The XXmotif web server for eXhaustive, weight matriX-based motif discovery in nucleotide sequences.

Abstract

The discovery of regulatory motifs enriched in sets of DNA or RNA sequences is fundamental to the analysis of a great variety of functional genomics experiments. These motifs usually represent binding sites of proteins or non-coding RNAs, which are best described by position weight matrices (PWMs). We have recently developed XXmotif, a de novo motif discovery method that is able to directly optimize the statistical significance of PWMs. XXmotif can also score conservation and positional clustering of motifs. The XXmotif server provides (i) a list of significantly overrepresented motif PWMs with web logos and E-values; (ii) a graph with color-coded boxes indicating the positions of selected motifs in the input sequences; (iii) a histogram of the overall positional distribution for selected motifs and (iv) a page for each motif with all significant motif occurrences, their P-values for enrichment, conservation and localization, their sequence contexts and coordinates. Free access: http://xxmotif.genzentrum.lmu.de.

Full text

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The XXmotif web server for eXhaustive,
weight matriX-based motif discovery
in nucleotide sequences
Sebastian Luehr, Holger Hartmann and Johannes So ¨ding*
Gene Center, Department of Biochemistry, and Center for Integrated Protein Science Munich (CIPSM),
Ludwig-Maximilians-Universita ¨t (LMU) Mu ¨nchen, Feodor-Lynen-Straße 25, 81377 Munich, Germany
Received March 5, 2012; Revised and Accepted May 29, 2012
ABSTRACT
The discovery of regulatory motifs enriched in sets of
DNA or RNA sequences is fundamental to the ana-
lysis of a great variety of functional genomics experi-
ments. These motifs usually represent binding sites
of proteins or non-coding RNAs, which are best
described by position weight matrices (PWMs). We
have recently developed XXmotif, a de novo motif
discovery method that is able to directly optimize
the statistical significance of PWMs. XXmotif can
also score conservation and positional clustering of
motifs. The XXmotif server provides (i) a list of sig-
nificantly overrepresented motif PWMs with web
logos and E-values; (ii) a graph with color-coded
boxes indicating the positions of selected motifs in
the input sequences; (iii) a histogram of the overall
positionaldistribution for
(iv) a page for each motif with all significant
motif occurrences, their P-values for enrichment,
conservation and localization, their sequence con-
texts and coordinates. Free access: http://xxmotif
.genzentrum.lmu.de.
selectedmotifs and
INTRODUCTION
To understand how cells read off information from the
genome at the right time at the right position, we have
to learn the sequence motifs that the regulatory factors
recognize and bind to. A large variety of experimental
methods yield sequences that are enriched in binding
sites of regulatory factors. Methods that can discover
these enriched motifs have therefore proven to be of
great practical importance for modern biological research
and a multitude of motif discovery methods have been
developed (1–4). Most of the tools can only be run on
the command line, making them inaccessible to the
majority of biologists. However, a few web services for
de novo motif discovery exist.
The most popular one is the MEME Suite server (5),
within which the position weight matrix (PWM)-based
MEME and GLAM2 motif discovery programs can be
run (6,7), alongside several related tools to compare the
discovered motifs with libraries of literature motifs and to
search for matches to the discovered motifs in sequence
databases. With a higher order background model to
describe sequences that should not carry the sought
motifs, MEME has shown state-of-the-art performance
(8). To use higher order models, users have to upload
theirownmodelfilegenerated
command line tool, which will limit most users to the
zero-order model with lower sensitivity. The SCOPE
web server combines three pattern-based motif discovery
tools, which are specialized to find non-degenerate, degen-
erate and gapped motifs, into a single prediction using a
‘winner takes all’ learning rule (9). The RegAnalyst server
runs a motif discovery method that searches for the most
enriched patterns using fixed thresholds for the maximum
number of allowed mismatches. It was originally de-
veloped for mycobacterial and yeast sequences, on which
it was reported to have higher sensitivity than SCOPE
(10). The WebMOTIFS server takes gene names from
human, mouse or Saccharomyces cerevisiae as input,
extracts promoter sequences, launches four motif discov-
ery programs and displays the results in a uniform format
(11). RSAT is a web toolbox for regulatory sequence
analysis that also offers several simple tools and Gibbs
sampling for motif discovery (12). Finally, AMADEUS
(13) is a software tool with a nicely designed graphical
user interface that presents an alternative to these web
services.
