in the post - genomic era , with the advent of rapid sequencing techniques , reliable and efficient functional annotation methods are needed .
routinely , a translated protein sequence is aligned towards a data base of already annotated sequences and by this it is endowed with different features depending on the level of sequence identity ( si ) . this similarity search is the basis for transfer of annotation by homology .
the uniprot knowledgebase ( uniprotkb ; http://www.uniprotkb.org/ ) is presently our major resource of information of protein sequences and of corresponding functions and structures , when available .
it provides links also to other resources / data bases , allowing a comprehensive knowledge of experimental and computational characteristics of known / putative proteins and genes .
however , only 4.4% of the all protein universe that presently ( uniprotkb release 2011_03 ; 8 march 2011 ) includes some 14 million of sequences has evidence at the protein and at the transcript level . with this scenario , inference of function and structure among related sequences requires the definition of rules to increase the reliability of annotation
this is routinely obtained with clustering methods by which sequences are included into sets of similarity .
examples of hierarchical clustering include systers ( 1 ) , picasso ( 2 ) and iproclass ( 3 ) .
clustr ( 4,5 ) and protonet ( 6,7 ) are the only web servers that comprise the large number of sequences made available by fully sequenced genomes and the entire uniprotkb .
both clustr and protonet cluster sequences according to different levels of si , as set by different e - value thresholds , and with different hierarchical algorithms . alternatively ,
non - hierarchical clustering partitions a sequence data set into disjoint clusters ( 8,9 ) .
however , neither hierarchical nor non - hierarchical methods consider explicitly proteins containing multiple domains or proteins that sharing common domains do not necessarily have the same function . proteins with different combinations of shared domains can have different molecular and biological functions , as recently re - discussed ( 10 ) . in order to address these problems , we developed bar ( 11 ) , an annotation procedure that relies on a non - hierarchical clustering method and a large - scale genome comparison where pairs of sequences are selected with very strict criteria of similarity and overlapping of the alignment as described in the next section .
we provided statistical validation that bar allows reliable functional and structural annotation in addition to that given by commonly used databases ( 11 ) . here , we introduce bar , an updated and extended version of bar that includes : ( i ) a 5-fold increase in sequences ; ( ii ) go terms from the three main roots ( molecular function , biological process and cellular localization ; http://www.geneontology.org/ ) ; ( iii ) pfam domains ( http://pfam.sanger.ac.uk/ ) ; ( iv ) known ligands and ( v ) for clusters containing pdb structure / s , a cluster hmm model and the corresponding alignment of the target sequence to the optimal template in the cluster for computing its 3d structure .
bar is constructed by performing an all - against - all pairwise alignment of all protein sequences ( collected from the entire uniprotkb 05_2010 , with the exclusion of fragments ( 9  399  063 sequences ) , and from the proteome of complete sequenced genomes available on the same date at the national center for biotechnology information ( ncbi ) [ www.ncbi.nlm.nih.gov/genomes/lproks.cgi ( prokaryotes ) ; www.ncbi.nlm.nih.gov/genomes/leuks.cgi ( eukaryotes ) ] and at ensembl ( http://www.ensembl.org/info/data/ftp/index.html ) for a total of 988 complete proteomes ( the list of the species is available at bar+ web site ) . for the sake of comparison
, we also used the entire swissprot 03_2011 ( 8 march ) . similarly to bar ( 11 ) , bar is also a non - hierarchical clustering method relying on a comparative large - scale genome analysis .
the method relies on a non - hierarchical clustering procedure characterized by a stringent metric that ensures a reliable transfer of features within clusters . in this new version ,
the method takes advantage of a larger scale pairwise sequence comparison than bar , including 13  495  736 protein sequences .
alignment is performed with blast ( 12 ) in a grid environment ( 11 ) . from this
we compute for each pair both the si and the coverage ( cov ) defined as the ratio of the length of the intersection of the aligned regions on the two sequences and the overall length of the alignment ( namely the sum of the lengths of the two sequences minus the intersection length ) . each protein
is then taken as a node and a graph is built allowing links among nodes only when the following similarity constraints are found among two proteins : their si is 40% and cov is 90% . by this , clusters are simply the connected components of the graph ( 11 ) .
