single nucleotide polymorphisms ( snps ) are the simplest and most frequent type of dna sequence variation among individuals and constitute one of the most powerful tools in the search for disease susceptibility genes , drug response - determining genes and the like ( 1,2 ) . with the introduction of large - scale genotyping techniques
the bottleneck in this type of experiments has moved towards the management and analysis of the data generated . in this context ,
one of the topics which has become a problem is the step of the selection of the optimal set of snps ( among several thousands of candidates in some cases ) for the genotyping experiment .
optimal snps must be the best possible markers for traits , which often are multigenic , usually reflecting disruptions in proteins that participate in a protein complex or a in a pathway ( 3 ) .
unfortunately , complex multigenic traits , for which markers display weak associations , still constitute a challenge .
factors such as linkage disequilibrium ( ld ) and minor allele frequency ( maf ) are of major importance for selecting optimal candidate snps .
recently , the predicted functional effect of an snp is gaining importance as a selection criterium because it constitutes a potential important factor for increasing the sensitivity of association tests significantly ( 36 ) . the availability of information on ld from projects such as hapmap ( 7 ) , on mafs ( 8) and improved methods for predicting function ( 5,6,9 ) , allow for a more sophisticated selection of candidate snps beyond the classical one - snp - at - a - time approach .
thus , snps can be selected taking into account the evolutionary constraints of the region analysed along with its likelihood of being the causative agent of any type of damage .
algorithms which use information to facilitate the posterior analysis of the results , such as the estimation of haplotype blocks ( 10 ) , combined with functional prediction of the effect of the snps , are expected to have a major impact on the efficiency of a large - scale genotyping study .
pupasuite combines the facilities offered by pupasnp ( 6 ) and pupasview ( 5 ) with new algorithms and visualisation procedures for functional haplotype prediction .
the pupasnp and pupasview programs are part of the pipeline of genotyping of the spanish national genotyping center ( cegen ; ) . both tools combined bear an average of 60 snp designs per day .
pupasuite combines the functionality of pupasnp ( 6 ) and pupasview ( 5 ) in a unique and more integrated interface , and adds new modules to facilitate the selection of the optimal set of snps for a large - scale genotyping study .
following the philosophy of pupasnp , the program allows to input either lists of genes or chromosomal regions , which would correspond to two common types of analysis : genes probably related to a disease because they are functionally related ( e.g. they belong to a pathway affected in the disease ) , or genes present in a chromosomal region linked to a disease .
pupasuite can also directly analyse lists of snps . in these three cases a list of snps with their putative functional effect is reported . in the case of chromosomal regions
it is also possible to find haplotype blocks ( 10 ) . for the list of snps , in addition to their putative functional effect , it is possible to retrieve information on maf in different populations from dbsnp ( 8) [ as annotated in the ensembl ( 11 ) ] , as well as ld parameters and haplotype blocks .
in addition to the analysis of lists of snps there is another new option : functional haplotypes . this option ( see below ) allows the user to test their own snp data and to find haplotypes ( 12 ) with the functional snps ( 5,6 ) and the tag snps ( 13 ) highlighted .
case - control studies can also be performed at this stage . the option display and filter snps for a single gene implements new functionalities in an environment a la pupasview ( 5 ) .
this option allows the sequential and interactive application of filters based on functionality , conservation , maf and the like ( 5 ) thus permitting an easy selection of a set of optimal snps for a particular gene .
here three important features of a snp have been taken into account in order to be considered as an optimal candidate for genotyping purposes : maf , ld with respect to other candidates ( 5 ) and putative functional effect .
maf values were taken from the ensembl ( 11 ) , which maps dbsnp ( 8) data onto the corresponding chromosomal coordinates .
the putative functional effect has been estimated in both coding and non - coding regions as described in ( 5 ) .
the following features have been used to report the putative functional effect of a polymorphism in non - coding nucleotides : 
 transcription factor binding sites from the transfac database ( 15).intron / exon border consensus sequences.exonic splicing enhancers ( 16).triplex - forming oligonucleotide target sequences ( 17 ) . 
 regarding the putative impact of a csnp , the following data and estimators are reported : 
 snps in exons causing an amino acid change ( purely a list of csnps)pmut ( 18,19 ) predictions.selective strengths (  parameter ) .
this estimator is new in this version of the program ( see below)snpeffect ( 9,20,21 ) predictions .
the likelihood of the predictions can be reinforced by looking simultaneously for human - mouse conserved regions ( 22 ) as reported in ensembl ( ) .
snps in exons causing an amino acid change ( purely a list of csnps ) pmut ( 18,19 ) predictions .
this estimator is new in this version of the program ( see below ) snpeffect ( 9,20,21 ) predictions .
