BACKGROUND
next-generation sequencing  technologies are widely used to answer key biological questions at the scale of the entire genome and with an unprecedented depth  <cit> .
whether determining genetic or genomic variations, cataloguing transcripts and assessing their expression levels, identifying dna-protein interactions or chromatin modifications, surveying the species diversity in an environmental sample, all these tasks are now tackled with large-scale sequencing and require computer intensive bioinformatic analyses  <cit> , although different.
identification of genetic variations can be addressed by whole genome sequencing  or whole exome sequencing  of single individuals.
wgs is particularly attractive because it allows to access the full spectrum of genetic variations, i.e.
coding and non coding single nucleotide variations  and short insertion-deletion variants , as well as copy number variants  and structural variants   <cit> .
in practice, out of major genome centers and a fortiori for the clinical routine translation, the development of this approach is still constrained by various difficulties such as the production organization, the yet expensive cost, the actual error rate of the technologies , the sheer volume of data to store and to transfer, requiring intensive informatics infrastructures and robust bioinformatics and filter procedures to retain only clinically relevant variants  <cit> .
as new genomes are sequenced, for example in the context of large projects like the  <dig> genomes project  <cit> , the number of expected variations may decrease.
but, first complete individual constitutional genome sequencing studies reported 3- <dig> million of snp per genome, 80-90% of which highly overlapped the national center for biotechnology information public snp database   <cit> , leaving anyway  <dig>  million novel variations to sift per genome  <cit> .
while wgs remains an appealing ultimate perspective, wes focusing on only the coding regions of the genome, has become in a few years the choice strategy to meet the challenge of identifying a coding allelic variant for rare human monogenic disorder  <cit> .
thanks to dna enrichment techniques, targeted sequencing of coding regions decreases the cost and improves the efficiency of large-scale coding variations discovery compared with what would require the entire human genome.
the human exome, made of ~ <dig>  exons for a size of ~ <dig> mbp, is  <dig> % of the total human genome.
thereby, not only targeted selection strategy reduces the cost but also accelerates the discovery of coding genetic variants that cause rare mendelian diseases.
in  <dig>  ng et al.
<cit> , by using an intersection recurrence strategy, showed the proof of the concept that identifying a gene responsible for a rare dominantly inherited disorder  was possible using wes of independant index cases.
since then, more and more papers confirmed the success of this strategy  <cit> .
up to now, classical approaches such as linkage analysis using genetic markers have been extensively used to identify the molecular basis for nearly  <dig>  mendelian disorders  <cit> .
but for over  <dig>  mendelian disorders, the gene remains unknown  <cit> .
the limited number of patients for rare diseases or the limited access to the related members of the family has been a frequent obstacle to conduct linkage analysis  <cit> .
as the ngs technologies have emerged, the long and fastidious classical linkage analysis for human mendelian disorders will be replaced by more direct identification of the causal variation and the corresponding gene.
moreover, in numerous cases there are no caryotypic nor cgh-array anomaly or negative result with sanger sequencing on known mutated genes or on neighbor genes in a pathway of interest, because of the low depth of this first generation sequencing technology  <cit> .
so, the exome-scale sequencing approach generates a technological breakthrough in medical genetics history in fundamental research for disease gene discovery and consequently in terms of new diagnostic methods and personalized medicine  <cit> .
numerous algorithms and software tools have been developed to efficiently manage terabytes of raw sequence variation data from wes.
commonly adopted variation discovery pipelines include successive bioinformatics steps for quality control of the short reads, alignment of the short reads to a reference sequence, variation calling and variation annotation  <cit> .
generally, ~ <dig>  variations per individual exome are obtained.
the challenge remains in efficient filtering strategies to find the causal variant and corresponding gene for a rare disease, among these thousands of candidates.
with this aim, additional analytical procedures which implicate various heuristic filtering strategies have emerged  <cit> .
usually, wide range common variations  are firstly excluded.
this is done by comparison to publicy available databases of human genetic variations and privately available variants from other exome sequencing projects.
to narrow down the search on remaining variations , other filters take into account the type of variations  and evaluate the functional effect of variations on gene products.
usually, various criteria are inspected for this task such as the physical properties of the wild-type and variant amino acids, the structural properties affecting protein dynamics and stability, the integrity of functional motifs and binding domains or sites implicated to posttranslational processing and cellular localization of proteins, evolutionary properties derived from a sequence alignment  <cit> .
beside these molecular nature and effects of the alternative allelic variants, filtering strategies also have to take into account the mode of inheritance of the disorder suggested by pedigree .
finally, taking advantages of multiple individuals, intersection or differential exome strategies can drastically reduce the remaining variations to several genes.
as the exome-scale sequencing is today positioned as a method of choice for disease gene discovery and personalized medicine, the success of the unavoidable filtering strategies of thousands variations lies in their implementation into convivial and versatile software tools.
end users with no computational skill have to be autonomous to conduct and combine themselves different filtering approaches, depending on their assumptions and of their study design, leading them to extract a limited list of likely candidate genes underlying a genetic disease.
with this aim, in partnership with and for medical geneticists, we developed eva , a user-friendly web-interfaced free software dedicated to filtering strategies for medical projects investigated with exome sequencing.
eva integrates the main filters dealing with common variations, molecular types, inheritance mode and multiple samples.
here we report a demonstrative case study with eva that allowed to identify a new candidate gene related to a rare form of alzeihmer disease  <cit> .
