cis - regulatory analysis is a key step in understanding and decoding transcriptional regulatory networks .
the researcher is interested in determining which transcription factors ( tfs ) regulate a gene ( or genes ) of interest , the locations of binding sites for those tfs , and , if the analysis has an evolutionary component , how those binding sites and regulatory influences evolve across species . for drosophila researchers ,
these tasks have been greatly facilitated by the availability of 12 drosophila genomes ( 1,2 ) and vast amounts of other genetic and genomic data ( 35 ) .
in addition , a variety of computational tools can nicely complement high throughput experimental approaches to the above tasks , and aid the biologist to efficiently design and conduct hypothesis - driven experiments .
for instance , available computational methods can summarize known binding specificities of tfs as  motifs and search the genome ( or genomic regions near a specific gene ) for matches to these motifs , thus identifying putative tf binding sites .
other more sophisticated methods can produce estimates of tf binding strength in a dna segment , by integrating all putative binding sites , both weak and strong , present in that segment .
application of these methods to multiple drosophila genomes , coupled with whole - genome alignments , can help describe the evolution of tf binding events .
cross species comparison can also improve the accuracy of predicting tf binding targets ( 68 ) .
computational methods have also been used to search for clusters of binding sites of multiple tfs , with the goal of identifying cis - regulatory modules ( crms , also called enhancers ) .
crms are 5001000-bp long regulatory elements that harbor multiple binding sites that together mediate a specific expression pattern of a neighboring gene ( 9 ) .
the identification of crms can provide a meaningful context in which the role of individual tf binding sites can be interpreted ; they may also help reduce false positives in predicting individual binding sites .
more recently , statistical methods have been demonstrated to recover functional crms without the prior knowledge of relevant tfs and/or their motifs .
such motif - blind approaches adopt the alternative paradigm of  supervised crm discovery , where a set of known crms with similar functionality ( expression patterns ) are used as  training data to locate other similar crms in the genome ( 10,11 ) .
genome surveyor 2.0 presents an easy - to - use , web - based graphical interface to many of the cis - regulatory analysis tools mentioned above .
it allows the user to perform tf target prediction and crm discovery using any motif(s ) from the flyfactorsurvey database ( 12 ) , the most comprehensive resource for drosophila motifs today .
tracks that profile matches to individual motifs or user - selected combinations of motifs , based on sequence information from a single genome or a combination of genomes .
it also provides tracks for  supervised crm prediction ( 10 ) , driven by a user - selected subset of known crms from the redfly database ( 13 ) .
additional tracks are available to visualize related information such as chromatin immunoprecipitation ( chip)-based profiles of tf occupancy , and previously characterized crms from the literature .
in addition to providing locus - centric visualization of cis - regulatory elements , genome surveyor 2.0 provides an interface to search for motif / chip - based binding site clusters genome - wide .
genome surveyor 2.0 provides users with the following components to perform cis - regulatory analysis in drosophila melanogaster ( figure 1a ) . 

a motif profile displays the estimated binding site presence for a user - selected tf motif as a function of genomic coordinates .
we obtain the single species profiles by running the program stubb ( 14 ) and multi - species profiles by averaging the profiles of orthologous regions from selected species.supervised crm discovery profiles .
this component allows the user to specify a set of known crms and search for novel crms that have a similar k - mer composition to the specified set .
supervised crm discovery methods do not require pre - selection of motifs , and provide a viable alternative to predicting functional crms , as explained in ( 10).profiles of other cis - regulatory information .
chip - based - binding profiles ( from bdtnp ) and experimentally validated crms ( from redfly ) can be displayed along with other profiles .
in addition to stubb - based motif profiles , the user may visualize binding site predictions by a more traditional method ( individual matches above a threshold).search for motif / chip clusters of binding sites .
this component provides the user with an ability to search the entire genome ( or list of loci ) for the most significant clusters of motif matches and/or chip sites . 