the combined effect of all the selective pressures causes the preservation of the functionally relevant parts of the genes . under this perspective
, comparative and evolutionary studies have been used to predict the putative functional effect of snps ( 19,23 ) although these have mainly ignored the underlying phylogeny . here
we present another more accurate estimator of functional effect , based on sequence comparison , but taking into account phylogenetic information ( 24 ) .
the selective pressures acting at a codon - level where non - synonymous csnps are found were evaluated by means of two alternative approaches : codon - based maximum likelihood ( ml ) models ( 25 ) implemented in paml ( 26 ) , and likelihood - ratio ( slr ) method ( 27 ) for testing deviations of neutrality . under the first approximation , an a priori statistical distribution describing the variation of  = dn / ds among sites is assumed for a number k of different classes of sites with k values at a proportion pk of the sequences representing the effects of purifying selection ( 0 < 0 < 1 ) , neutral evolution ( 1 = 1 ) , and positive selection ( 2 > 1 ) ( 25 ) .
the method involves two main steps : first , the adjustment by maximum likelihood of the evolutionary parameters to the sequences of the species compared considering two different models ; and second , the use of the bayes theorem to compute the posterior probability that each site belongs to a specific site class k defined under an a priori distribution ( 28 ) . two different models ( m2a and m8 ) were evaluated by maximum likelihood on the sequences ( 29 ) . under the sitewise likelihood - ratio method ( slr ) a site - by - site approach to test for neutrality is used .
in contrast to similar approaches developed previously ( 30 ) , slr uses the entire alignment of the sequence to determine parameters common to all sites , such as evolutionary distances . using this approach
there is no need to specify a model of how  varies along the sequence .
a correction for multiple testing in order to obtain statistical confidence for inferences on deviations from neutrality on each site is also performed .
the snpeffect database ( 9 ) describes the effect of coding non - synonymous snps on several phenotypic properties of human proteins using either sequence - based or structural bioinformatics tools .
molecular phenotypes are grouped in three categories : structure and dynamics , functional sites and cellular processing .
next to various external tools snpeffect uses algorithms developed at the collaborating research groups , among which tango ( 20 ) to predict -aggregation regions in protein sequences and foldx ( 21 ) to predict the stability change caused by the single amino acid variation .
in addition to using already available data , the users can input their own data to use the predictions on possible functional effects in combination with haplotype analysis .
data must be provided to the program in linkage pedigree format ( pre makepred , ) .
the pupasuite estimates blocks by three methods : confidence intervals ( 10 ) , four gamete rule ( 31 ) and solid spine of ld ( 14 ) and reconstruct haplotypes using the em algorithm ( 12 ) as implemented in haploview ( 14 ) .
the haplotypes found in this way are represented with the corresponding functional information on all the snps included in it and all the ld values .
this representation provides a very intuitive picture of the possible functional impact of any of the haplotypes beyond the individual effect of each snp . for case / control data a chi - square test
is performed and the corresponding p - value for the allele frequencies in cases versus control is reported .
the combination of functional haplotype information with case / control tests allows to easily ascribe cases to haplotypes with functional alterations .
we have presented an integrated resource for helping in the selection of optimal sets of snps oriented to large - scale genotyping assays .
the program merges the functionalities of other two previous resources , pupasnp ( 6 ) and pupasview ( 5 ) , and expand the capabilities of the program with new information and new facilities .
the snpeffect database ( 9 ) as well as a new , unpublished prediction method has been included to improve the estimation of the putative pathological effect of snps .
moreover , in addition to use publicly available data on snps , users can analyse their own experiments .
what is novel and unique to tools of this type is the possibility of analysing functionally haplotypes , beyond the classical analysis one - snp - at - a - time which ignores interactions between the mutations .
the usefulness of this type of resources is proven by the use made by the cegen in its pipeline of genotyping .
the previous tools , which have been running for more than two years , have now an approximate average of 60 daily snp designs ( and ) .
output with the graphic representation of snps with putative functional effect in the gene brca2 , along with ld maps .
 we have developed a web tool , pupasuite , for the selection of single nucleotide polymorphisms ( snps ) with potential phenotypic effect , specifically oriented to help in the design of large - scale genotyping projects . 
 pupasuite uses a collection of data on snps from heterogeneous sources and a large number of pre - calculated predictions to offer a flexible and intuitive interface for selecting an optimal set of snps . 
 it improves the functionality of pupasnp and pupasview programs and implements new facilities such as the analysis of user 's data to derive haplotypes with functional information . 
 a new estimator of putative effect of polymorphisms has been included that uses evolutionary information . 
 also snpeffect database predictions have been included . 
 the pupasuite web interface is accessible through   and through . 
INTRODUCTION
OUTLINE OF THE PROGRAM
CRITERIA TO SELECT SNPS AS A GOOD CANDIDATES FOR GENOTYPING
EVOLUTION AT WORK: THE SELECTIVE STRENGTHS ON CSNPS
SNPEFFECT DATABASE
FUNCTIONAL HAPLOTYPES
DISCUSSION
Figures and Tables