notransmembrane proteins were included in the negative set (the situation for which
SignalP-noTM is optimized).
SignalP 4.0 is best at the prediction of signal peptides, measured by correlation
coefficient on the entire data set, for all three organism types. However, the cleavage site
sensitivity is not as high as that of SignalP 3.0. In general, we believe that the much lower
level of false positives from transmembrane regions is more important than the few
percent worse cleavage site sensitivity. However, we have chosen to give the user an
opportunity of running SignalP-noTM instead of SignalP 4.0 by telling the server that thesubmitted sequences do not contain transmembrane regions.
SignalP-noTM should be directly comparable to SignalP 3.0 NN (the neural network partof SignalP 3.0). For the bacterial datasets, SignalP-noTM is the best method according tocorrelation coefficient when there are no transmembrane sequences in the data, while theeukaryotic SignalP-noTM is slightly worse than SignalP 3.0 NN. When measured bycleavage site sensitivity, SignalP-noTM is better than SignalP 3.0 NN only for Grampositive bacteria.
If the values for SignalP 3.0 are compared to those given in the SignalP 3.0 article,9 itmust be taken into account that we have changed the way cleavage site performance iscalculated in order to be able to benchmark SignalP against other methods. In the SignalP
3.0 article, it was the percentage of cleavage sites predicted by the position of the
maximal Y-score, regardless of whether the D-score was above threshold or not (seeNature Methods: doi:10.1038/nmeth.1701

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i: Eukaryotic sequences
All Sequences
Only TM
No TM
MethodSP corr. CS sens. (%) CS prec. (%) FP-rate (%) SP corr.
SignalP 4.0
68.3
65.9
6.1
0.919
0.874
SignalP-TM
0.871
66.2
0.909
68.2
3.3
SignalP-noTM
0.674
71.3
44.2
38.1
0.960
SignalP 3.0 NN
0.686
45.1
39.5
72.9
0.968
SignalP 3.0 HMM
0.762
72.1
51.8
23.2
0.932
PrediSi
0.561
66.0
37.6
52.6
0.909
SPEPlip
0.717
66.5
46.4
30.7
0.953
Signal-CF*
0.475
62.3
28.0
74.8
0.878
Signal-3L*
0.432
53.3
24.5
73.7
0.821
SignalBlast SP1
0.690
29.4
19.1
27.3
0.851
SignalBlast SP2
0.739
30.0
24.3
16.0
0.843
SignalBlast SP3
0.736
24.6
26.5
7.2
0.774
SignalBlast SP4
0.546
29.5
14.7
51.3
0.794
Phobius
0.811
62.4
50.8
15.3
0.931
Philius
0.800
65.8
54.3
14.0
0.902
MEMSAT3
0.252
0.2
0.2
26.7
0.468
MEMSAT-SVM
0.381
0.8
1.0
16.6
0.488
SPOCTOPUS
0.776
34.7
30.2
13.8
0.880
ii: Gram-positive bacterial sequences
All Sequences
Only TM
No TM
MethodSP corr. CS sens. (%) CS prec. (%) FP-rate (%) SP corr.
SignalP 4.0
72.9
0.894
0.851
83.3
2.6
SignalP-TM
72.9
0.894
0.851
83.3
2.6
SignalP-noTM
0.556
77.1
36.3
47.9
0.948
SignalP 3.0 NN
0.618
72.9
41.7
35.0
0.934
SignalP 3.0 HMM
0.558
77.1
38.1
43.5
0.895
PrediSi
0.607
60.4
38.7
28.2
0.880
SPEPlip
0.488
60.4
28.7
50.4
0.866
Signal-CF*
0.415
23.4
29.8
0.814
78.3
Signal-3L*
0.420
34.8
10.5
84.6
0.799
SignalBlast SP1
0.575
22.9
11.8
33.3
0.804
SignalBlast SP2
0.684
22.9
15.9
19.7
0.868
SignalBlast SP3
0.711
22.9
16.9
17.1
0.881
SignalBlast SP4
0.419
22.9
7.3
62.4
0.659
Phobius
0.704
60.4
43.9
18.8
0.894
Philius
0.726
52.1
41.7
12.8
0.854
MEMSAT3
0.101
0.0
0.0
10.3
0.261
MEMSAT-SVM
0.675
0.0
0.0
11.1
0.812
SPOCTOPUS
0.625
35.4
34.0
14.5
0.807