( a ) general scheme of genome surveyor 2.0 . users may investigate the region of interest ( defined through gbrowse ) for potential cis - regulatory activity using a variety of tracks . to find the targets of specific motifs
, users may activate the corresponding single species , multi - species and/or chip profiles .
they may search for crms that are similar to a set of previously validated crms , using the  supervised crm discovery tracks .
the location of strong binding sites in a region can be visualized with the  binding sites above threshold track .
( green , red and blue boxes show inputs , built - in tracks and tracks that are computed on - the - fly , respectively . ) ( b ) an instance of genome surveyor output .
thirteen different tracks are shown : ( numbered from 1 to 13 top to bottom ) redfly crms and predicted genes .
motif profiles for bicoid ( bcd_solexa ) in d.mel and d.pse as two separate tracks ( selected from the  stubb : individual motifs , individual species plugin ) .
multi - species profiles for bicoid ( bcd_solexa ) and kruppel ( kr_nar ) motifs ( selected from the  stubb : individual motifs , multi - species avg. plugin ) .
average of motif profiles for bicoid ( bcd_solexa ) and kruppel ( kr_nar ) motifs in d.mel and d.pse as two separated tracks ( selected from the  stubb : multi motif avg .
average of multi - species profiles for the bcd_solexa and kr_nar motifs ( selected from the  stubb : multi - motif , multi - species avg. plugin ) .
motif profile for user provided pwm ; here , the bicoid ( bcd ) motif from the jaspar database ( selected from the  stubb : your motifs plugin ) .
the similarity profile to a set of known crms active in blastoderm - stage development ( selected from the  supervised crm discovery plugin ) .
individual high scoring sequence motif matches ( blue codes for the highest score ) for bcd_solexa ( selected from the  binding sites above threshold ) .
a motif profile displays the estimated binding site presence for a user - selected tf motif as a function of genomic coordinates .
we obtain the single species profiles by running the program stubb ( 14 ) and multi - species profiles by averaging the profiles of orthologous regions from selected species .
this component allows the user to specify a set of known crms and search for novel crms that have a similar k - mer composition to the specified set .
supervised crm discovery methods do not require pre - selection of motifs , and provide a viable alternative to predicting functional crms , as explained in ( 10 ) .
chip - based - binding profiles ( from bdtnp ) and experimentally validated crms ( from redfly ) can be displayed along with other profiles .
in addition to stubb - based motif profiles , the user may visualize binding site predictions by a more traditional method ( individual matches above a threshold ) .
this component provides the user with an ability to search the entire genome ( or list of loci ) for the most significant clusters of motif matches and/or chip sites .
users may investigate the region of interest ( defined through gbrowse ) for potential cis - regulatory activity using a variety of tracks . to find the targets of specific motifs , users may activate the corresponding single species , multi - species and/or chip profiles .
they may search for crms that are similar to a set of previously validated crms , using the  supervised crm discovery tracks .
the location of strong binding sites in a region can be visualized with the  binding sites above threshold track .
( green , red and blue boxes show inputs , built - in tracks and tracks that are computed on - the - fly , respectively . ) ( b ) an instance of genome surveyor output .
thirteen different tracks are shown : ( numbered from 1 to 13 top to bottom ) redfly crms and predicted genes .
motif profiles for bicoid ( bcd_solexa ) in d.mel and d.pse as two separate tracks ( selected from the  stubb : individual motifs , individual species plugin ) .
multi - species profiles for bicoid ( bcd_solexa ) and kruppel ( kr_nar ) motifs ( selected from the  stubb : individual motifs , multi - species avg. plugin ) .
average of motif profiles for bicoid ( bcd_solexa ) and kruppel ( kr_nar ) motifs in d.mel and d.pse as two separated tracks ( selected from the  stubb : multi motif avg .
average of multi - species profiles for the bcd_solexa and kr_nar motifs ( selected from the  stubb : multi - motif , multi - species avg. plugin ) .
motif profile for user provided pwm ; here , the bicoid ( bcd ) motif from the jaspar database ( selected from the  stubb : your motifs plugin ) .
the similarity profile to a set of known crms active in blastoderm - stage development ( selected from the  supervised crm discovery plugin ) .
individual high scoring sequence motif matches ( blue codes for the highest score ) for bcd_solexa ( selected from the  binding sites above threshold ) .
the first three components are implemented as plugins for gbrowse ( 15 ) , and their outputs are  tracks that may be added to the current view of gbrowse . note that all of these tracks / profiles can be displayed simultaneously , as illustrated in figure 1b .
we have pre - computed the motif profiles of a large collection of experimentally validated tfs for d. melanogaster ( 12 ) using the hidden markov model - based program stubb ( 14 ) .