Although various published tools can score conserva-
tion in multiple sequence alignments of related species and
a few can exploit the positional clustering of motifs, to our
usingaMEME
*To whom correspondence should be addressed. Tel: +49 89 2180 76742; Fax: +49 89 2180 76797; Email: soeding@genzentrum.lmu.de
W104–W109
doi:10.1093/nar/gks602
Nucleic Acids Research, 2012, Vol. 40, Web Server issue Published online 12 June 2012
? The 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/
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knowledge, none of the web services offers this useful
functionality. In contrast, the XXmotif web server can
combine enrichment P-values for PWMs with P-values
for sequence conservation and for positional clustering
of motif occurrences.
METHOD SUMMARY
The binding site motifs of regulatory factors are described
either with PWMs or with patterns, such as consensus
sequences with degenerate IUPAC characters, sometimes
allowing for mismatches (14). A PWM is a statistical
model – represented by 4?l matrix – that has weights
for the four bases at each of the l binding sites positions.
In contrast to patterns that either do or do not match, a
PWM gives a more nuanced description of the binding
affinity landscape. From a thermodynamic point of
view, a PWM approximates the binding energy under
the assumption that each position contributes independ-
ently of the others. Although it is straightforward to
calculate enrichment P-values for patterns, this is more
challenging to do for PMWs and usually involves time-
consuming random sampling. Therefore, all PWM-based
methods to date have taken a likelihood-based approach
for finding enriched PWMs. XXmotif is the first
PWM-based method to directly optimize the motif enrich-
ment P-value in its PWM stage.
XXmotif consists of three stages: a masking stage, a
pattern stage, and a PWM stage. In the masking stage,
repeat regions, compositionally biased segments and hom-
ologous segment pairs are masked out.
When parts of sequences in the input set are identical or
similar over longer regions, this region can give rise to a
significant motif even if just these two occurrences are
observed. The reason is that the motif is so long and in-
formative that it would be very unlikely to observe even
two such motifs by chance. Hence, to avoid reporting false
motifs stemming from regions of local homology,
XXmotif masks regions of local homology found by an
all-against-all sequence comparison using BLAST. For
similar reasons, XXmasker also masks perfect repeats of
length 50 or more base pairs.
In the pattern stage, XXmotif calculates enrichment
P-values for seed patterns, consisting of all 5-mers with
up to two degenerate IUPAC characters, and all palin-
dromic and tandemic 6-mer seeds with gaps up to size
11. For each seed pattern, an enrichment P-value is
calculated using a binomial distribution and a length-
and gap-dependent Bonferroni correction factor. For
each non-degenerate seed (i.e. without IUPAC charac-
ters), the five most significant matching IUPAC seed
patterns are extended, allowing gaps of up to 3, until the
P-value cannot be improved anymore. IUPAC strings are
then converted to PWMs by counting the nucleotides at
each position in the matching sequence segments. In the
PWM stage, thousands of candidate PWMs are iteratively
optimized: similar PWMs are merged, and PWMs are
extended (allowing gaps up to 2) or shortened, until
their enrichment P-value cannot be improved anymore.
Enrichment P-values give the statistical significance of
the enrichment of a PWM in the positive sequence set
compared with the expectation derived from the back-
ground model. Enrichment P-values are calculated from
the single-site P-values for each possible motif position. A
single-site P-value quantifies the significance of the match
of a single site to the PWM. It is the probability that a
random site (generated from the background model) will
obtain at least the same score. Hence, the better the PWM
score of the single site, the more significant and the nearer
to zero is its single-site P-value. We developed an efficient
branch-and-bound algorithm to compute the single-site
P-values for all sites in the positive sequence set. The en-
richment P-value is calculated from all single-site P-values
in the input sequences using order statistics: the enrich-
ment P-value is the probability to obtain by chance on a
same-size set of background sequences at least K out of N
possible motif positions with better single-site P-values
than the Kth single-site P-value actually observed. We
chooseKthatoptimizes
Finally, the enrichment P-value can be combined with
the P-values for conservation and localization into a
total P-value. E-values are obtained by multiplying the
total motif P-values with a Bonferroni-like correction
factor, which penalizes model complexity similar to the
Akaike information criterion. For a detailed description,
see (H. Hartmann, E.W. Guthoehrlein, M. Siebert,
S. Luehr and J. So ¨ ding, submitted for publication).
XXmotif has been compared with various versions of
five state-of-the-art methods for motif discovery (MEME
(7), Weeder (15), PRIORITY (16), AMADEUS (5) and
ERMIT (17)) on a standard benchmark set containing 352
datasets of ChIP-enriched sequences from S. cerevisiae
(18), and the other containing 34 sets of metazoan se-
quences obtained with a wide range of experimental
approaches (5). XXmotif showed 20–50% higher sensitiv-
ity (number of correctly identified motifs) than the other
tools on the Harbison datasets (18) and 15–300% on the
metazoan datasets. The quality of the reported PWMs was
measured in a partial area under receiver operating char-
acteristic curve (pAUC) analysis and showed between
30 and 75%higher values
(H. Hartmann, E.W. Guthoehrlein, M. Siebert, S. Luehr
and J. So ¨ ding, submitted for publication).
