the alignment of two or more sequences has become an essential sequence analysis technique in biological research .
state - of - the - art multiple sequence alignment ( msa ) methods , such as t - coffee ( 1 ) and muscle ( 2 ) , as well as other msa methods available to date , perform alignments by only using the sequences in the given set .
although they use profile technology to match distant sequence sets , they do not use further homology information for the sequences that are available in current sequence databases .
the benefit of using homologous information to align distant sequences has been shown in a number of studies ( 3 ) , while the use of profiles to represent the additional homologous information has been shown to have many advantages ( 4,5 ) .
for this reason , the praline toolbox ( 6,7 ) has been recently re - designed to include homology - extended multiple alignment ( 8) , where as an initial step a profile for each sequence in a given set is built by using psi - blast ( 9,10 ) and the progressive alignment then proceeds using the psi - blast profiles instead of the given sequences . this approach has been previously applied with success to local pairwise alignment methods for homology modelling ( 1115 ) and is extended in praline for global msa . the recently updated mafft alignment tool ( 3,16 ) also uses homologous sequences to improve the alignment quality of distant sequences .
however , in the mafft approach , the additional information is not incorporated in profiles for each of the query sequences , but homologous sequences are added to the original set and then aligned together using the various mafft alignment strategies . in the end ,
the homologous sequences are removed , leaving the aligned original sequences to form the final alignment . in this paper
we present the new web server for the praline toolbox ( 6,7 ) , where we have added two new alignment features : homology - extended multiple alignment ( 8) and the integration of predicted secondary structure information with iteration capabilities ( v. a. simossis and j. heringa , submitted for publication ) .
we show results for the cytochrome p450 homstrad ( 17 ) sequence set as an example to demonstrate how the homology - extended strategy and integrating secondary structure information , in combination with the visualization possibilities of the server output can lead to meaningful interpretations .
the homology - extended msa strategy enriches the information for each of the sequences in a given set by collecting putative homologous sequences .
each sequence is submitted as a query to psi - blast over a database of choice [ default : non - redundant ( nr ) ] .
the resulting psi - blast alignments are then filtered for redundancy ( 100% sequence identity ) . in the event that no hits or only redundant hits are detected
, the psi - blast e - value threshold is automatically adjusted to a 10-fold less stringent setting ( e.g. from 10  10 to 10  10 ) and the query is re - submitted . once all the sequences to be aligned have at least one additional putative homologue , each psi - blast alignment is converted into a profile and progressively aligned . a more detailed account of the praline homology - extended multiple alignment algorithm and its performance is available in ref .
the advantage of this strategy is that it uses a much larger amount of position - specific information in the homology - extended profiles to score the alignment of two or more positions . as a result ,
the cases that benefit the most are those that evolution has changed so extensively ( < 30% identity ) that the homology ( common ancestry ) between them is almost undetectable when compared directly ( 8) . in table 1
, the performance of the homology - extended alignment strategy on 254 homstrad ( 17 ) multiple alignment cases has been compared with the state - of - the - art methods t - coffeev2.03 and musclev3.51 .
the results show that for the strictest quality measure , column scoring , the overall improvement of the pralinepsi strategy is > 3.5% relative to t - coffee and muscle . moreover , the improvement is > 5% for the most distant and difficult test cases with sequences < 30% sequence identity .
in addition , pralinepsi has also been compared with the praline standard global progressive alignment strategy ( pralinebasic ) ( 6 ) and the pralinebasic and pralinepsi strategies with integrated predicted [ psipred ( 19 ) and yaspin ( 20 ) ] secondary structure information , respectively , named as pralinebasic - psipred , pralinebasic - yaspin , pralinepsi - psipred and pralinepsi - yaspin .
the latter secondary structure - guided alignment strategies of praline are discussed in the next section . as shown in table 1 ,
the improvement in alignment quality achieved by homology - extended alignment ( pralinepsi ) as compared with other methods is significant in the more difficult alignment cases with average sequence identity percentages < 60% . as would be expected , in the easier alignment cases that share > 60% sequence identity , all the alignments are of comparable high quality .
when used as an option on the server , the homology - extended alignment strategy can further be customized by manually entering the desired iteration count , starting e - value cut - off and database to be searched by psi - blast for the building of the homology - extended profiles ( default : 3 iterations , starting with a cut - off of 10  10 on the nr database ) .
the default parameters have been optimized by testing different settings on the homstrad database of structural alignments ( 8) .