( stubb examines each 500-bp window and computes a score for the presence of one or more strong or weak binding sites in that window , without imposing arbitrary thresholds on what constitute a motif match . )
we have also generated motif profiles for 11 other drosophila species and mapped them to the d.mel coordinates .
users may select from the following options related to motif profiles : 
 individual species , individual motif : this option displays the profiles of the selected motif(s ) in the selected species .
given this option , users might easily check , for example , whether a specific potential binding event is conserved between d.mel and d.pse by turning on the tracks of the corresponding motif for both species .
also , they may easily assess the similarity between the targets of two or more tfs .
all tracks are directly linked to ( and just a click away from ) the flyfactorsurvey database ( 12 ) that provides detailed information about the binding site 's specificity and the method used to characterize it.individual species , multi - motif : this option averages the profiles of selected motifs for each selected species .
this provides a convenient way to look for clusters of binding sites of several tfs , as a means to discover novel crms .
for example , a user searching for enhancers regulating dorsal / ventral ( d / v ) patterning may choose to select the motifs involved in this process ( e.g. those for the tfs dl , twi , sna ) and examine their average profile .
the user may repeat this process for other species as well , to examine if the predicted crm in d. melanogaster is independently supported by predictions at orthologous locations in those species.multi-species , individual motif : this option combines the profiles of a selected motif from different species , using simple averaging or a phylogenetic tree - based averaging ( 7 ) .
the peaks in this profile represent the tf targets that are conserved across species.multi-species , multi - motif : this option averages all the profiles from selected motifs and species to create a single track .
the peaks in this profile represent the strong clusters of binding sites that are conserved across species , and may thus correspond to functional crms.user-defined motif : this option allows users to input their own position weight matrices ( pwms ) , rather than selecting from a pre - defined list of motifs .
although there has been an intense effort to characterize the binding specificities of all tfs in d. melanogaster ( 16 ) , there remain many tfs with unknown binding specificity .
the user - defined motif option allows motifs that are not part of the publically available database to be used .
individual species , individual motif : this option displays the profiles of the selected motif(s ) in the selected species . given this option
, users might easily check , for example , whether a specific potential binding event is conserved between d.mel and d.pse by turning on the tracks of the corresponding motif for both species .
also , they may easily assess the similarity between the targets of two or more tfs .
all tracks are directly linked to ( and just a click away from ) the flyfactorsurvey database ( 12 ) that provides detailed information about the binding site 's specificity and the method used to characterize it .
individual species , multi - motif : this option averages the profiles of selected motifs for each selected species .
this provides a convenient way to look for clusters of binding sites of several tfs , as a means to discover novel crms .
for example , a user searching for enhancers regulating dorsal / ventral ( d / v ) patterning may choose to select the motifs involved in this process ( e.g. those for the tfs dl , twi , sna ) and examine their average profile .
the user may repeat this process for other species as well , to examine if the predicted crm in d. melanogaster is independently supported by predictions at orthologous locations in those species .
multi - species , individual motif : this option combines the profiles of a selected motif from different species , using simple averaging or a phylogenetic tree - based averaging ( 7 ) . the peaks in this profile represent the tf targets that are conserved across species .
multi - species , multi - motif : this option averages all the profiles from selected motifs and species to create a single track . the peaks in this profile represent the strong clusters of binding sites that are conserved across species , and may thus correspond to functional crms . user - defined motif : this option allows users to input their own position weight matrices ( pwms ) , rather than selecting from a pre - defined list of motifs .
although there has been an intense effort to characterize the binding specificities of all tfs in d. melanogaster ( 16 ) , there remain many tfs with unknown binding specificity .