we discuss our development choices and the position of eva among other filtering tools recently published.
methods
implementation of eva
exomedb was developed under mysql .
the main tables are variation, gene and individual in which data are integrated from a list of variants  associated with their annotations .
currently, eva works with the ncbi  <dig>  build version of the human genome but integrates an archive for the previous version .
each new project is subject to a remote loading using an online variation integration module that accepts txt files and xls files.
the web interface was developed under php .
for the implemented filtering strategies , a combination of criteria selected by the user, is transformed into an sql query and sent to the exomedb database.
then, eva's interface displays the remaining variations in table browsers .
the variation statistics module proposes interactive bar and pie vizualizations of exome data implemented with the free javascript charting library highcharts .
to assure the confidentiality of the exome data, evas integrates an authentication module requiring a login and a password given by an administrator.
each login/password is strickly project specific.
users can only see and manage their own exome projects.
some performance statistics are described in the result section.
at the time of this writing, eva's interface is accessible at the web address http://plateforme-genomique-irib.univ-rouen.fr/eva/index.php through the described authentication process.
eva's current and update versions will be freely available under a creative commons attribution-noncommercial-noderivs  <dig>  unported license  and will be downloaded from the same web site.
data
the input file  of the variation integration module of eva is a list of variants  obtained from an independant variant calling procedure  and then annotated .
although actually, the format of these files is a proprietary format, it includes classical annotations for the detected variations and the affected genes.
for the detected variations main information are: the chromosome and the genomic position, the number of the read bases for each nucleotide, the reference base and the modified base deduced from an allelic count procedure and annotated with the genotype homozygosis or heterozygosis status, the number of total sequenced bases and the number of used bases for the detection variant, the score of the variation depending on the quality and coverage, the type of variation , the rs name if known in dbsnp , cigar format and length for the indels.
for the affected genes the main information are: the gene name , the ncbi refseq accession number  <cit>  for all mrna variants expressed by the gene, the type of affected position , 5' or 3' utr) and the corresponding number of the exon or intron along the gene structure, the functional categories of variations , the exon or intron start and stop positions included the variation and finally position of the variations in the corresponding protein sequence with the description of the codon and corresponding amino-acid for both the reference protein and the detected variations.
case study: the alzheimer disease
thanks to a nationwide recruitment ), exome sequencing was performed in fourteen autosomal dominant early-onset alzheimer disease  unrelated index cases without mutation on known genes , presenilin <dig> and  <dig> ) and also without known copy number variants of app gene and genes involved in amyloid beta  peptide processing or signaling.
integragen company  performed exome sequencing.
three micrograms of genomic dna from each individual, extracted from peripheral blood lymphocytes and sheared by sonication to obtain an average fragment size of 150- <dig> bp, were used for the construction of a shotgun sequencing library using paired-end adapters.
exome capture was performed using the sureselect human all exon kits  <dig> mb version  <dig>   or sureselect human all exon kits  <dig> mb version  <dig>  for a second batch .
sequencing was realised on an illumina genome analyser gaiix  or on an illumina hiseq  <dig> .
raw image files were processed by using the illumina pipeline .
for the genetics variant detection, the  <dig> bp sequencing reads were aligned to the ncbi human reference genome  or ncbi  <dig> ), using elandv <dig>  means coverage were of 65-fold  and 80-fold  with a percentage of aligned reads ranging between 88% and 95%.
only high quality variations having a qphred threshold >  <dig> were conserved .
the annotation procedure of the detected variations only concerned those included in the coordinates given by the exon kits capture extended to +/-  <dig> pb in the flanking intron.
the description of the annotated files is explained in the methods section, 'data' subheadings.
each annotated file corresponding to the project  was integrated in exomedb using the variation integration module of eva.
RESULTS
overview: exomedb and eva web interface
for a given wes project, corresponding to several individuals and their respective variations, eva manages data thanks to six modules .
in input, after authentication , an online variation integration module takes the variations files  obtained from an independant variant calling bioinformatics pipeline.
annotated variations are stored in a relational database exomedb which main tables are the annotated variations, the corresponding genes and the individuals .
the web interface integrates four other modules for exome mining.
the variation statistics module allows through a guided mode selection of individuals, chromosomes, genomic regions, genes, genic region, type of snv or indel, to summarize in tables and to graphically represent the global or selected distribution of variations of a given wes project .
the table browser module allows to precisely explore data by project, individual, gene or variation through rigorous and sortable categorized tables   the search module can be used for a direct and quick access to a specific region, gene, variation for a given project, and finally  the filtering strategy module, which is the major element in exome mining to discover potential canditate genes, offers to combine filters for common variations, molecular types, inheritance mode and multiple samples to drastically narrow down variations .
the selected combination is transformed into a sql query and sent to the exomedb database.
query results of the table browser module, quick search module and filtering strategy module can be explored by five elements types presented in interactive tables: 'variation overview' , 'gene list', 'gene details' , 'variation list'  and 'variation details' .
in the 'variation overview' tables , the set of all the variations is divided in known and unknown variations according to the information in dbsnp.
due to the molecular process of the exome capture kit, most variations occur in exons but some detected variations also occur in splice sites.
even if exomedb integrates variations extended to +/-  <dig> pb in the flanking intron, we choose to show on the table only variations extended to +/-  <dig> pb in the intron, corresponding to the dinucleotide splicing site.