this enrichmentP-value.
than theother tools
INPUT
On the ‘Data upload page’ (Figure 1A), users can enter the
input sequence set and an optional background sequence
set (up to 25 MB per file). The background sequences are
used to learn the statistical background model, which de-
scribes how ‘normal’ sequences look like. XXmotif will
then try to find motifs that are enriched in the input set
in comparison to the expectation derived from the back-
ground model. When no background sequences are
supplied, a second-order background model is trained
from the input sequences.
It is not trivial to supply a suitable background set. It
should have a trinucleotide distribution similar to the
positive sequences while not being enriched for the
motifs we seek. More concretely, the background set
should have a similar mono-, di- and trinucleotide
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composition as the positive set. If this is not the case,
XXmotif may run very slowly – because it tries to
extend sequences too much – and it may produce falsely
significant motifs. If in doubt, it is better to omit the back-
ground set altogether and to let XXmotif learn the back-
ground model from the positive set. We are about to add
an automatic quality test that will warn the user if the
background set is not well chosen.
To increase the sensitivity of the motif search, XXmotif
can calculate motif conservation P-values during the
search,which arecombined
P-values. In this case, the user can upload a set of input
and background multiple sequence alignments, using the
‘multiple FASTA’ format.
On the ‘Options’ page, the suggested default options
can be modified (Figure 1B). First, the user can specify
how many motif occurrences per input sequence are
expected. For most transcription factor and microRNA
binding sites, we would expect multiple occurrences, for
example. For core promoter motifs or splice sites, we
would expect zero or one occurrence per sequence.
When selecting the latter option, only the best occurrence
per sequence is scored, whereas with the former option, all
occurrences above a certain single-site significance P-value
are scored. Searching on both strands is recommended for
all motifs that should occur with similar probabilities on
both strands (i.e. as reverse complements of each other).
This is true for most transcription factor and microRNA
binding sites, for example, but not for core promoter or
splice site motifs. The order of the background model
specifies how long the patterns are that XXmotif learns
from the background sequence set. An eighth-order model
learns the frequencies of 9-mer nucleotides to model
the correlations between nearby nucleotides. This is the
default option selected when a background set is
supplied by the user. When the background model is
learned from the positive set, the default order is set to
2. If we were to train a model of order 8 from the positive
set, no motif shorter than 10 nucleotides could become
significant.
Under ‘Advanced options’, the user can first specify one
of three similarity thresholds for merging motifs (low,
medium and high). Setting this threshold to ‘high’ will
produce longer lists of motifs consisting of groups of
similar, partially redundant motifs, which were not
similar enough to be merged with each other. Setting the
threshold to ‘low’ will produce shorter, non-redundant
lists of motifs, as similar motifs are merged into a single
PWM. However, to be able to discern PWMs of factors
with similar binding affinities, the ‘high’ threshold is pref-
erable, as it prevents XXmotif from merging the similar
but distinct motifs.
The user can further specify which 5-mer and 6-mer
patterns are evaluated as seed patterns to initiate the
search. The number of uninformative (gap) positions in
the 5-mer seeds can also be set. When setting this param-
eter to 1, all seeds of the types XXXXX, XNXXXX,
XXNXXX, XXXNXX and XXXXNX will be assessed,
for example, where X stands for an informative position
and N stands for ‘any nucleotide’. Usually, it is sufficient
to choose zero here. XXmotif also allows changing the
with theenrichment
Figure 1. Pages for submitting a job to the XXmotif web server:
(A) upload input and background sequence sets, (B) set options for
the motif search and (C) verify and submit.
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Figure 2. Sample results with boxes that can be expanded with the orange buttons on the left. (A) Summary list of discovered motifs sorted by
significance (E-value). (B) The ‘multi distribution plot’ depicts positions and strand of motif occurrences on the input sequences. Motifs can be
selected in the upper part. The single-site P-values are represented by the height of the box, their length corresponds to the motif length. (C) The
‘localization plot’ is a histogram view of the positional distribution of selected motifs relative to an anchor point. All plots can be downloaded in
PDF format.
