explicitly visualizing sequences and their relationships as a network provides concise and intuitive exploration possibilities . in this respect ,
crosslink nicely complements the software clans ( 1 ) which uses a network to visualize sequence similarity between amino acid sequences .
crosslink delegates the determination of sequence relationships to the established tools blast ( 2 ) , vmatch ( 3 ) and rnahybrid ( 4 ) .
users versed with these tools will appreciate that ( almost ) all tool - specific parameters may be set from within crosslink .
furthermore , crosslink allows relationships determined by distinct tools to be visualized within the same network .
both blast and vmatch can detect local sequence similarity in both sense and antisense directions and are suitable for a wide range of scenarios .
vmatch employs a suffix array - based approach that permits constraints on the match length and on the number of mismatched bases within a match .
rnahybrid is a specialized tool that can predict potential binding sites of micrornas in large target rnas using an extension of the classical rna secondary structure prediction algorithm ( 5 ) . in general ,
rnahybrid finds the energetically most favorable hybridization sites of a small rna in a large rna .
although crosslink can be put to use in many scenarios amenable to the above tools , it can be especially useful in a microrna context : micrornas interact with target transcripts by complementary base - pairing and can be classified into families on the basis of sequence similarity  relationships that can be detected by using vmatch / rnahybrid and blast , respectively ( cf . examples below ) .
balancing flexibility and complexity , crosslink allows the user to independently specify three different kinds of relationship searches , each with its own strategy ( blast , vmatch and rnahybrid ) and a set of parameters . to this end ,
crosslink 's input consists of two sets of rna , a and b , each provided in the fasta format .
the first kind of similarity search , saa , is performed between all sequences of set a , yielding the set of matches maa .
likewise , similarity searches sab and sbb are performed to yield the set of matches mab ( between all sequences of set a and all sequences of set b ) and the set of matches mbb ( between all sequences of set b ) , respectively . for clarity , a color scheme is associated with each kind of match : reddish colors frame the parameter input controls for saa as well as the match representations of maa in the network , corresponding alignment windows and histograms .
similarly , sab and mab are associated with greenish colors and sbb and mbb are associated with bluish colors ( figure 1 ) . within each color scheme , shades indicate the orientation of each match : a dark shade is associated with matches in sense orientation and a light shade is associated with matches in antisense orientation .
in addition to orientation , each match has the following attributes : e - value , length and the proportion of identical nucleotides within the alignment when the match was determined by using blast or vmatch ; minimal free energy ( mfe ) , length and the proportion of paired nucleotides within the alignment when the match was determined by using rnahybrid . for each match set , a visualization option panel ( figure 2 )
is provided that uses a histogram for each match attribute to display the corresponding value distribution .
note that the e - value and mfe attribute histograms run on a logarithmic scale and the length and identity / paired proportion histograms run on a linear scale .
serving a 2-fold purpose , the visualization option panel also allows manipulation of the network : a threshold may be set for each attribute and a specified combination of thresholds then determines which matches will be considered for analysis and represented as links in the network and which will be suppressed .
a threshold is set by adjusting a slider for each attribute and selecting a combination mode .
two combination modes are available : in conjunction mode ( logical  and ) only matches that pass all thresholds will be displayed . in disjunction mode ( logical 
or ) only matches that pass at least one of the thresholds will be displayed .
whether the threshold acts as a cutoff for smaller or higher values of an attribute is specified by a radio button setting located on the left and right of each attribute histogram .
in addition , all sense and/or antisense matches may be suppressed for a given match set .
exploration can further be focused on an arbitrary selection of sequences by removing all remaining sequences ( along with their relationships ) from the exploration session using the menu bar ( viewremove all unselected nodes ) .
an exploration session involves three phases that occur in order : first , during a parametrization phase , the two input files are chosen and for each of the three relationship searches a strategy ( blast , vmatch or rnahybrid ) is selected and corresponding parameters are specified .
next , in the search phase , crosslink uploads all necessary information to the server and the search is performed remotely . upon completion
the results are passed back . during the final exploration phase the resulting network is visualized and
a reset button permits the user to jump back to the parametrization phase with the current parameters .
representing each match by its own link may clutter up the network visualization when many sequence pairs each yield a multitude of local matches .
therefore , each match set can independently be displayed in either  single match representative mode or 
multiple match representative mode. in  single match representative mode each link between two network nodes represents a single match between the corresponding sequences . in the case of several matches between this pair of sequences each
is represented by its own link running side by side between the two nodes . in 
multiple match representative mode a link between a pair of sequences represents all corresponding matches .
one can select whether the representative of this match set should be the one with the smallest e - value / mfe , greatest length or highest identity / paired proportion  as this may be relevant for the mentioned attribute histograms .
clicking on a node or link of the network spawns a separate window displaying detail information about the corresponding sequence or match(es ) ( figure 3 ) , respectively .
note that the alignment is displayed in text form exactly as output by the originating tool .