variations in exons can be snv or indels.
we categorized single variations into four functional classes: synonymous, miss sense, stop loss and non sense.
for indels we classified into two categories: frameshift or non frameshift.
in output, eva offers export files .
eva also provides several cross-links with a selection of relevant external international databases and softwares for further functional and pathogenic effect inspection of the sorting variation and gene candidates .
filtering strategy module
the filtering strategy module integrates the current main categories of filters based on common variations, molecular type of the variants, modes of inheritance, homozygous or heterozygous nature of the allelic variant and multiple individuals.
first, eva compares the data to international catalogues of variations.
in a constitutive sorting, the set of all the variations is divided in known and unknown variations according to the information in the dbsnp .
eva also offers to reduce the number of variations by confronting them to the hapmap project  <cit> , the  <dig> genomes project  <cit> , complete genomics public data  <cit> , integragen public data  <cit>  or the exome sequencing project  <cit> .
in addition, other filters or table browsers offer to sift variations depending on their:  functional categories for snv  and indels ;  genic region  or genomic region;  quality score and coverage.
finally, one of the strengths of eva is the implementation of inheritance filters considering intersection or conversely differential exome strategies:  recurrence strategy for dominant or recessive independent familial cases ;  filters for homozygous, heterozygous or composite cases in intra-familial studies ;  and de novo strategy for sporadic cases .
for each strategy the displayed result is a list of potential candidate genes associated with the number of affected individuals .
again, it consists on an interactive table that could be readily explored.
the user can get 'gene details'  containing interactive links to other tables 'variation overview' , 'variation list' , and 'variation details' .
to ensure a rapid execution of eva  implemented in priority to focus on filtering strategies, we made the choice not to include variant effect prediction functionalities.
nevertheless, to facilitate the further prioritization of remained variations and genes, external functional and pathogenicity interpretation tools  are cross-linked as well as useful external international databases of genes, proteins, pathways, diseases and literature and genome browsers.
case study: alzheimer disease
after screening more than one hundred autosomal dominant early-onset alzheimer disease  families for known mutations  the molecular basis of this rare disorder still remained unexplained in several of them.
moreover, the lack of dna for affected relatives precluding a linkage analysis in these cases, a full exome sequencing strategy was decided to identify new candidate gene with unknown mutations.
exome sequencing, variation detection and annotation were performed by integragen company  for fourteen adeoad unrelated index cases.
the annotated variant files were subjected to exomedb to a remote loading using the online variation integration module of eva.
then, the intersection recurrence filtering strategy was applied with eva.
here the main steps of our filtering procedure are summarized:
firstly we displayed the full project data.
figure  <dig> corresponds to the raw 'variation overview' of this exome project integrated in eva and is obtained with the table browser module.
in this interactive table, variations are displayed by individuals and divided into two groups on the dbsnp <dig> referencing basis.
'known' means variations referenced in dbsnp, while 'unknown' means variations not referenced in dbsnp.
within those groups the variations are rigourously and usefully displayed by two functional classes 'exon' and 'intron' ).
exonic variations are classified into six sub-categories, 'synonym', 'missense', 'stop loss' and 'nonsense' for snv and frameshift  and no frameshift  for indels.
in total,  <dig>   to  <dig>   genetic variants were identified per exome according to the capture protocol .
among these,  <dig> % in average are unknown variations .
secondly, thanks to the filtering strategy module we applied a stringent primary screening based on .
figure  <dig> corresponds to the 'variation overview' after this one: variations retained were previously 'unknown'  but then filtered against hapmap exome projects, and against  <dig> integragen exome projects from unrelated individuals with non-neurodegenerative diseases, the other filters parameters were 'non-synonym' snv, 'frameshift coding' indels, 'splice acceptor and donor site' and 'heterozygous'.
finally, the number of unknown variations by individual drastically decreases from  <dig> in average for batch # <dig> and  <dig> in average for batch # <dig>  to  <dig> and  <dig> respectively.
so, remaining unknown variations after this primary screening with eva represented only 2% of total genetic variants identified per exome versus  <dig> % in the raw data.
thirdly, a secondary screening of the remaining variations based on the inheritance mode assumption of the disease was applied with an intersection recurrence procedure.
table  <dig> summarizes the number of genes harboring at least one of these variants classified according to their recurrence in the patient sample.
the  <dig> patients did not have in common a single altered gene, indicating that, within this sample, the disease was genetically heterogeneous.
nevertheless, we observed that the number of candidate genes drastically decreased with the increasing number of concerned individuals.
so, eva enabled geneticists to focus further investigations on the affected genes shared by a minimum of  <dig> patients, representing a short list of less than  <dig> genes.
number of
with remaining
finally, after wet investigations  combined with in silico analysis , one gene  containing unknown mutations in 5/ <dig> exomes  or missense ) has become a new strong candidate gene for the adeoad  <cit> .
performance
to date, exomedb stores wgs projects  corresponding to a total of  <dig> individuals and contains also targeted resequencing projects corresponding to  <dig> genes for  <dig> individuals.
as showed on table  <dig>  the size of exomedb is about  <dig> mb, mainly due to the tables variation  and individual_variation .
tests of eva have been performed on the adeoad exome project  with one user logged in.
the server is running linux with four  <dig> ghz processors,  <dig> gb ram and  <dig> gb hd.
we use the "mysqli::prepare" mode which speeds up the request time once the first request has been treated.
table  <dig> shows request times in both cases.
while it is clear that performances depend on the number of users logged in simultaneously and on the number of variants in the database , eva works with a reasonable time of execution compatible with the regular needs of a medical genetics laboratory.
tests of eva have been performed on the adeoad exome project  with one user logged in.
