Seq2Logo: A method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion

Abstract

Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in
a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino
acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often
compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation
focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to
amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts
to correct for low number of observations and different logotype representations each capturing different aspects related
to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally
conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM.
Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed).

Full text

Seq2Logo: a method for construction and
visualization of amino acid binding motifs and
sequence profiles including sequence weighting,
pseudo counts and two-sided representation of
amino acid enrichment and depletion
Martin Christen Frølund Thomsen and Morten Nielsen*
Center for Biological Sequence Analysis, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
Received January 6, 2012; Revised April 30, 2012; Accepted May 2, 2012
ABSTRACT
Seq2Logo is a web-based sequence logo generator.
Sequence logos are a graphical representation of
the information content stored in a multiple
sequence alignment (MSA) and provide a compact
and highly intuitive representation of the position-
specific amino acid composition of binding motifs,
active sites, etc. in biological sequences. Accurate
generation of sequence logos is often compromised
by sequence redundancy and low number of obser-
vations. Moreover, most methods available for
sequence logo generation focus on displaying the
position-specific enrichment of amino acids,
discarding the equally valuable information related
to amino acid depletion. Seq2logo aims at resolving
these issues allowing the user to include sequence
weighting to correct for data redundancy,
pseudo counts to correct for low number of obser-
vations and different logotype representations
each capturing different aspects related to amino
acid enrichment and depletion. Besides
allowing input in the format of peptides and
MSA, Seq2Logo accepts input as Blast sequence
profiles, providing easy access for non-expert
end-users to characterize and identify functionally
conserved/variable amino acids in any given
protein of interest. The output from the server is a
sequence logo and a PSSM. Seq2Logo is available at
http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May
2012, date last accessed).
INTRODUCTION
The idea of generating a logo from aligned sets of sequences
was introduced in 1990 by Schneider and Stephens (1). The
intention of a sequence logo is to concentrate into a single
plot the general consensus, the order of predominance
of residues at every position, the relative frequencies of
every residue at every position, the amount of information
present at every position and significant locations. This
logo is then able to present all of the relevant information
to the viewer in a fast and concise manner.
Several webservers exist to generate sequence logos from
MSA’s (2–5). All these servers suffer from different limita-
tions in the handling sequence redundancy and low number
of observations. Moreover, to the best of our knowledge, all
public sequence logo servers, with the exception of the
Icelogo (4) and two-sample logo (5) methods, focus on dis-
playing the position-specific enrichment of amino acids, dis-
carding the equally valuable information related to amino
acid depletion. Seq2logo aims at resolving these issues
allowing the user to include sequence weighting to correct
for data redundancy, pseudo counts to correct for low
number of observations (6–8) and five different logotype
representations each capturing different aspects related to
amino acid enrichment and depletion. In addition to the
usual Shannon logo (9), Seq2Logo includes the option to
create Kullback–Leibler (KL) (10) logos where the
depleted (under-represented) amino acids are represented
on the negative y-axis. Besides the conventional KL logo,
Seq2Logo can also display a weighted KL logo, where the
relative height of each amino acid is proportional to the
log-odds ratio and a probability weighted KL logo, where
the relative height of each amino acid is proportional to the
product of the probability and log-odds ratio. Finally,
*To whom correspondence should be addressed. Tel: +45 4525 2425; Fax: +45 4593 1585; Email: mniel@cbs.dtu.dk
Published online 25 May 2012 Nucleic Acids Research, 2012, Vol. 40, Web Server issue W281–W287
doi:10.1093/nar/gks469
ßThe Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

inspired by the work of Fujii et al. (11), Seq2Logo also
includes an option to visualize PSSM (position-specific
scoring matrix) logos, where the height of a bar is given by
the sum of the absolute value of the PSSM weight matrix
values and the height of a given amino acids is proportional
to the absolute value of the weight matrix score. In particu-
lar, the weighted KL logo provides a visual and highly in-
tuitive representation of both amino acid enrichment and
depletion in for instance receptor binding motifs. Besides
allowing input in the format of peptides and MSAs, the
Seq2Logo server accepts inputs such as Blast sequence
profiles, providing easy access for non-expert end-users to
characterize and identify functionally conserved/variable
amino acids in any given protein of interest.
MATERIALS AND METHODS
Seq2Logo implements two strategies to improve the
accuracy of the estimated sequence logo. The first
strategy is sequence weighting which corrects for data re-
dundancy. The second strategy is pseudo counts which
correct for a low number of observations. Sequence
weighting is implemented as described in (6,8) and
pseudo counts as described in (7). For details, see
Supplementary Data.