the user - defined motif option allows motifs that are not part of the publically available database to be used .
the redfly database catalogs over 800 experimentally characterized crms in d. melanogaster , along with their spatial / temporal expression patterns ( 13 ) .
this extensive resource can be used as  training data to computationally predict novel crms genome - wide , through  supervised crm discovery methods .
these methods score a genomic segment for sequence similarity to any given set of known crms .
the similarity score is based on frequencies of short words in the sequences , and can detect the presence of shared binding sites without relying on prior knowledge of motifs . as such , this is a pragmatic approach to crm discovery when the likely transcriptional regulators of a gene are not known in advance , or their binding specificities have not been characterized .
genome surveyor 2.0 allows the user to profile any genomic region with two different scores [ hexmcd and imm ( m. kazemian , q. zhu , m. s. halfon , s. sinha , manuscript under preparation ) ] ( 10 ) .
the training set of crms may be selected as one of over 30 different subsets of redfly crms , defined by the tissue / stage of development that they help regulate ( 11 ) .
users may select from the following three tracks for additional information to aid their analysis : 
 binding sites above a threshold .
it displays individual binding sites predicted based on how well they match the selected / provided motifs.chip profiles
. this track displays chip - based measurements of tf occupancy ( 17 ) . at this time , these profiles are available for a limited number of tfs.redfly crms .
it helps user to check the availability of any known enhancer in their region of interest .
each crm is linked back to the redfly database for detailed information ( e.g. crm expression pattern , the evidence for the element , the source , binding sites ) .
it displays individual binding sites predicted based on how well they match the selected / provided motifs . chip profiles
. this track displays chip - based measurements of tf occupancy ( 17 ) . at this time ,
it helps user to check the availability of any known enhancer in their region of interest .
each crm is linked back to the redfly database for detailed information ( e.g. crm expression pattern , the evidence for the element , the source , binding sites ) .
crms are known to harbor binding sites for several tfs , which act together to achieve specific regulatory functions . as such , computational tools for
genome - wide crm discovery typically search for clusters of binding sites with suitably chosen collections of tf motifs .
genome surveyor 2.0 provides an interface for users to search for the most significant clusters of binding sites in the d. melanogaster genome for any user - specified combination of tfs ( figure 2a ) . 

( a ) an interface to search for motif / chip - based binding site clusters .
there are three main panels : search options , motif collection and chip data collection .
search options : here , the user may select the types of profiles (  sorting criteria ) to use in the search and also the search region , which is either the entire genome or a list of genomic loci .
more advanced search options are also provided ( e.g. the number of top results ) .
motifs collection and chip data collection : here , the user may select the specific motifs and chip profiles to use in the search . ( b ) sample output of the search tool .
a genome - wide search was conducted with five tfs involved in anterior patterning ( bcd , kr , hb , tll and hkb ) , retrieving segments ( of 500  bp each ) where a significant cluster of binding sites for one or more of these tfs was found .
the second column reports the location of the segment , along with a link to the genome surveyor view of this locus .
it also provides the names of known crms that overlap the segment ( e.g.  eve_stripe1 for segment 1 ) .
the third and fourth columns show the strength ( z - score relative to the genome - wide average ) of binding site clustering in the segment , based on single species [  stubb(ss) ] and multi - species [  stubb(ms) ] analysis respectively .
the fifth column reports the scores of each selected motif , separately for single species (  ss ) and multi - species (  ms ) analysis .
a ) an interface to search for motif / chip - based binding site clusters .
there are three main panels : search options , motif collection and chip data collection .
search options : here , the user may select the types of profiles (  sorting criteria ) to use in the search and also the search region , which is either the entire genome or a list of genomic loci .
more advanced search options are also provided ( e.g. the number of top results ) .
motifs collection and chip data collection : here , the user may select the specific motifs and chip profiles to use in the search .
a genome - wide search was conducted with five tfs involved in anterior patterning ( bcd , kr , hb , tll and hkb ) , retrieving segments ( of 500  bp each ) where a significant cluster of binding sites for one or more of these tfs was found .