CONCLUSIONS
eva is developed to be a user-friendly, versatile, efficient-filtering and free assisting software for whole exome sequencing, providing a response to new needs at the expanding era of medical genomics investigated by these targeted next-generation sequencing technologies, for fundamental research, clinical diagnostics and personalized medicine  <cit> .
interfacing various now commonly adopted filtering criteria and strategies on whole exome data, eva thereby makes non-programmer medical geneticists autonomous to pinpoint themselves among ~ <dig>  variations per individual exome, few candidate variations and genes related to a rare disease, depending of their specific assumptions and study design.
eva constitutes a platform for exome sequencing data storage and for drastic screening of clinical relevant genetics variations.
thanks to different modules  it integrates and stores annotated exome variation data as strictly confidential to the project owner,  for the analytical process, it proposes to combine the main filters dealing with common human variations , homozygous or heterozygous nature of the allelic variant, inheritance modes and multiple samples considering intersection or conversely differential exome strategies , quality of the variations  it offers quick searching or advanced browsing of annotated data and filtered results thanks to various interactive categorized or sortable tables and useful graphical visualizations  finally it offers export files and cross-links to external relevant databases and softwares for further functional effects inspection  <cit>  of the small subset of sorted candidate variations and genes.
eva has been used to successfully identify a new candidate gene, sorl <dig>  related to a rare form of alzeihmer disease , despite a genetics heterogeneity  <cit> .
sorl <dig> encodes the sortilin-related receptor lr11/sorla, a protein involved in the control of amyloid beta peptide production, the same pathway as previously known genes app, and presenilin  <dig> and  <dig>  in this case study, the primary screening with eva  reduced unknown variations to only 2%  of total genetic variants identified per exome.
the secondary screening implementing the intersection recurrence strategy led to a short list of genes  on which geneticists focused for further in silico and wet experiments and among which they discovered one.
in  <dig> patients of the  <dig> independant index cases investigated, we found that the sorl <dig> gene harbored unknown nonsense  or missense  mutations.
performance tests showed that eva run with a reasonable time of execution compatible with the regular needs of a medical genetics laboratory.
for the case study it takes between  <dig> s  to  <dig> s  to load and execute the selected filters  from the currently  <dig> mb size of exomedb.
the commonly assumption for wes mining is that causal variants related to a mendelian disorder under investigation will not be present in public databases of genetic variations or other exome sequencing projects  <cit> .
that is why, the more variation data available the more the filtering strategies in exome mining would be successful.
to enhance its filtering performances, eva confronts exome data currently to  <dig> external public data  <cit>  and will be regularly updating as new large-scale variations data will be published.
some polymorphisms of these ressources  are not associated with their allelic frequency and lack experimental annotation of their functional impact.
so, projects like the snp database of effects   <cit> , storing computationally annotated functional impacts of non synonymous snps or the annotation of  <dig> top human cancer genes frequently mutated  <cit>  could be of interest for eva improvement.
alternative tools designed for the similar task as eva have been recently published  <cit> .
varsifter  <cit>  is a graphical java program for desktop computers.
it is designed to read exome-scale variation data in either a tab-delimited text file with header, or an uncompressed vcf file.
it proposes numerous filtering options but doesn't propose graphic visualization nor statistical summaries of a wes project.
sva  <cit>  is largely based on a genome browser to deal with wge as well as wes and sifts small and large variants.
while it proposes many manipulations of data, it is not clear if inherihance filtering are implemented.
more, sva is a java program requiring a recommend hardware equipped with at least  <dig> gb of ram and 1tb of free hard disk, which are substantial computational resources, in practice not very compatible to all individual laboratory.
finally, var-md  <cit>  is a family based tool.
it analyzes wgs and wes variants exclusively in small human pedigrees with mendelian inheritance excluding the scope of the differential exome analysis.
as perspectives are concerned, the input format for eva for the variation integration module, which is currently a proprietary format will be soon standardized in order to offer a wide use of this tool; we retained the variant call format  format, generated by the  <dig> genomes project.
the variation integration modulewill also allow the annotation of the raw variations by both annovar  <cit>  and the variant effect predictor ensembl api  <cit> .
regular updates are made concerning build version of the human genome, international variation catalogues and improvement of filtering functionalities as well as organization of results tables and graphics.
future developments include a graphical representation of a candidat gene with its variations and a more specific filtering strategy for somatic mutations.
list of abbreviations used
adeoad: autosomal dominant early-onset alzheimer disease; wgs: whole genome sequencing; wes: whole exome sequencing; snv: single nucleotide variation.
competing interests
the authors declare that they have no competing interests.
authors' contributions
dc expressed the need of the tools, supervised the project and its design, oriented the filtering strategy functionality and realized the case study.
sc realized the implementation of eva, co-realized the case study, contributed to the manuscript.
al, ml, epg and tl co-supervised the implementation of exomedb and the eva interface and contributed to the manuscript.
cc implemented the variation statistics module and contributed to the manuscript.
hd co-supervised the project and its design, oriented the categorization of variations and the different views of results on interface.