seventy percent of the whole data set ( 9  401  223 sequences ) falls into 913  962 clusters .
the number of sequence in the clusters ranges from two up to 87  893 in the most populated ( molecular function : abc transporter ) .
given our stringent criteria , 87% of the clusters contain sequences whose standard deviation ( sd ) of the protein length is 5 residues . the remaining sequences ( 30% of the total ) originate singletons ( containing just one sequence ) .
well annotated sequences are characterized by functional and structural annotations derived from uniprotkb entries ( figure 1 ) .
these include go , pfam , pdb and scop ( http://scop.mrc-lmb.cam.ac.uk/scop/ ) ( when available ) . to assess whether
go and pfam terms are significant in a cluster , we compute p - values and given the multiplicity of the terms , we applied the bonferroni correction ( 11 ) .
we evaluated the cumulative distribution of bonferroni corrected p - values by adopting a bootstrapping procedure . from this
we set the threshold p - value at 0.01 in order to discriminate among random and significant ( cluster associated ) features ( 11 )
. validated features ( significant for the cluster ) are those endowed with p    0.01 . according to our procedure when hypothetical and or putative proteins fall into an annotated and validated cluster
, they can safely inherit go terms and pfam domain / s even in the case of very low si with the most annotated proteins .
these sequences can therefore be labelled as distantly related homologues and inherit function and structure ( when available ) in a validated manner .
we previously discussed that this procedure can increase the level of annotation of uniprotkb ( 11 ) . here
we increase the level of structural and functional annotations of cluster - included sequences by 54% ( figure 2a ) .
they can anyway carry along information ( figure 2b ) , provided that each singleton is endowed with pdb and/or pfam and/or go annotation . 

our method collects sequences from the protein universe ( uniprotkb ) including also some 988 genomes . by this , all the features [ pdb (   scop classification ) ( red circles ) , go terms ( including molecular function , biological process and cellular localization ) and pfam models ( blue circles ) are also included .
an extensive blast alignment is performed of all the 13  495  736 sequences in a grid environment .
the sequence similarity network is built by connecting two sequences only if their si is 40% with an overlapping cov    90% .
about 913  762 clusters are obtained by splitting of the connected components . by this
, any cluster may contain from 2 up to 87  893 sequences ( one cluster containing abc transporters from prokaryotes , eukaryotes and archaea ) .
when clusters are endowed with pdb template / s , a cluster - hmm is generated by considering all the sequences that have an identity    40% and a cov    90% with the structure / s ( pink subset ) .
the cluster - hmm can be used to align all the other sequences in the cluster to template / s . 

figure 2.different types of annotations are possible with bar . after clustering and depending on the features ( structure , domains and function ) annotated in the cluster ,
the percentage of sequences endowed with a given annotation type and inheriting validated annotation ( p  <  0.01 ) is indicated .
percentage is computed with respect to 9  401  223 comprised in 913  762 clusters .
inherited : sequences that inherit annotations by falling into a cluster . without validated annotation : the slice comprises sequences with no annotation and not validated annotations .
our method collects sequences from the protein universe ( uniprotkb ) including also some 988 genomes . by this , all the features [ pdb (   scop classification ) ( red circles ) , go terms ( including molecular function , biological process and cellular localization ) and pfam models ( blue circles ) are also included .
an extensive blast alignment is performed of all the 13  495  736 sequences in a grid environment .
the sequence similarity network is built by connecting two sequences only if their si is 40% with an overlapping cov    90% .
about 913  762 clusters are obtained by splitting of the connected components . by this
, any cluster may contain from 2 up to 87  893 sequences ( one cluster containing abc transporters from prokaryotes , eukaryotes and archaea ) .
when clusters are endowed with pdb template / s , a cluster - hmm is generated by considering all the sequences that have an identity    40% and a cov    90% with the structure / s ( pink subset ) .