the second column reports the location of the segment , along with a link to the genome surveyor view of this locus .
it also provides the names of known crms that overlap the segment ( e.g.  eve_stripe1 for segment 1 ) .
the third and fourth columns show the strength ( z - score relative to the genome - wide average ) of binding site clustering in the segment , based on single species [  stubb(ss) ] and multi - species [  stubb(ms) ] analysis respectively .
the fifth column reports the scores of each selected motif , separately for single species (  ss ) and multi - species (  ms ) analysis .
users first select the type of binding site profiles that will be used for search ( single / multi species motif profiles or chip profiles ) .
next , they may choose to scan the entire genome , or provide a list of genomic loci where the search will be performed .
advanced options ( e.g. the number of top hits or the minimum number of different tfs in a predicted cluster ) are available , but default settings are provided and help pages provide guidance for changing them . finally , the user selects the motif or chip profiles of interest and begins the search .
the output of the search tool is a table of predicted regulatory sequences ( 500-bp segments with clusters of binding sites ) in the d. melanogaster genome , with links to appropriate gbrowse views ( figure 2b ) .
the results are sorted based on the average value of the selected profiles in the segments .
single as well as multi - species scores are reported for each segment . moreover , a score representing each motif 's presence in the segment is shown separately , to help the user determine which motifs contribute significantly to the cluster .
the output also includes information about the nearest neighboring genes and their distances from the binding site cluster .
stubb is a popular crm discovery tool that has been tested by multiple groups in different species ( 1820 ) .
we have shown previously that regions with high stubb scores are highly enriched for experimentally observed tf binding ( chip ) , and that the enrichment improves significantly upon incorporating multi - species information ( 7 ) .
stubb score profiles can be utilized to investigate the binding site composition of any genomic region .
the strategy of combining the stubb profiles of multiple tfs and identify the segments with highest average scores ( figure 3 ) has been demonstrated to recover known crms ( 16 ) .
genome - wide predictions of the  supervised crm prediction methods included in genome surveyor 2.0 have been assessed statistically and validated experimentally ( 10 ) . 

eight different tracks are shown : the  stubb individual species profiles for seven tfs ( cad , kr , bcd , gt , hb , kni , tll ) involved in initial stages of anterior  posterior ( a / p ) patterning in the embryo , and the  stubb multi - motif avg. profile over tll , kr , bcd and gt .
blue shaded regions represent two known a / p related crms , h_stripe1 and h_stripe0 , that are both known to be bcd - activated crms ( 21 ) . the presence ( high peaks in score profile ) of this known activator , as well as specific transcriptional repressors ( tll and kr for h_stripe1 and gt for h_stripe0 ) , is consistent with the crm expression patterns .
the bottom profile ( red ) illustrates how combining stubb profiles from multiple motifs might help in crm discovery .
eight different tracks are shown : the  stubb individual species profiles for seven tfs ( cad , kr , bcd , gt , hb , kni , tll ) involved in initial stages of anterior  posterior ( a / p ) patterning in the embryo , and the  stubb multi - motif avg. profile over tll , kr , bcd and gt .
blue shaded regions represent two known a / p related crms , h_stripe1 and h_stripe0 , that are both known to be bcd - activated crms ( 21 ) .
the presence ( high peaks in score profile ) of this known activator , as well as specific transcriptional repressors ( tll and kr for h_stripe1 and gt for h_stripe0 ) , is consistent with the crm expression patterns .
the bottom profile ( red ) illustrates how combining stubb profiles from multiple motifs might help in crm discovery .
we have pre - computed the motif profiles of a large collection of experimentally validated tfs for d. melanogaster ( 12 ) using the hidden markov model - based program stubb ( 14 ) .