hd and tl coordonned the collaboration between the two teams inserm u <dig>  and tibs-litis  and wrote the paper.
acknowledgements and funding
this work has been partially supported by grant phrc gmaj, centre national de référence malades alzheimer jeunes.
the authors thank the litis for the host of eva on one of its server.
this article has been published as part of bmc bioinformatics volume  <dig> supplement  <dig>  2012: selected articles from research from the eleventh international workshop on network tools and applications in biology .
the full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/13/s14

 next-generation sequencing technologies are widely used to answer key biological questions at the scale of the entire genome and with an unprecedented depth. whether determining genetic or genomic variations, cataloguing transcripts and assessing their expression levels, identifying dna-protein interactions or chromatin modifications, surveying the species diversity in an environmental sample, all these tasks are now tackled with large-scale sequencing and require computer intensive bioinformatic analyses, although different. identification of genetic variations can be addressed by whole genome sequencing or whole exome sequencing of single individuals. wgs is particularly attractive because it allows to access the full spectrum of genetic variations, i.e. coding and non coding single nucleotide variations and short insertion-deletion variants, as well as copy number variants and structural variants. in practice, out of major genome centers and a fortiori for the clinical routine translation, the development of this approach is still constrained by various difficulties such as the production organization, the yet expensive cost, the actual error rate of the technologies, the sheer volume of data to store and to transfer, requiring intensive informatics infrastructures and robust bioinformatics and filter procedures to retain only clinically relevant variants. as new genomes are sequenced, for example in the context of large projects like the genomes project, the number of expected variations may decrease. but, first complete individual constitutional genome sequencing studies reported 3- million of snp per genome, 80-90% of which highly overlapped the national center for biotechnology information public snp database, leaving anyway million novel variations to sift per genome. while wgs remains an appealing ultimate perspective, wes focusing on only the coding regions of the genome, has become in a few years the choice strategy to meet the challenge of identifying a coding allelic variant for rare human monogenic disorder. thanks to dna enrichment techniques, targeted sequencing of coding regions decreases the cost and improves the efficiency of large-scale coding variations discovery compared with what would require the entire human genome. the human exome, made of ~ exons for a size of ~ mbp, is % of the total human genome. thereby, not only targeted selection strategy reduces the cost but also accelerates the discovery of coding genetic variants that cause rare mendelian diseases. in ng et al., by using an intersection recurrence strategy, showed the proof of the concept that identifying a gene responsible for a rare dominantly inherited disorder was possible using wes of independant index cases. since then, more and more papers confirmed the success of this strategy. up to now, classical approaches such as linkage analysis using genetic markers have been extensively used to identify the molecular basis for nearly mendelian disorders. but for over mendelian disorders, the gene remains unknown. the limited number of patients for rare diseases or the limited access to the related members of the family has been a frequent obstacle to conduct linkage analysis. as the ngs technologies have emerged, the long and fastidious classical linkage analysis for human mendelian disorders will be replaced by more direct identification of the causal variation and the corresponding gene. moreover, in numerous cases there are no caryotypic nor cgh-array anomaly or negative result with sanger sequencing on known mutated genes or on neighbor genes in a pathway of interest, because of the low depth of this first generation sequencing technology. so, the exome-scale sequencing approach generates a technological breakthrough in medical genetics history in fundamental research for disease gene discovery and consequently in terms of new diagnostic methods and personalized medicine. numerous algorithms and software tools have been developed to efficiently manage terabytes of raw sequence variation data from wes. commonly adopted variation discovery pipelines include successive bioinformatics steps for quality control of the short reads, alignment of the short reads to a reference sequence, variation calling and variation annotation. generally, ~ variations per individual exome are obtained. the challenge remains in efficient filtering strategies to find the causal variant and corresponding gene for a rare disease, among these thousands of candidates. with this aim, additional analytical procedures which implicate various heuristic filtering strategies have emerged. usually, wide range common variations are firstly excluded. this is done by comparison to publicy available databases of human genetic variations and privately available variants from other exome sequencing projects. to narrow down the search on remaining variations, other filters take into account the type of variations and evaluate the functional effect of variations on gene products. usually, various criteria are inspected for this task such as the physical properties of the wild-type and variant amino acids, the structural properties affecting protein dynamics and stability, the integrity of functional motifs and binding domains or sites implicated to posttranslational processing and cellular localization of proteins, evolutionary properties derived from a sequence alignment. beside these molecular nature and effects of the alternative allelic variants, filtering strategies also have to take into account the mode of inheritance of the disorder suggested by pedigree. finally, taking advantages of multiple individuals, intersection or differential exome strategies can drastically reduce the remaining variations to several genes. as the exome-scale sequencing is today positioned as a method of choice for disease gene discovery and personalized medicine, the success of the unavoidable filtering strategies of thousands variations lies in their implementation into convivial and versatile software tools. end users with no computational skill have to be autonomous to conduct and combine themselves different filtering approaches, depending on their assumptions and of their study design, leading them to extract a limited list of likely candidate genes underlying a genetic disease. with this aim, in partnership with and for medical geneticists, we developed eva, a user-friendly web-interfaced free software dedicated to filtering strategies for medical projects investigated with exome sequencing. eva integrates the main filters dealing with common variations, molecular types, inheritance mode and multiple samples. here we report a demonstrative case study with eva that allowed to identify a new candidate gene related to a rare form of alzeihmer disease. we discuss our development choices and the position of eva among other filtering