the rule - of - thumb that structure is more conserved than sequence is a well - documented fact ( 2124 ) . as a result
, many studies have shown that its use to guide sequence alignment improves alignment quality , especially between distant sequences ( 68,1115,25 ) . to this end , we have devised a secondary structure scoring scheme for the alignment algorithm that combines exchange weights from four types of matrices : sequence or profile positions that have not been assigned the same secondary structure class are scored using a generic matrix ( default : blosum62 ) , otherwise the positions that have matching helix , strand or coil assignments use the lthy ( 26 ) helix- , strand- and coil - specific matrices , respectively .
the use of the secondary structure information significantly improves the pralinebasic alignment quality and also boosts the pralinepsi alignments in the very difficult alignment cases < 20% sequence identity ( v. a. simossis and j. heringa , submitted for publication ) . in table 1
, it is clearly shown that the use of the secondary structure is beneficial for pralinebasic ( > 4% improvement in cases with < 60% identity ) , albeit not as significant as the improvements seen with pralinepsi .
the secondary structure integration options of praline involve the use of any one of the seven prediction methods that are listed [ phdpsi ( 27 ) , profsec ( b. rost , unpublished data ) , sspro 2.01 ( 28 ) , yaspin ( 20 ) , psipred ( 19 ) , jnet ( 29 ) and predator ( 30,31 ) ] to predict the secondary structure of the input sequences .
in addition , the user can optionally select to also search the protein data bank ( pdb ) to find 3d structure information for the input sequences and use the dssp - derived secondary structure for the alignment .
if both dssp and a prediction method are selected , the predictions will only be integrated into the alignment for those sequences that do not have a pdb entry .
finally , in the same list as the seven prediction methods , an optimally segmented ( 24 ) or majority voting consensus can be alternatively used that currently combines the predictions of profsec , yaspin and psipred .
praline provides a number of alignment strategies , such as profile pre - processing and iterative alignment optimization ( 6,7 ) .
the secondary structure - guided strategies using phd , profsec , jnet and sspro , and the profile pre - processing strategies can be set to use consistency information to drive subsequent alignment rounds ( iterations ) , each time drawing upon the theoretically higher quality information from the previous cycle .
a detailed account of these strategies can be found in previously published work ( 6,7,18,25,32,33 ) .
the praline program is designed to use two or more input protein sequences in the fasta format ( 34 ) .
the proposed maximum number of sequences that should be submitted to the server is set to 500 with length 2000 , but this is mainly to limit the server load and is not the limit of the praline program .
in addition , owing to the long running time needed for strategies , such as pralinepsi , an optional email notification can be requested that is delivered upon a completion of the job and contains the link to the results and some statistics on the resulting alignment .
similar to the previous version of the server ( 18 ) , the gap opening and gap extension penalties and the amino acid substitution matrix can be manually set if needed [ default : 12 , 1 with blosum62 ( 35 ) ] for any of the praline alignment strategies .
the results page is automatically displayed once the job is complete and contains various sections depending on the options selected ( figure 1 ) . in order to provide
all generated files for the user , there is a link to download a compressed file with all the results in the job directory [ figure 1 , ( d ) ] and also individual links that allow the user to download specific files related to each sequence in the set ( e.g. a psi - blast profile or a secondary structure file ) [ figure 1 , ( e ) ] .
if the iteration number selected is > 0 , a subtitle informs the user which iteration cycle results are presented on the page [ figure 1 , ( a ) ] .
the alignment from each iteration cycle is presented on a different page and is accessible by the corresponding links [ figure 1 , ( c ) ] .
in addition , it informs the user of the total time taken for the process to complete , provides some statistics related to the visible alignment [ figure 1 , ( b ) ] and if the iterations were halted due to alignment convergence or limit cycle convergence and which iteration was the last ( not applicable in the figure 1 example ) . in the case of iteration - specific output , such as alignment of the iteration or secondary structure prediction ,
if profile pre - processing is selected the user has the option of viewing the profile pre - processing scores for all pairwise alignments for deriving an optimum cut - off value [ figure 1 , ( g ) ] .
finally , depending on the selected parameters of the job , a series of buttons allows switching between the available colour - coded views [ figure 1 , ( h ) ] [ details about the colour schemes are described in ( 18 ) ] . at any point
, the visible alignment can be converted into a pdf for printing or further manipulation [ figure 1 , ( i ) ] .