( stubb examines each 500-bp window and computes a score for the presence of one or more strong or weak binding sites in that window , without imposing arbitrary thresholds on what constitute a motif match . )
we have also generated motif profiles for 11 other drosophila species and mapped them to the d.mel coordinates .
users may select from the following options related to motif profiles : 
 individual species , individual motif : this option displays the profiles of the selected motif(s ) in the selected species . given this option
, users might easily check , for example , whether a specific potential binding event is conserved between d.mel and d.pse by turning on the tracks of the corresponding motif for both species .
also , they may easily assess the similarity between the targets of two or more tfs .
all tracks are directly linked to ( and just a click away from ) the flyfactorsurvey database ( 12 ) that provides detailed information about the binding site 's specificity and the method used to characterize it.individual species , multi - motif : this option averages the profiles of selected motifs for each selected species .
this provides a convenient way to look for clusters of binding sites of several tfs , as a means to discover novel crms .
for example , a user searching for enhancers regulating dorsal / ventral ( d / v ) patterning may choose to select the motifs involved in this process ( e.g. those for the tfs dl , twi , sna ) and examine their average profile .
the user may repeat this process for other species as well , to examine if the predicted crm in d. melanogaster is independently supported by predictions at orthologous locations in those species.multi-species , individual motif : this option combines the profiles of a selected motif from different species , using simple averaging or a phylogenetic tree - based averaging ( 7 ) .
the peaks in this profile represent the tf targets that are conserved across species.multi-species , multi - motif : this option averages all the profiles from selected motifs and species to create a single track .
the peaks in this profile represent the strong clusters of binding sites that are conserved across species , and may thus correspond to functional crms.user-defined motif : this option allows users to input their own position weight matrices ( pwms ) , rather than selecting from a pre - defined list of motifs .
although there has been an intense effort to characterize the binding specificities of all tfs in d. melanogaster ( 16 ) , there remain many tfs with unknown binding specificity .
the user - defined motif option allows motifs that are not part of the publically available database to be used .
individual species , individual motif : this option displays the profiles of the selected motif(s ) in the selected species .
given this option , users might easily check , for example , whether a specific potential binding event is conserved between d.mel and d.pse by turning on the tracks of the corresponding motif for both species .
also , they may easily assess the similarity between the targets of two or more tfs .
all tracks are directly linked to ( and just a click away from ) the flyfactorsurvey database ( 12 ) that provides detailed information about the binding site 's specificity and the method used to characterize it .
individual species , multi - motif : this option averages the profiles of selected motifs for each selected species .
this provides a convenient way to look for clusters of binding sites of several tfs , as a means to discover novel crms .
for example , a user searching for enhancers regulating dorsal / ventral ( d / v ) patterning may choose to select the motifs involved in this process ( e.g. those for the tfs dl , twi , sna ) and examine their average profile .
the user may repeat this process for other species as well , to examine if the predicted crm in d. melanogaster is independently supported by predictions at orthologous locations in those species .
multi - species , individual motif : this option combines the profiles of a selected motif from different species , using simple averaging or a phylogenetic tree - based averaging ( 7 ) .
multi - species , multi - motif : this option averages all the profiles from selected motifs and species to create a single track .
the peaks in this profile represent the strong clusters of binding sites that are conserved across species , and may thus correspond to functional crms . user - defined motif
: this option allows users to input their own position weight matrices ( pwms ) , rather than selecting from a pre - defined list of motifs .
although there has been an intense effort to characterize the binding specificities of all tfs in d. melanogaster ( 16 ) , there remain many tfs with unknown binding specificity .
the user - defined motif option allows motifs that are not part of the publically available database to be used .
the redfly database catalogs over 800 experimentally characterized crms in d. melanogaster , along with their spatial / temporal expression patterns ( 13 ) .
this extensive resource can be used as  training data to computationally predict novel crms genome - wide , through  supervised crm discovery methods .
these methods score a genomic segment for sequence similarity to any given set of known crms .
the similarity score is based on frequencies of short words in the sequences , and can detect the presence of shared binding sites without relying on prior knowledge of motifs . as such , this is a pragmatic approach to crm discovery when the likely transcriptional regulators of a gene are not known in advance , or their binding specificities have not been characterized .
genome surveyor 2.0 allows the user to profile any genomic region with two different scores [ hexmcd and imm ( m. kazemian , q. zhu , m. s. halfon , s. sinha , manuscript under preparation ) ] ( 10 ) .