In a sequence logo, the height of the bar is equal to
the information content at each amino acid position. The
information content is calculated using the relation
I¼P
a
palog2pa=qa, where p
a
and q
a
are the observed prob-
ability (calculated from the data) and background probabil-
ity, respectively, of the amino acid a. If an equiprobable
background amino acid distribution is applied, a conven-
tional Shannon sequence logo is displayed. If a background
amino acid distribution reflecting the prevalence of the dif-
ferent amino acids is applied, a Kullback–Leibler sequence
logo is displayed. The choice of the Kullback–Leibler
logotype in Seq2Logo not only provides correction for the
uneven distribution of amino acids, but also expresses the
depleted amino acids (where p
a
<q
a
) on the negative side of
the y-axis. This enables the user to quickly identify enriched
and depleted (under-represented) amino acids. To enhance
the identification and information of the depleted
amino acids, Seq2Logo includes another logotype called
weighted Kullback–Leibler. This logo type presents each
individual amino acid proportional to its relative log-odds
score [log
2
(p
a
/q
a
)]. Another logotype is included called
probability weighted Kullback–Leibler, where the relative
height of each individual amino acid is proportional to p
a
·
log
2
(p
a
/q
a
). Finally, Seq2Logo includes an option to display
PSSM-logos (11), where the height of a bar is equal to the
sum of the absolute value of the PSSM weight matrix values
and the height of each amino acid is proportional to the
absolute value of the weight matrix score (with negative
values displayed on the negative y-axis).
THE WEB SERVER
The Seq2Logo server has a simple interface that allows
non-expert users to generate and customize accurate
logos from any amino acid sequence data of interest.
Input
The interface is split in two parts for easy overview. The
first and the most important part is submission (Figure 1,
left panel). Here, the user can upload or paste in the input
data in addition to specifying the logotype (Shannon,
Kullback–Leibler, Weighted Kullback–Leibler, probabil-
ity weighted Kullback–Leibler or PSSM-logo) and condi-
tions for handling the input data (sequence weighting and
pseudo counts). Seq2Logo can read sequence data in the
following formats: Fasta, ClustalW, Raw peptide
sequences and Weight/Blast matrix (for details on each
format refer to Supplementary Data). The detection of
the format happens automatically through the identifica-
tion of key elements from each format. In the submission
part, the user further specifies which output files should be
created. In the graphical layout (Figure 1, right panel), the
user can customize the graphical layout of the logo plot.
Page size sets the resolution of the image and stacks per
line and lines per page determine how the logo should
look. Assigning each amino acid symbol to a color
defines the amino acid colors. There are six colors to
choose from: Red, green, blue, yellow, purple or orange.
All amino acids left out will be black. Several predefined
color-schemes are available. The user can also rotate the
position numbers on the x-axis and hide various features
of the graph.
Output
An example of the output from Seq2Logo generated using
the input specifications from Figure 1 is shown in
Figure 2. The figure shows on the positive y-axis, the
amino acids enriched at each peptide position and on
the negative y-axis the corresponding depleted amino
acids. In this case, the logo is calculated from a set of 13
artificial peptide sequences proposed to bind the
HLA-A*02:01 class I major histocompatibility complex
(MHC) molecule. This molecule has a binding motif
with strong interactions at P2 and P9 both positions
with prevalence for hydrophobic amino acids (12).
One of the distinct powers of Seq2Logo is its ability to
deal with data redundancy and low number of observa-
tions. To the best of our knowledge, no other public
sequence logo servers share this ability. In Figure 3, the
cruciality of these features for the generation of accurate
sequence logos describing a binding motif is illustrated.
The figure displays Shannon sequence logos generated
by Seq2Logo, using different option to improve the
accuracy, as well as sequence logos generated by
Weblogo (2) and EnoLOGOS (3). When comparing the
logos calculated from the small sample data set with the
logo obtained from the larger data set, it is apparent that
the inclusion of sequence weighting and pseudo counts
have a significant positive impact on the overall
accuracy of the binding motif description.
The other distinct feature of Seq2Logo compared
to most other public sequence logo server is the display
of depleted amino acids on the negative y-axis in
Kullback–Leibler logos. Most sequence logo servers
display the relative height of the different amino acids
in a manner proportional to their frequency, thus
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Figure 2. Output from Seq2Logo. The upper panel shows the sequence logo calculated from a set of 13 artificial peptide sequences using the
specification defined in Figure 1 (sequence weighting using clustering, pseudo count with a weight of 200 and logotype as Kullback–Leibler).
Enriched amino acids are shown on the positive y-axis and depleted amino acids on the negative y-axis. The lower panel gives the position-specific
(log-odds) scoring matrix (PSSM) calculated by Seq2Logo. Each line corresponds to a position and gives the consensus amino acid and the log-odds
scores for the 20 amino acids.