the cluster - hmm can be used to align all the other sequences in the cluster to template / s .
different types of annotations are possible with bar . after clustering and depending on the features ( structure , domains and function ) annotated in the cluster ,
the percentage of sequences endowed with a given annotation type and inheriting validated annotation ( p  <  0.01 ) is indicated .
percentage is computed with respect to 9  401  223 comprised in 913  762 clusters .
inherited : sequences that inherit annotations by falling into a cluster . without validated annotation : the slice comprises sequences with no annotation and not validated annotations .
in bar , when pdb templates are present within a cluster ( with or without their scop classification ) , profile hmms are computed on the basis of sequence to structure alignment and are cluster associated ( cluster - hmm ) ( figure 1 ) . when different templates are present in a cluster the structural alignment among them is computed with mustang ( 13 ) .
multiple alignments comprising all the overlapping templates and the sequences similar to them ( with si    40% and cov   90% ) are computed with muscle ( 14 ) and fed to hmmer 2.3 ( 15 ) in order to train the profile - hmm . by this , a library of 10 858 hmms is made available for aligning even distantly related sequences to a given pdb template / s .
the server also provides the pairwise query sequence  structural target alignment computed with the viterbi decoding implemented in hmmer from the correspondent cluster - hmm and useful for further processing and/or computing the corresponding 3d structure .
bar allows 35 possible fine grain types of annotations ( plus no annotation ) ( table 1 ) .
the most complete type of annotation is the one with pdb ( with and without scop annotation ) and go terms and pfam domains with p    0.01 ( validated ) ( first row in table 1 ) .
interestingly , enough 0.11% of the total sequences in our database are sufficient to annotate in a validated manner and with the most complete annotation another 21.99% sharing common clusters ( 8251 ; 0.90% of the total ) , with an annotation gain factor higher than 200 .
summing up ( along the first row of table 1 ) , we can conclude that validated functional annotation is possible within 10% of the clusters .
eleven percent of the sequences remains without annotation and are included in 45% of the clusters . about 57% of singletons ( corresponding to 17% of the total set )
table 1.the fine grain types of annotation with barpdb ( % ) scop monoscop multiwithout pdbgo validated  pfam validated  clusters8251 ( 0.90)3613 ( 0.40)1461 ( 0.16)83  266 ( 9.11 )  sequences2  982  449 ( 22.10)1  408  542 ( 10.44)1  028  565 ( 7.62)2  903  431 ( 21.51 )  inherited2  967  743 ( 21.99)1  404  011 ( 10.40)1  026  154 ( 7.60)1  382  310 ( 10.24 )  pfam  clusters8334 ( 0.91)3647 ( 0.40)1463 ( 0.16)85  886 ( 9.40 )  sequences2  984  057 ( 22.11)1  409  647 ( 10.45)1  028  569 ( 7.62)2  922  876 ( 21.66 )  inherited2  969  285 ( 22.00)1  405  095 ( 10.41)1  026  156 ( 7.60)1  398  603 ( 10.36 )  without pfam  clusters320 ( 0.04)123 ( 0.01)256251 ( 0.68 )  sequences42  202 ( 0.31)15  415 ( 0.11)7363 ( 0.05)143  533 ( 1.06 )  