the remaining of the results page consists of a short description of the visible colour scheme with a key to the colours , after which the colour - coded alignment follows ( an example of the conservation and the secondary structure colour - coding is shown in figure 2 ) .
owing to the large number of possible outputs , we have provided a set of nine representative sample outputs for the p450 alignment on the server , each one representing a different combination of praline strategies and settings .
these examples are intended as supplementary material to this article and can be accessed through a dedicated link on the server pages or directly at .
they can also be used as an indication of cpu times needed by each of the praline strategies . in figure 2
, we illustrate sections of the pralinepsi alignment of the  p450 homstrad sequence set ( 21% average sequence identity ) using both dssp ( 36 ) and profsec secondary structure integration settings .
the secondary structure information for each sequence in this alignment has been derived by using dssp , since all the sequences have a corresponding pdb structure .
the cytochrome p450 enzymes primarily act as oxidases in multi - component electron transport chains to break down naturally occurring toxins and mutagens .
the structure is almost triangular , with the c - terminal part being mostly helical , while the n - terminal part is more -sheet rich .
the signature motif of p450 enzymes is the haem - binding site , which is often represented as fxxgxxxcxg ( figure 2c ) .
/ g)x(e / d)t ( figure 2a ) where the threonine ( t ) residue is part of the oxygen - binding site and an invariant exxr sequence ( figure 2b ) .
the exxr and the c residue at the haem - binding site are the only completely conserved amino acids in p450s .
these well - documented details are straightforwardly visualized in the praline output conservation colour scheme , while the secondary structure view allows us to relate them in a structural context .
as stated in the literature ( 37 ) , the oxygen binding and exxr motifs are each part of two distinct c - terminal helices , while the haem - binding motif flanks the n - terminal end of the last helix . owing to space limitations the alignment has been sectioned to concentrate on these regions , but the full alignment can be viewed online in example 9 of the supplementary material .
the praline results page headers . a : the subtitle indicating which iteration results are presented on this page ( only available if iteration > 0 is selected ) .
b : the time taken to run the job and statistics related to the visible alignment .
c : the links to all other available iteration cycle results ( only available if iteration > 0 is selected ) .
f : links to iteration - specific output files ( only available if iteration > 0 is selected ) .
g : the button that hides / reveals the profile pre - processing scores of the sequence set ( only available if profile pre - processing is selected ) .
i : the button that generates and opens a pdf version of the alignment in the visible colour scheme .
the alignment has been sectioned to focus on the regions containing the conserved motifs of the cytochrome p450 enzymes ( signified by the black bars above the rulers ) .
( a ) the oxygen - binding motif , ( b ) the exxr motif and ( c ) the haem - binding motif . for each section
, the top colour scheme shows conservation levels according to the colour key and the bottom one shows the secondary structure each residue belongs to ( red : helix ; green : strand ; and clear : coil ) .
the ruler on top of each alignment block shows which parts of the alignment are visible .
the quality assessment of 254 homstrad multiple alignment cases generated by different alignment strategies the significance of the results ( p - value from kolmogorov 
the column scores are the percentage correctly aligned columns with regard to the homstrad structure alignment .
 profile alignement ( praline ) is a fully customizable multiple sequence alignment application . 
 in addition to a number of available alignment strategies , praline can integrate information from database homology searches to generate a homology - extended multiple alignment . 
 praline also provides a choice of seven different secondary structure prediction programs that can be used individually or in combination as a consensus for integrating structural information into the alignment process . 
 the program can be used through two separate interfaces : one has been designed to cater to more advanced needs of researchers in the field , and the other for standard construction of high confidence alignments . the web - based output is designed to facilitate the comprehensive visualization of the generated alignments by means of five default colour schemes based on : residue type , position conservation , position reliability , residue hydrophobicity and secondary structure , depending on the options set . 
 a user can also define a custom colour scheme by selecting which colour will represent one or more amino acids in the alignment . 
 all generated alignments are also made available in the pdf format for easy figure generation for publications . 
 the grouping of sequences , on which the alignment is based , can also be visualized as a dendrogram . 
 praline is available at . 
INTRODUCTION
HOMOLOGY-EXTENDED MULTIPLE ALIGNMENT
INTEGRATION OF SECONDARY STRUCTURE
PROFILE PRE-PROCESSING AND ITERATION
THE NEW PRALINE SERVER
SAMPLE OUTPUTS
Figures and Tables