Figure 1. The submission (left) and graphical layout (right) part of the web interface. In the submission part the user specifies the input file, the
format of output files, the logotype and the conditions for the handling of the input data. In the Graphical Layout part, the user customizes the
graphical layout of the logo plot; page size, stacks per line, lines per page, colours, bars, rotation of position numbers and title.
Nucleic Acids Research, 2012, Vol. 40, Web Server issue W283

displaying only the position-specific enrichment of amino
acids, discarding the equally valuable information related
to amino acid depletion. To improve on this issue,
Seq2Logo includes a series of distinct logotypes (see
Figure 4). In addition to the usual Shannon logo,
Seq2Logo includes the option to create Kullback–Leibler
(KL) logos where depleted amino acids are represented on
the negative y-axis. Besides the conventional KL logo,
Seq2Logo can also display a weighted KL logo, where
the relative height of each amino acid is proportional to
the log-odds ratio and a probability weighted KL logo,
where the relative height of each amino acid is propor-
tional to the product of the probability and log-odds
ratio. In particular, the weighted KL logo provides a
visual and highly intuitive representation of both amino
acid enrichment and depletion in for instance receptor
binding motifs. Besides these information-based logo-
types, Seq2Logo offers the possibility of displaying
PSSM-logos calculated either from a log-odds weight
matrix derived by Seq2Logo from a multiple sequence
alignment or from a user-defined PSSM. In the
PSSM-logo, the height of the bar and amino acid at
each position is proportional to the absolute value of the
PSSM weight matrix values. This logotype is particularly
powerful when illustrating depletion of a small set of
amino acids form otherwise variable positions in a
sequence motif. One such example is N-linked
glycosylation sites that are known to have the motif
N-X-S/T where X can be any amino acid but
P. Visualizing this motif as an information-based
sequence logo will not capture the depletion of P at the
position between N and S/T as all amino acids except
P are found at this position, hence making the overall
information content very small. On the other hand,
visualizing the motif as a PSSM-logo, the strong depletion
of P at the position between N and S/T becomes apparent
(see Figure 5).
A powerful way to characterize sequence conservation/
variation within a protein family is by use of sequence
profiles. Such sequence profiles can be obtained using
Psi-Blast (7). Seq2Logo accepts input of such sequence
profile in the Blast profile format allowing easy access
for non-expert end-users to characterize and identify func-
tionally conserved/variable amino acids in any given
protein of interest. Blast sequence profile can be generated
either in-house using a command like ‘blastpgp ddbe
0.00001 j4Q blastprofile i fasta o out’, where dbis
the sequence database used to search by Blast, edefines
the e-value cut-off for significant hits, jdefines the
number of Psi-blast iterations, iis the input file in
FASTA format, Qis the output file for the blast
profile (the file to be used by Seq2Logo to visualized the
sequence profile) and o is the file for the blast output.
Alternatively, the Blast2logo webserver (www.cbs.dtu.dk/
biotools/Blast2logo (14 May 2012, date last accessed)) can
be used to obtain the sequence profile. Figure 6 demon-
strates the use of Seq2Logo to display a sequence profile
for Rhamnogalacturonan acetylesterase (PDBid 1K7C,
chain A). The active site of 1K7C.A is defined by the
residues S9, G42, N74, D192 and H195 (13). All these
residues are highly conserved in the sequence logo (in
fact they are among the 10 residues with the highest infor-
mation content, data not shown). Another striking obser-
vation from the logo is the lack of sequence information in
the area between positions 75 and 105, suggesting that this
part of the protein is highly variable (most likely an inser-
tion) within the protein family. Both these observations
illustrate the power of sequence profiles combined with
Seq2Logo as a simple tool to identify functionally import-
ant residues and insertions in protein sequences.
Figure 3. Sequence logos generated from small sequence samples. All logos except the right logo in the lower row were calculated from a set of 13
artificial peptide sequences proposed to bind HLA-A*02:01 (see Figure 1). The upper row shows logos calculated by Seq2Logo using: (i) without
sequence weighting and pseudo count correction, (ii) sequence weighting by clustering and no pseudo count correction and (iii) sequence weighting by
clustering and pseudo count correction with a weight on prior of 200. The lower row shows logos calculated using: (i) Weblogo with ‘small sample
correction’, (ii) EnoLOGOS and (iii) Seq2Logo from a set of 229 HLA-A*02:01 9mer ligands downloaded from the SYFPEITHI database (12) with
sequence weighting by clustering and pseudo count correction with a weight on prior of 200.