inherited41  825 ( 0.31)15  303 ( 0.11)7331 ( 0.05)93  568 ( 0.69)go  pfam validated  clusters8938 ( 0.98)3887 ( 0.43)1504 ( 0.16)133  895 ( 14.65 )  sequences3  042  649 ( 22.55)1  450  437 ( 10.75)1  029  707 ( 7.63)3  311  421 ( 24.54 )  inherited3  026  916 ( 22.43)1  445  521 ( 10.71)1  027  219 ( 7.61)1  617  763 ( 11.99 )  pfam  clusters9357 ( 1.02)4033 ( 0.44)1526 ( 0.17)322  937 ( 35.34 )  sequences3  045  465 ( 22.57)1  451  928 ( 10.76)1  029  755 ( 7.63)3  739  076 ( 27.71 )  inherited3  029  337 ( 22.45)1  446  890 ( 10.72)1  027  247 ( 7.61)1  852  223 ( 13.72 )  singletons2608 ( 0.02)1051  515  720 ( 11.23 )  without pfam  clusters452 ( 0.05)176 ( 0.02)3045  539 ( 4.98 )  sequences46  311 ( 0.34)17  020 ( 0.13)7400 ( 0.05)330  354 ( 2.45 )  inherited45  803 ( 0.34)16  862 ( 0.12)7362 ( 0.05)226  500 ( 1.68 )  singletons27922129  212 ( 0.96)without go  pfam validated  clusters679 ( 0.07)345 ( 0.04)1554  314 ( 5.94 )  sequences44  172 ( 0.33)27  775 ( 0.21)654547  459 ( 4.06 )  inherited43  416 ( 0.32)27  410 ( 0.20)633221  585 ( 1.64 )  pfam  clusters779 ( 0.09)377 ( 0.04)16122  236 ( 13.38 )  sequences44  582 ( 0.33)27  983 ( 0.21)656695  684 ( 5.15 )  inherited43  735 ( 0.32)27  592 ( 0.20)634301  792 ( 2.24 )  singletons20510702  834 ( 5.21 )  without pfam  clusters270 ( 0.03)83 ( 0.01)5412  192 ( 45.11 )  sequences5308 ( 0.04)1771 ( 0.01)1541  494  443 ( 11.07 )  inherited5023 ( 0.04)1689 ( 0.01)149  singletons129101  743  526 ( 12.92)percentage is evaluated with respect to the total number of sequences in the data base ( 13  495  736 sequences ) .
bold character : sequences that inherit the annotation typevalues are negligible . validated : p    0.01 ( see text for details , 11 ) . within bar clusters , 35 different types of annotations
are possible : ( i ) + go+pfam+pdb [ with or without scop ( monodomain , multidomain ) * ] ; go and pfam are or not validated ( no . of levels  =  12 ) .
( ii ) + pfam+pdb ( with or without scop ) * ( no . of levels  =  6 ) .
( iii ) + go+pdb ( with or without scop ) * ( number of levels  =  6 ) .
( iv ) + pfam+go ( no . of levels  =  4 ) . ( v ) + pdb ( with or without scop ) * ( number of levels  =  3 ) .
( vi ) + go ( no . of levels  =  2 ) .
( vii ) + pfam ( no . of levels  =  2 ) .
seventy percent of the initial set fall into clusters ( 913  962 ) and 53% in validated clusters .
some 6% of the sequences are annotated without validation and the remaining 11% are not annotated ( rightmost bottom cell ) .
about 17 and 13% of the sequences are singletons with and without annotations , respectively .
the fine grain types of annotation with bar percentage is evaluated with respect to the total number of sequences in the data base ( 13  495  736 sequences ) .
bold character : sequences that inherit the annotation type values are negligible . validated : p    0.01 ( see text for details , 11 ) . within bar clusters , 35 different types of annotations
are possible : ( i ) + go+pfam+pdb [ with or without scop ( monodomain , multidomain ) * ] ; go and pfam are or not validated ( no . of levels  =  12 ) .
( ii ) + pfam+pdb ( with or without scop ) * ( no . of levels  =  6 ) .
( iii ) + go+pdb ( with or without scop ) * ( number of levels  =  6 ) .