tools recently published. methods implementation of eva exomedb was developed under mysql. the main tables are variation, gene and individual in which data are integrated from a list of variants associated with their annotations. currently, eva works with the ncbi build version of the human genome but integrates an archive for the previous version. each new project is subject to a remote loading using an online variation integration module that accepts txt files and xls files. the web interface was developed under php. for the implemented filtering strategies, a combination of criteria selected by the user, is transformed into an sql query and sent to the exomedb database. then, eva's interface displays the remaining variations in table browsers. the variation statistics module proposes interactive bar and pie vizualizations of exome data implemented with the free javascript charting library highcharts. to assure the confidentiality of the exome data, evas integrates an authentication module requiring a login and a password given by an administrator. each login/password is strickly project specific. users can only see and manage their own exome projects. some performance statistics are described in the result section. at the time of this writing, eva's interface is accessible at the web address http://plateforme-genomique-irib.univ-rouen.fr/eva/index.php through the described authentication process. eva's current and update versions will be freely available under a creative commons attribution-noncommercial-noderivs unported license and will be downloaded from the same web site. data the input file of the variation integration module of eva is a list of variants obtained from an independant variant calling procedure and then annotated. although actually, the format of these files is a proprietary format, it includes classical annotations for the detected variations and the affected genes. for the detected variations main information are: the chromosome and the genomic position, the number of the read bases for each nucleotide, the reference base and the modified base deduced from an allelic count procedure and annotated with the genotype homozygosis or heterozygosis status, the number of total sequenced bases and the number of used bases for the detection variant, the score of the variation depending on the quality and coverage, the type of variation, the rs name if known in dbsnp, cigar format and length for the indels. for the affected genes the main information are: the gene name, the ncbi refseq accession number for all mrna variants expressed by the gene, the type of affected position, 5' or 3' utr) and the corresponding number of the exon or intron along the gene structure, the functional categories of variations, the exon or intron start and stop positions included the variation and finally position of the variations in the corresponding protein sequence with the description of the codon and corresponding amino-acid for both the reference protein and the detected variations. case study: the alzheimer disease thanks to a nationwide recruitment ), exome sequencing was performed in fourteen autosomal dominant early-onset alzheimer disease unrelated index cases without mutation on known genes, presenilin and ) and also without known copy number variants of app gene and genes involved in amyloid beta peptide processing or signaling. integragen company performed exome sequencing. three micrograms of genomic dna from each individual, extracted from peripheral blood lymphocytes and sheared by sonication to obtain an average fragment size of 150- bp, were used for the construction of a shotgun sequencing library using paired-end adapters. exome capture was performed using the sureselect human all exon kits mb version or sureselect human all exon kits mb version for a second batch. sequencing was realised on an illumina genome analyser gaiix or on an illumina hiseq. raw image files were processed by using the illumina pipeline. for the genetics variant detection, the bp sequencing reads were aligned to the ncbi human reference genome or ncbi ), using elandv means coverage were of 65-fold and 80-fold with a percentage of aligned reads ranging between 88% and 95%. only high quality variations having a qphred threshold > were conserved. the annotation procedure of the detected variations only concerned those included in the coordinates given by the exon kits capture extended to +/- pb in the flanking intron. the description of the annotated files is explained in the methods section,'data' subheadings. each annotated file corresponding to the project was integrated in exomedb using the variation integration module of eva.  overview: exomedb and eva web interface for a given wes project, corresponding to several individuals and their respective variations, eva manages data thanks to six modules. in input, after authentication, an online variation integration module takes the variations files obtained from an independant variant calling bioinformatics pipeline. annotated variations are stored in a relational database exomedb which main tables are the annotated variations, the corresponding genes and the individuals. the web interface integrates four other modules for exome mining. the variation statistics module allows through a guided mode selection of individuals, chromosomes, genomic regions, genes, genic region, type of snv or indel, to summarize in tables and to graphically represent the global or selected distribution of variations of a given wes project. the table browser module allows to precisely explore data by project, individual, gene or variation through rigorous and sortable categorized tables the search module can be used for a direct and quick access to a specific region, gene, variation for a given project, and finally the filtering strategy module, which is the major element in exome mining to discover potential canditate genes, offers to combine filters for common variations, molecular types, inheritance mode and multiple samples to drastically narrow down variations. the selected combination is transformed into a sql query and sent to the exomedb database. query results of the table browser module, quick search module and filtering strategy module can be explored by five elements types presented in interactive tables:'variation overview','gene list','gene details','variation list' and'variation details'. in the'variation overview' tables, the set of all the variations is divided in known and unknown variations according to the information in dbsnp. due to the molecular process of the exome capture kit, most variations occur in exons but some detected variations also occur in splice sites. even if exomedb integrates variations extended to +/- pb in the flanking intron, we choose to show on the table only variations extended to +/- pb in the intron, corresponding to the dinucleotide splicing site. variations in exons can be snv or indels. we categorized single variations into four functional classes: synonymous, miss sense, stop loss and non sense. for indels we classified into two categories: frameshift or non frameshift. in output, eva offers export files. eva also provides several cross-links with a selection of relevant external international databases and softwares for further functional and pathogenic effect inspection of the sorting variation and gene candidates. filtering strategy module the filtering strategy module integrates the current main