the training set of crms may be selected as one of over 30 different subsets of redfly crms , defined by the tissue / stage of development that they help regulate ( 11 ) .
users may select from the following three tracks for additional information to aid their analysis : 
 binding sites above a threshold .
it displays individual binding sites predicted based on how well they match the selected / provided motifs.chip profiles
. this track displays chip - based measurements of tf occupancy ( 17 ) . at this time , these profiles are available for a limited number of tfs.redfly crms .
it helps user to check the availability of any known enhancer in their region of interest .
each crm is linked back to the redfly database for detailed information ( e.g. crm expression pattern , the evidence for the element , the source , binding sites ) .
it displays individual binding sites predicted based on how well they match the selected / provided motifs .
chip profiles . this track displays chip - based measurements of tf occupancy ( 17 ) . at this time , these profiles are available for a limited number of tfs . redfly crms .
it helps user to check the availability of any known enhancer in their region of interest .
each crm is linked back to the redfly database for detailed information ( e.g. crm expression pattern , the evidence for the element , the source , binding sites ) .
crms are known to harbor binding sites for several tfs , which act together to achieve specific regulatory functions . as such , computational tools for
genome - wide crm discovery typically search for clusters of binding sites with suitably chosen collections of tf motifs .
genome surveyor 2.0 provides an interface for users to search for the most significant clusters of binding sites in the d. melanogaster genome for any user - specified combination of tfs ( figure 2a ) . 

( a ) an interface to search for motif / chip - based binding site clusters .
there are three main panels : search options , motif collection and chip data collection .
search options : here , the user may select the types of profiles (  sorting criteria ) to use in the search and also the search region , which is either the entire genome or a list of genomic loci .
more advanced search options are also provided ( e.g. the number of top results ) .
motifs collection and chip data collection : here , the user may select the specific motifs and chip profiles to use in the search . ( b ) sample output of the search tool .
a genome - wide search was conducted with five tfs involved in anterior patterning ( bcd , kr , hb , tll and hkb ) , retrieving segments ( of 500  bp each ) where a significant cluster of binding sites for one or more of these tfs was found .
the second column reports the location of the segment , along with a link to the genome surveyor view of this locus .
it also provides the names of known crms that overlap the segment ( e.g.  eve_stripe1 for segment 1 ) .
the third and fourth columns show the strength ( z - score relative to the genome - wide average ) of binding site clustering in the segment , based on single species [  stubb(ss) ] and multi - species [  stubb(ms) ] analysis respectively .
the fifth column reports the scores of each selected motif , separately for single species (  ss ) and multi - species (  ms ) analysis .
( a ) an interface to search for motif / chip - based binding site clusters .
there are three main panels : search options , motif collection and chip data collection .
search options : here , the user may select the types of profiles (  sorting criteria ) to use in the search and also the search region , which is either the entire genome or a list of genomic loci .
more advanced search options are also provided ( e.g. the number of top results ) .
motifs collection and chip data collection : here , the user may select the specific motifs and chip profiles to use in the search . ( b ) sample output of the search tool .
a genome - wide search was conducted with five tfs involved in anterior patterning ( bcd , kr , hb , tll and hkb ) , retrieving segments ( of 500  bp each ) where a significant cluster of binding sites for one or more of these tfs was found .
the second column reports the location of the segment , along with a link to the genome surveyor view of this locus .
it also provides the names of known crms that overlap the segment ( e.g.  eve_stripe1 for segment 1 ) .
the third and fourth columns show the strength ( z - score relative to the genome - wide average ) of binding site clustering in the segment , based on single species [  stubb(ss) ] and multi - species [  stubb(ms) ] analysis respectively .
the fifth column reports the scores of each selected motif , separately for single species (  ss ) and multi - species (  ms ) analysis .
users first select the type of binding site profiles that will be used for search ( single / multi species motif profiles or chip profiles ) .
next , they may choose to scan the entire genome , or provide a list of genomic loci where the search will be performed .