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iii: Gram-negative bacterial sequences
All Sequences
Only TM
No TM
MethodSP corr. CS sens. (%) CS prec. (%) FP-rate (%) SP corr.
SignalP 4.0
65.4
70.8
1.5
0.882
0.848
SignalP-TM
0.815
61.5
0.839
75.3
1.1
SignalP-noTM
0.497
71.2
26.1
35.8
0.948
SignalP 3.0 NN
0.542
74.0
30.8
28.5
0.925
SignalP 3.0 HMM
0.477
26.1
39.2
0.931
76.9
PrediSi
0.479
75.0
27.2
35.6
0.901
SPEPlip
0.429
70.2
21.4
45.1
0.891
Signal-CF*
0.288
73.1
13.8
78.1
0.698
Signal-3L*
0.287
73.1
13.5
81.1
0.714
SignalBlast SP1
0.530
39.4
14.6
25.4
0.767
SignalBlast SP2
0.252
18.3
3.2
72.8
0.543
SignalBlast SP3
0.642
34.6
22.8
11.5
0.836
SignalBlast SP4
0.387
39.4
9.4
46.1
0.635
Phobius
0.586
73.1
33.6
23.3
0.920
Philius
0.639
26.1
15.7
0.872
76.9
MEMSAT3
0.084
0.0
0.0
17.8
0.312
MEMSAT-SVM
0.497
1.0
0.6
16.4
0.780
SPOCTOPUS
0.510
33.7
18.6
20.5
0.848
Table E. Benchmarking of signal peptide and cleavage site predictions on the comparison dataset for all
three organism groups. ‘SP corr.’ denotes signal peptide correlation, while ‘CS sens.’ denotes cleavage site
sensitivity (the percentage of actual cleavage sites that are predicted correctly) and ‘CS prec.’ denotes
cleavage site precision (the percentage of predicted cleavage sites that are correct). ‘FP-rate’ (false positiverate) is the percentage of transmembrane sequences that are incorrectly predicted as a signal peptide. ‘No
TM’ denotes the test where there were no transmembrane sequences in the data, i.e. only the first negative
set was used. Note that performance for SignalP 4.0 and SignalP-TM are identical for Gram-positivebacterial sequences, since SignalP 4.0 does not use the combination scheme for this organism group. The
methods indicated with a star (*) can only make predictions for sequences longer than 50 aa. For thosemethods the evaluation sets were reduced by 4, 2, and 22 sequences for the Eukaryote, Gram-positive andGram-negative sets, respectively.

Supplementary Methods for definitions). In other words, a cleavage site could be scoredas correct even though the sequence was not predicted to be a signal peptide. This
necessarily gives a higher number than the cleavage site sensitivity (Table E). In
addition, our use of the nested cross-validation (Supplementary Methods) results in aslightly more conservative performance estimation than the plain cross-validation used in
the SignalP 3.0 article.
The other methods that were designed to discriminate between signal peptides andtransmembrane sequences (Philius, Phobius and SPOCTOPUS) have also highcorrelation coefficients for eukaryotic data, while they are somewhat worse for thebacterial sequences. This may be due to the fact that these three methods did not divide

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their training data into different organism groups but pooled them all together, resulting
in methods that are optimized for the most abundant organism group in the data, theeukaryotes. The cleavage site performance is rather high for Phobius and Philius,especially regarding cleavage site sensitivity on the Gram-negative bacterial sequences,where Philius shares the winning position with SignalP 3.0 HMM (the Hidden Markov
Model part of SignalP 3.0), while SPOCTOPUS is not as good at locating cleavage sites
correctly. The number of false-positive signal peptide predictions from transmembranesequences is lower for these methods than for most of the dedicated signal peptide
predictors, but still much higher than for SignalP 4.0.
MEMSAT3 and MEMSAT-SVM do surprisingly bad, especially regarding cleavage sites
where they are almost always wrong. In all fairness, it should be said that the authors ofthese two methods never promoted them as signal peptide prediction tools. However,since they do provide predictions of signal peptides in their output, users may be led into
believing that these predictions are useful, which our analysis indicates that they are not.
Signal-CF and Signal-3L have fairly high cleavage site sensitivities (except for Signal-3L
for Gram-positive bacteria, where Signal-CF is the winner), but this should be seen incontext of their low cleavage site precisions and very high false positive rates ontransmembrane regions. On eukaryotic data, these two methods predict a signal peptide
for almost 75% of the transmembrane helices. Signal-3L had a peculiar problem with its
cleavage site assignment, since it in some cases produced an output stating that there wasa signal peptide from position 1 to –1. In these cases, we scored it as a positive prediction
with wrong cleavage site. This happened 18 times in the eukaryotic set and 22 times in
the Gram-negative set.
Signal-BLAST differs from all the other methods, since it uses alignments to known
signal peptides for prediction instead of machine learning or statistical methods. SignalBLAST can be run in four modes optimized for different situations, and we have tested
all four. The best mode according to signal peptide correlation is SP3 (optimized for
accuracy and specificity) which almost matches Phobius and Philius in correlation
coefficient and has very few false positives. However, the cleavage site performance ofall Signal-BLAST versions is rather low. This is an interesting observation, since itsuggests that cleavage sites are much less conserved in evolution than signal peptides are.
In contrast to earlier versions of SignalP, we have measured the performance of SignalP
4.0 using nested cross-validation (see Supplementary Methods for details), which
ensures total separation between the data used for optimizing the neural networks anddata used for testing the performance. However, we have shown that the overestimationof performance due to early stopping and architecture selection is not large. On the other
hand, our use of the nested cross-validation procedure has shown that averaging over
several networks is very efficient for improving performance.
As described in the main text, the data were divided into eukaryotic, Gram-positivebacterial, and Gram-negative bacterial sets. Since some differences are also known tooccur between mammalian and yeast signal peptides,20 we tested a further subdivision of

Nature Methods: doi:10.1038/nmeth.170111the eukaryotic data into animals, fungi, and plants, and trained separate methods for thesethree groups. However, this did not give any improvement, and performance for all threegroups was better when using the method trained on all eukaryotic sequences (results notshown).
When comparing the SignalP-noTM part of SignalP 4.0 with SignalP 3.0 NN, one would
expect to see an improvement in performance, since the data set has grown. This is not
the case for the eukaryotic data, where both the cleavage site performances and thecorrelation coefficients are slightly worse. One reason for this might be that we did notdiscard sequences with rare amino acids at the –1 position relative to the cleavage site, as
was done in the data set of SignalP 3.0. Our reasoning was that since all signal peptides
are annotated as experimentally verified, the method should be able to recognize eventhose with atypical cleavage sites; but maybe these outliers actually disturb the training
and make the method perform slightly worse. This should be thoroughly tested in thenext SignalP version.

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