categories of filters based on common variations, molecular type of the variants, modes of inheritance, homozygous or heterozygous nature of the allelic variant and multiple individuals. first, eva compares the data to international catalogues of variations. in a constitutive sorting, the set of all the variations is divided in known and unknown variations according to the information in the dbsnp. eva also offers to reduce the number of variations by confronting them to the hapmap project, the genomes project, complete genomics public data, integragen public data or the exome sequencing project. in addition, other filters or table browsers offer to sift variations depending on their: functional categories for snv and indels; genic region or genomic region; quality score and coverage. finally, one of the strengths of eva is the implementation of inheritance filters considering intersection or conversely differential exome strategies: recurrence strategy for dominant or recessive independent familial cases; filters for homozygous, heterozygous or composite cases in intra-familial studies; and de novo strategy for sporadic cases. for each strategy the displayed result is a list of potential candidate genes associated with the number of affected individuals. again, it consists on an interactive table that could be readily explored. the user can get'gene details' containing interactive links to other tables'variation overview','variation list', and'variation details'. to ensure a rapid execution of eva implemented in priority to focus on filtering strategies, we made the choice not to include variant effect prediction functionalities. nevertheless, to facilitate the further prioritization of remained variations and genes, external functional and pathogenicity interpretation tools are cross-linked as well as useful external international databases of genes, proteins, pathways, diseases and literature and genome browsers. case study: alzheimer disease after screening more than one hundred autosomal dominant early-onset alzheimer disease families for known mutations the molecular basis of this rare disorder still remained unexplained in several of them. moreover, the lack of dna for affected relatives precluding a linkage analysis in these cases, a full exome sequencing strategy was decided to identify new candidate gene with unknown mutations. exome sequencing, variation detection and annotation were performed by integragen company for fourteen adeoad unrelated index cases. the annotated variant files were subjected to exomedb to a remote loading using the online variation integration module of eva. then, the intersection recurrence filtering strategy was applied with eva. here the main steps of our filtering procedure are summarized: firstly we displayed the full project data. figure corresponds to the raw'variation overview' of this exome project integrated in eva and is obtained with the table browser module. in this interactive table, variations are displayed by individuals and divided into two groups on the dbsnp referencing basis.'known' means variations referenced in dbsnp, while'unknown' means variations not referenced in dbsnp. within those groups the variations are rigourously and usefully displayed by two functional classes'exon' and'intron' ). exonic variations are classified into six sub-categories,'synonym','missense','stop loss' and'nonsense' for snv and frameshift and no frameshift for indels. in total, to genetic variants were identified per exome according to the capture protocol. among these, % in average are unknown variations. secondly, thanks to the filtering strategy module we applied a stringent primary screening based on. figure corresponds to the'variation overview' after this one: variations retained were previously'unknown' but then filtered against hapmap exome projects, and against integragen exome projects from unrelated individuals with non-neurodegenerative diseases, the other filters parameters were'non-synonym' snv,'frameshift coding' indels,'splice acceptor and donor site' and'heterozygous'. finally, the number of unknown variations by individual drastically decreases from in average for batch # and in average for batch # to and respectively. so, remaining unknown variations after this primary screening with eva represented only 2% of total genetic variants identified per exome versus % in the raw data. thirdly, a secondary screening of the remaining variations based on the inheritance mode assumption of the disease was applied with an intersection recurrence procedure. table summarizes the number of genes harboring at least one of these variants classified according to their recurrence in the patient sample. the patients did not have in common a single altered gene, indicating that, within this sample, the disease was genetically heterogeneous. nevertheless, we observed that the number of candidate genes drastically decreased with the increasing number of concerned individuals. so, eva enabled geneticists to focus further investigations on the affected genes shared by a minimum of patients, representing a short list of less than genes. number of with remaining finally, after wet investigations combined with in silico analysis, one gene containing unknown mutations in 5/ exomes or missense ) has become a new strong candidate gene for the adeoad. performance to date, exomedb stores wgs projects corresponding to a total of individuals and contains also targeted resequencing projects corresponding to genes for individuals. as showed on table the size of exomedb is about mb, mainly due to the tables variation and individual_variation. tests of eva have been performed on the adeoad exome project with one user logged in. the server is running linux with four ghz processors, gb ram and gb hd. we use the "mysqli::prepare" mode which speeds up the request time once the first request has been treated. table shows request times in both cases. while it is clear that performances depend on the number of users logged in simultaneously and on the number of variants in the database, eva works with a reasonable time of execution compatible with the regular needs of a medical genetics laboratory. tests of eva have been performed on the adeoad exome project with one user logged in.  eva is developed to be a user-friendly, versatile, efficient-filtering and free assisting software for whole exome sequencing, providing a response to new needs at the expanding era of medical genomics investigated by these targeted next-generation sequencing technologies, for fundamental research, clinical diagnostics and personalized medicine. interfacing various now commonly adopted filtering criteria and strategies on whole exome data, eva thereby makes non-programmer medical geneticists autonomous to pinpoint themselves among ~ variations per individual exome, few candidate variations and genes related to a rare disease, depending of their specific assumptions and study design. eva constitutes a platform for exome sequencing data storage and for drastic screening of clinical relevant genetics variations. thanks to different modules it integrates and stores annotated exome variation data as strictly confidential to the project owner, for the analytical process, it proposes to combine the main filters dealing with common human variations, homozygous or heterozygous nature of the allelic variant, inheritance modes and multiple samples considering intersection or conversely differential