clicking on a subset of selected nodes spawns a separate window displaying the corresponding sequences in the fasta format .
this enables export of sequence subsets for further scrutiny using other tools . by default ,
sequences of set a and set b are displayed as red and blue nodes , respectively , in the network .
arbitrary colors may be assigned to subsets of sequences using the following strategy : a color can be associated with a text pattern .
each sequence , which contains the text pattern literally as a substring in its fasta header , will be colored accordingly .
any number of such pattern  color associations may be specified ( figure 4 ) . a sequence thus associated with several colors
any subsequent exploration task can be based on such a configuration template either  as is or after modification .
each configuration template contains the following parameters : each of the three sequence similarity search strategies including all parameters , the custom pattern  color associations and the two sequence input file names ( as associated default file names ) .
note that , for consistency , selecting a different configuration template does not change the currently stated input file names .
however , the default file names associated with the current template can be chosen explicitly .
a visualization window offers fast and powerful navigation of the network shown in the main view area ( figure 5 ) : an overview area displays the currently visible clipping as a gray rectangle , which can be dragged , focussing the main view area accordingly .
double - clicking on a sequence in the sequence selection pane ( figure 5 , lower left ) centers the view onto this sequence .
the default layout algorithm is a fruchterman  reingold ( 6 ) spring - embedding , similar to the one used in the biolayout ( 7 ) library , where each link acts as a spring pulling at the sequences it is attached to .
reset node positions button undoes all node movements performed since the last application of a layout algorithm .
crosslink 's visualization component is based on the yfiles ( 8) graph library which provides the spring - embedding implementation .
crosslink provides three example configuration templates along with the corresponding sequence files . to try out crosslink ,
one merely has to select one of the examples and press the  run button .
the following example scenarios are provided : example 1 : sequence set a consists of all rice micrornas of families 440446 available from mirbase ( 9 ) .
sequence set b contains a subset of repetitive rice sequences downloaded from the tigr rice genome annotation database .
it is immediately visible that , for example , the rice microrna family 445 exhibits very close sequence similarity to a family of repetitive rice sequences . initially displaying a multitude of links in a tangle ,
example 2 : sequence set a consists of all arabidopsis microrna precursors available at mirbase .
sequence set b contains all ( 2000 ) sequences contained in the arabidopsis small rna project database ( 10 ) to date .
setting these two sets in relationship with each other allows one to assess which microrna families have been sequenced by the asrp project .
this example also demonstrates crosslink 's ability to handle large sets of sequences and also shows the power of the spring - embedding algorithm in clustering micrornas into families .
example 3 : sequence set a consists of the drosophila micrornas dme - mir-3 , dme - mir-4 and dme - mir-5 .
sequence set b contains all corresponding targets which have been predicted ( with an e - value < 1 ) in a study by rehmsmeier et al .
( 4 ) , plus some randomly picked sequences from the same study that have not been predicted as potential targets of these micrornas .
this example demonstrates the use of rnahybrid , for example , revealing that one sequence ( accession no .
furthermore , the capability of custom pattern  color associations is shown as each predicted target set of the rehmsmeier et al .
( 4 ) study is associated with its own color ( yellow , magenta and cyan for the targets of dme - mir-3 , dme - mir-4 and dme - mir-5 , respectively ) and the non - targets are shown in blue .
both versions require an installed java runtime environment ( jre1.4.2 or later ) . to prevent overload of our server ,
the local version requires locally installed ncbi blast , vmatch and rnahybrid tools and a tcsh command line .
the crosslink website at   provides a user manual including a quick start guide plus detailed descriptions of the example input data that crosslink supplies .
network of sequence sets a ( red nodes ) and b ( blue nodes ) with corresponding matches of set maa , mab and mbb represented by links in reddish , greenish and bluish colors , respectively .
the visualization options panel for matches in set maa displaying the histograms associated with each match attribute .
the visualization window , with an overview area on the top left and a sequence selection panel on the lower left .
 crosslink is a versatile tool for the exploration of relationships between rna sequences . after a parametrization phase 
 , crosslink delegates the determination of sequence relationships to established tools ( blast , vmatch and rnahybrid ) and then constructs a network . 
 each node in this network represents a sequence and each link represents a match or a set of matches . 
 match attributes are reflected by graphical attributes of the links and corresponding alignments are displayed on a mouse - click . 
 the distributions of match attributes such as e - value , match length and proportion of identical nucleotides are displayed as histograms . 
 sequence sets can be highlighted and visibility of designated matches can be suppressed by real - time adjustable thresholds for attribute combinations . 
 powerful network layout operations ( such as spring - embedding algorithms ) and navigation capabilities complete the exploration features of this tool . 
 crosslink can be especially useful in a microrna context since vmatch and rnahybrid are suitable tools for determining the antisense and hybridization relationships , which are decisive for the interaction between micrornas and their targets . 
 crosslink is available both online and as a standalone version at . 
INTRODUCTION
DESIGN
EXAMPLES
AVAILABILITY
Figures and Tables