advanced options ( e.g. the number of top hits or the minimum number of different tfs in a predicted cluster ) are available , but default settings are provided and help pages provide guidance for changing them . finally , the user selects the motif or chip profiles of interest and begins the search .
the output of the search tool is a table of predicted regulatory sequences ( 500-bp segments with clusters of binding sites ) in the d. melanogaster genome , with links to appropriate gbrowse views ( figure 2b ) .
the results are sorted based on the average value of the selected profiles in the segments .
single as well as multi - species scores are reported for each segment . moreover , a score representing each motif 's presence in the segment is shown separately , to help the user determine which motifs contribute significantly to the cluster .
the output also includes information about the nearest neighboring genes and their distances from the binding site cluster .
stubb is a popular crm discovery tool that has been tested by multiple groups in different species ( 1820 ) .
we have shown previously that regions with high stubb scores are highly enriched for experimentally observed tf binding ( chip ) , and that the enrichment improves significantly upon incorporating multi - species information ( 7 ) .
stubb score profiles can be utilized to investigate the binding site composition of any genomic region .
the strategy of combining the stubb profiles of multiple tfs and identify the segments with highest average scores ( figure 3 ) has been demonstrated to recover known crms ( 16 ) .
genome - wide predictions of the  supervised crm prediction methods included in genome surveyor 2.0 have been assessed statistically and validated experimentally ( 10 ) . 

eight different tracks are shown : the  stubb individual species profiles for seven tfs ( cad , kr , bcd , gt , hb , kni , tll ) involved in initial stages of anterior  posterior ( a / p ) patterning in the embryo , and the  stubb multi - motif avg. profile over tll , kr , bcd and gt .
blue shaded regions represent two known a / p related crms , h_stripe1 and h_stripe0 , that are both known to be bcd - activated crms ( 21 ) .
the presence ( high peaks in score profile ) of this known activator , as well as specific transcriptional repressors ( tll and kr for h_stripe1 and gt for h_stripe0 ) , is consistent with the crm expression patterns .
the bottom profile ( red ) illustrates how combining stubb profiles from multiple motifs might help in crm discovery .
eight different tracks are shown : the  stubb individual species profiles for seven tfs ( cad , kr , bcd , gt , hb , kni , tll ) involved in initial stages of anterior  posterior ( a / p ) patterning in the embryo , and the  stubb multi - motif avg. profile over tll , kr , bcd and gt .
blue shaded regions represent two known a / p related crms , h_stripe1 and h_stripe0 , that are both known to be bcd - activated crms ( 21 ) . the presence ( high peaks in score profile ) of this known activator , as well as specific transcriptional repressors ( tll and kr for h_stripe1 and gt for h_stripe0 ) , is consistent with the crm expression patterns .
the bottom profile ( red ) illustrates how combining stubb profiles from multiple motifs might help in crm discovery .
funding for open access charge : this work was supported in part by grants by the national institute of health ( grant r01hg004744 - 01 to m.h.b . , grant r01gm085233 - 01 to s.s . ) .
the funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript .
 genome surveyor 2.0 is a web - based tool for discovery and analysis of cis - regulatory elements in drosophila , built on top of the gbrowse genome browser for convenient visualization . 
 genome surveyor was developed as a tool for predicting transcription factor ( tf ) binding targets and cis - regulatory modules ( crms / enhancers ) , based on motifs representing experimentally determined dna binding specificities . since its first publication 
 , we have added substantial new functionality ( e.g. phylogenetic averaging of motif scores from multiple species , and a novel crm discovery technique ) , increased the number of supported motifs about 4-fold ( from 100 to 400 ) , added provisions for evolutionary comparison across many more drosophila species ( from 2 to 12 ) , and improved the user - interface . 
 the server is free and open to all users , and there is no login requirement . 
 address : http://veda.cs.uiuc.edu/gs . 
INTRODUCTION
WEB INTERFACE
Single/multi-species motif profiles
Supervised CRM discovery profiles
Binding sites, ChIP and REDfly profiles
Search interface for Motif/ChIP clusters of binding sites
Methods validation
FUNDING