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amount of pseudocounts added to the nucleotide counts in
motif occurrences. The addition of pseudocounts ensures
that the PWM constructed from the motif occurrences in
the positive set can predict motif occurrences in new
datasets better than without pseudocounts. This param-
eter does not normally need to be changed. With a check
box, the XXmasker tool can be switched off, which masks
repeat regions and regions of local homology (see Method
Summary section).
Upon pressing ‘next step’, a summary of all selected
options is presented (Figure 1C), and corrections can be
made using the ‘back’ button. After job submission, the
user is directed to a status page, which can be bookmarked
and automatically redirects to the results page when the
job is finished. If the user has provided an email address, a
notification with the result page URL is sent. XXmotif
runs around 5min on 100 sequences of length 1000. Run
time scales approximately linearly with the average
sequence length and the number of sequences in the
positive sequence set.
OUTPUT
The results page lists the web logos, E-values and number
of sites of matched motifs found up to an E-value of 100
(Figure 2A). When both strands were searched, the reverse
complement versions of the motifs are also plotted. More
detailed results are hidden behind expandable boxes.
The ‘multi distribution plot’ (Figure 2B) depicts with
colored boxes the position and strand of significant
motif occurrences within the input sequences. The motifs
to display in this plot can be selected by the user in the
upper part of the plot. This allows plotting clustered
binding sitesmarking,for
elements, co-occurring pairs of motifs and other positional
biases. Setting the mouse over a particular motif site will
show the site’s sequence, strand, start and end position,
the single-site P-value measuring the match quality with
the PWM and a conservation P-value (if multiple
sequence alignments had been supplied). Only sequences
with at least one motif site are shown. Most significant
motifs are drawn last and may hide less significant ones.
example, cis-regulatory
Figure 3. Detailed motif view. The first box (motif distribution plot) plots the position of significant motif matches within the input sequences. The
second box (motif site table) gives detailed information on all significant motif matches.
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When the input sequences are all of the same length, a
‘localization plot’ can be displayed (Figure 2C). This
graph is useful to analyze positional preferences with
respect to the fixed-length sequence window of the input
sequences. It shows in a histogram view the positional
distributions of all user-selected motif occurrences with
each motif in a different color. For instance, Motif 1
(TATA-Box) in Figure 2B is exactly positioned between
?33 to ?27bp with respect to the transcription start site
(TSS) at Position 0, whereas Motif 2 (YY1) is located
mainly downstream of the TSS. Mouse-over in the histo-
gram provides the position with respect to the anchor
point and the number of counts of the motif. For
instance, Motif 4 in Figure 2C has 15 counts sharply
peaked at Position ?6 with respect to the TSS and a
‘CA’ dinucleotide at Position ?1, indicating an initiator
like function.
Detailed information about each motif can be obtained
by clicking the expand buttons in the motif summary list.
Two single motif graphs can then be viewed (Figure 3).
The ‘motif distribution plot’ is similar to the ‘multi distri-
bution plot’ and indicates the positions of significant
matches of the selected motif on the input sequences.
The ‘motif site table’ lists all significant matches with
their sequence identifiers, strands, positions, the single-site
P-values and the sequence contexts of the motif.
All plots can be downloaded with the buttons below
them. All data files generated by the XXmotif program,
such as lists of motifs with their occurrence positions,
P-values and site sequences, PWM weight coefficients
and images of motif logos can be downloaded by expand-
ing the box ‘Download XXmotif output files’.
DOCUMENTATION
Two sample input sets and pre-computed results allow the
user to get a quick overview of the server’s usage and
results. Help buttons and mouse-over explanations are
available for all input options. More general help is
listed on the FAQ page.
IMPLEMENTATION
The XXmotif web server runs on an Apache server and is
implemented using PHP, PERL and R scripts. The user
interface is dynamically generated HTML content with
JavaScripts from the jQuery library. Submitted jobs are
processed on a Scientific Linux computer cluster.
CONCLUSION
With the XXmotif web server, we aim to make a very
sensitive and reliable motif discovery method easily ac-
cessible to non-expert users. The server has clearly
structured input and results pages and offers various
useful interactive analyses. It is unique in being able to
include evidence from motif conservation and positional
clustering in the motif search.
FUNDING
Funding for open access charge: Deutsche Forschungsge-
meinschaft (DFG) [SFB646]. We acknowledge financial
support by the DFG, the Center for Protein Science
Munich (CIPSM), and a research professorship from the
Ludwig-Maximilians-Universita ¨ t
through theExcellence Initiative
Bundesministerium fu ¨ r Bildung und Forschung.
Mu ¨ nchen,
of
financed
Germanthe
Conflict of interest statement. None declared.
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