( iv ) + pfam+go ( no . of levels  =  4 ) . ( v ) + pdb ( with or without scop ) * ( number of levels  =  3 ) . ( vi )
( vii ) + pfam ( no . of levels  =  2 ) .
seventy percent of the initial set fall into clusters ( 913  962 ) and 53% in validated clusters .
some 6% of the sequences are annotated without validation and the remaining 11% are not annotated ( rightmost bottom cell ) .
about 17 and 13% of the sequences are singletons with and without annotations , respectively .
when a query sequence is submitted , there are three possible outcomes ( figure 3 ) .
the sequence can match a sequence already present in the cluster ( or in a singleton ) . by this ,
non - annotated proteins can inherit functional and structural annotation from other proteins within the same cluster .
validated annotations are inherited when clusters are endowed with validated go and pfam ( p  <  0.01 ) .
the query sequence may then align with any other sequence in bar with the stringent criteria of our procedure and , therefore , find a cluster from where it can safely inherit all the corresponding structural and functional features .
alternatively , when the criteria are not met , all the blast matches are returned .
provided that the sequence after running blast has a level of si    40% with a cov    90% to any sequence of bar , it is included into a cluster . in the above example
, the cluster is well annotated and the sequence inherits all the possible annotations from the cluster including go terms ( 203 ) , pdb / s , ligands , scop and pfam annotations and the cluster - hmm .
furthermore in pir format alignment / alignments of the query sequence to the cluster template / s with cluster hmm is / are also provided .
provided that the sequence after running blast has a level of si    40% with a cov    90% to any sequence of bar , it is included into a cluster . in the above example
, the cluster is well annotated and the sequence inherits all the possible annotations from the cluster including go terms ( 203 ) , pdb / s , ligands , scop and pfam annotations and the cluster - hmm .
furthermore in pir format alignment / alignments of the query sequence to the cluster template / s with cluster hmm is / are also provided .
this is based on the notion that indeed the bar annotation system increases its capacity only when we add information .
this is achieved when proteins with evidence at the transcript and protein level ( e.g. : pdb new files and/or proteins with go / pfam terms ) are included in the system .
for example , by comparing uniprotkb 05_2010 with swissprot 03_2011 , we collected some 2445 sequences carrying information according to our criteria ( evidence at protein / transcript level ) . by aligning this set towards bar clusters ,
about 8% aligns with singletons and only 0.03% of the total number of bar singletons become new clusters ( with two protein sequences ) .
another 7% fall into non - validated clusters without affecting the statistical significance of the cluster - specific annotation .
we are currently planning to include other annotation resources in order to extend our annotation process with more protein domains and their interactions .
d.p . is the recipient of a miur ( ministero istruzione universit ricerca ) fellowship supporting his ph.d
. program ; miur - firb ( fondo per gli investimenti della ricerca di base ) 2003/libi - international laboratory for bioinformatics delivered ( to r.c .
funding for open access charge : fondo ordinario per le universit ( ffo ) 2010 delivered ( to r.c . and
 we introduce bar - plus ( bar+ ) , a web server for functional and structural annotation of protein sequences . 
 bar+ is based on a large - scale genome cross comparison and a non - hierarchical clustering procedure characterized by a metric that ensures a reliable transfer of features within clusters . in this version , 
 the method takes advantage of a large - scale pairwise sequence comparison of 13  495  736 protein chains also including 988 complete proteomes . 
 available sequence annotation is derived from uniprotkb , go , pfam and pdb . 
 when pdb templates are present within a cluster ( with or without their scop classification ) , profile hidden markov models ( hmms ) are computed on the basis of sequence to structure alignment and are cluster - associated ( cluster - hmm ) . 
 therefrom , a library of 10  858 hmms is made available for aligning even distantly related sequences for structural modelling . 
 the server also provides pairwise query sequence  structural target alignments computed from the correspondent cluster - hmm . 
 bar+ in its present version allows three main categories of annotation : pdb [ with or without scop ( * ) ] and go and/or pfam ; pdb ( * ) without go and/or pfam ; go and/or pfam without pdb ( * ) and no annotation . 
 each category can further comprise clusters where go and pfam functional annotations are or are not statistically significant . 
 bar+ is available at http://bar.biocomp.unibo.it/bar2.0 . 
INTRODUCTION
BAR
CLUSTER-HMMs
DIFFERENT ANNOTATIONS with BAR
SUBMITTING A PROTEIN SEQUENCE TO BAR
BAR
FUNDING