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Figure 4. The different logotype representations covered by Seq2Logo. Sequence logos generated from at set of 13 artificial peptide sequences
proposed to bind HLA-A*02:01 (see Figure 1). All logos were calculated using clustering and pseudo counts with a weight on prior at 200.
Upper row, left panel: Shannon, right panel: Kullback–Leibler. Lower row left panel: weighted Kullback–Leibler, right panel: probability
weighted Kullback–Leibler.
Figure 5. PSSM-logo for the N-linked glycosylation motif. The motif was calculated from a set of 2128 unique experimentally verify N-glycosylation
sites downloaded from the UniprotKB protein database. Only peptide fragments of length 11 (5 before and 5 after the N) were included in the
analysis.
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INTEGRATING SEQ2LOGO WITH OTHER
PREDICTION SERVERS
To improve the usability and make Seq2Logo able to co-
operate with other programs and servers, a form-handler
was implemented on the server that makes it possible to
send input data directly to Seq2Logo. This simple
form-handler allows a quick and easy transfer of data to
Seq2Logo and defines a platform for using Seq2Logo as a
visualization tool for other programs. The form data sent
to Seq2Logo is inserted directly into the input field.
An instruction of how to implement this transfer can be
found at: http://www.cbs.dtu.dk/biotools/Seq2Logo-1.0/
bin/easytransferbutton.html (14 May 2012, date last
accessed).
DISCUSSION AND CONCLUSION
Sequence logos provide a powerful way to visualize amino
acid preferences in a receptor binding motif, as well as
sequence conservation/variation and the location of func-
tionally essential residues in multiple sequence alignments.
Accurate estimation of a sequence motif is often
compromised by data redundancy and low number of
observations. Inappropriate handling of these issues can
lead to inaccurate estimation of the sequence motif and
subsequent poor sequence logo representation. Moreover,
the majority of sequence logo webservers have a poor
visualization of the information related to amino acid de-
pletion since they focus on displaying the position-specific
enrichment of amino acids.
Here, we have proposed a novel sequence logo generator,
Seq2Logo that aims at addressing these shortcomings and
allow non-expert end-users, via an easy to use web-interface,
to generate accurate sequence logos from protein sequence
data. We have demonstrated that Seq2Logo can deal with
sequence redundancy and low number of observations in a
manner superior to that of other public available sequence
logo generators like Weblogo and ENOlogos. Besides
the conventional Shannon sequence logo, Seq2Logo also
incorporates distinct logotypes where depleted amino
acids are displayed on the negative y-axis. These logotypes
offer a unique possibility for Seq2Logo to display for
instance receptor-binding motifs in a format that highlights
both favored and disfavored amino acids at the different
positions in the motif.
Figure 6. Seq2Logo visualization of a Blast sequence profile for 1K7C chain A. The Blast profile was obtained using Blast2logo (www.cbs.dtu.dk/
biotools/Blast2logo (14 May 2012, date last accessed)) searching against the nr70 sequence database with default options. The active site of 1K7C:A
is defined by the residues S9, G42, N74, D192 and H195 (13). All these residues show up as highly conserved in the sequence logo.
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A sequence profile is a powerful way to capture pos-
ition-specific information about sequence conservation/
variation within a protein family. Seq2Logo accepts
sequence profiles in the Blast format as input and can in
a very simple and intuitive manner be used in combination
with Blast as a tool to visualize sequence profiles and
identify functionally conserved/variable amino acids in
any given protein of interest.
Finally, to allow other servers dealing with multiple
sequence alignments and binding motifs to directly co-
operate with Seq2Logo and benefit from its improved
features, the server includes a form-handler that enables
communication with Seq2Logo via a simple html form.
This feature has allowed for a simple and effective im-
provement to two of our own webservers NNAlign (14)
and Blast2logo (www.cbs.dtu.dk/biotools/Blast2logo
(14 May 2012, date last accessed)), and we believe this
to be an additional feature that will become very useful
for other webserver developers within the field of for
instance receptor-binding motif characterization.
In its current form, Seq2Logo can only handle amino
acid input data. The reason for this limitation is that most
of its unique features like pseudo count estimates from
Blosum substitution matrices and sequence weighting of
are specific for amino acid data. The ability to also handle
nucleic acids will be a part of a future update for the
method.
In conclusion, we believe Seq2Logo to be an important
and novel tool for non-expert users to construct accurate
sequence logos describing receptor binding motifs and
sequence variations in multiple sequence alignments.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online:
Supplementary Methods and Supplementary References
[6–8,15,16].
FUNDING
Funding for open access charge: National Institutes of
Health (NIH) [contract nos HHSN272200900045C and
HHSNN26600400006C].
Conflict of interest statement. None declared.
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