exome strategies, quality of the variations it offers quick searching or advanced browsing of annotated data and filtered results thanks to various interactive categorized or sortable tables and useful graphical visualizations finally it offers export files and cross-links to external relevant databases and softwares for further functional effects inspection of the small subset of sorted candidate variations and genes. eva has been used to successfully identify a new candidate gene, sorl related to a rare form of alzeihmer disease, despite a genetics heterogeneity. sorl encodes the sortilin-related receptor lr11/sorla, a protein involved in the control of amyloid beta peptide production, the same pathway as previously known genes app, and presenilin and in this case study, the primary screening with eva reduced unknown variations to only 2% of total genetic variants identified per exome. the secondary screening implementing the intersection recurrence strategy led to a short list of genes on which geneticists focused for further in silico and wet experiments and among which they discovered one. in patients of the independant index cases investigated, we found that the sorl gene harbored unknown nonsense or missense mutations. performance tests showed that eva run with a reasonable time of execution compatible with the regular needs of a medical genetics laboratory. for the case study it takes between s to s to load and execute the selected filters from the currently mb size of exomedb. the commonly assumption for wes mining is that causal variants related to a mendelian disorder under investigation will not be present in public databases of genetic variations or other exome sequencing projects. that is why, the more variation data available the more the filtering strategies in exome mining would be successful. to enhance its filtering performances, eva confronts exome data currently to external public data and will be regularly updating as new large-scale variations data will be published. some polymorphisms of these ressources are not associated with their allelic frequency and lack experimental annotation of their functional impact. so, projects like the snp database of effects, storing computationally annotated functional impacts of non synonymous snps or the annotation of top human cancer genes frequently mutated could be of interest for eva improvement. alternative tools designed for the similar task as eva have been recently published. varsifter is a graphical java program for desktop computers. it is designed to read exome-scale variation data in either a tab-delimited text file with header, or an uncompressed vcf file. it proposes numerous filtering options but doesn't propose graphic visualization nor statistical summaries of a wes project. sva is largely based on a genome browser to deal with wge as well as wes and sifts small and large variants. while it proposes many manipulations of data, it is not clear if inherihance filtering are implemented. more, sva is a java program requiring a recommend hardware equipped with at least gb of ram and 1tb of free hard disk, which are substantial computational resources, in practice not very compatible to all individual laboratory. finally, var-md is a family based tool. it analyzes wgs and wes variants exclusively in small human pedigrees with mendelian inheritance excluding the scope of the differential exome analysis. as perspectives are concerned, the input format for eva for the variation integration module, which is currently a proprietary format will be soon standardized in order to offer a wide use of this tool; we retained the variant call format format, generated by the genomes project. the variation integration modulewill also allow the annotation of the raw variations by both annovar and the variant effect predictor ensembl api. regular updates are made concerning build version of the human genome, international variation catalogues and improvement of filtering functionalities as well as organization of results tables and graphics. future developments include a graphical representation of a candidat gene with its variations and a more specific filtering strategy for somatic mutations. list of abbreviations used adeoad: autosomal dominant early-onset alzheimer disease; wgs: whole genome sequencing; wes: whole exome sequencing; snv: single nucleotide variation. competing interests the authors declare that they have no competing interests. authors' contributions dc expressed the need of the tools, supervised the project and its design, oriented the filtering strategy functionality and realized the case study. sc realized the implementation of eva, co-realized the case study, contributed to the manuscript. al, ml, epg and tl co-supervised the implementation of exomedb and the eva interface and contributed to the manuscript. cc implemented the variation statistics module and contributed to the manuscript. hd co-supervised the project and its design, oriented the categorization of variations and the different views of results on interface. hd and tl coordonned the collaboration between the two teams inserm u and tibs-litis and wrote the paper. acknowledgements and funding this work has been partially supported by grant phrc gmaj, centre national de référence malades alzheimer jeunes. the authors thank the litis for the host of eva on one of its server. this article has been published as part of bmc bioinformatics volume supplement 2012: selected articles from research from the eleventh international workshop on network tools and applications in biology. the full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/13/s14
thereby, it provides a response to new needs at the expanding era of medical genomics investigated by wes for both fundamental research and clinical diagnostics. it constitutes a platform for data storage and for drastic screening of clinical relevant genetics variations by non-programmer geneticists. thanks to different modules, eva integrates and stores annotated exome variation data as strictly confidential to the project owner, allows to combine the main filters dealing with common variations, molecular types, inheritance mode and multiple samples, offers the browsing of annotated data and filtered results in various interactive tables, graphical visualizations and statistical charts, and finally offers export files and cross-links to external useful databases and softwares for further prioritization of the small subset of sorted candidate variations and genes. this approach is a revolution in medical genetics history, impacting both fundamental research, and diagnostic methods leading to personalized medicine. to deal with the expansion of wes in medical genomics individual laboratories, new convivial and versatile software tools have to implement these filtering steps. we report a demonstrative case study that allowed to identify a new candidate gene related to a rare form of alzheimer disease. for this purpose, commonly adopted procedures which implicate various filtering strategies have emerged: exclusion of common variations, type of the allelics variants, pathogenicity effect prediction, modes of inheritance and multiple individuals for exome comparison. whole exome sequencing has become the strategy of choice to identify a coding allelic variant for a rare human monogenic disorder. they generally lead to ~ variations that have to be narrow down to find the potential pathogenic allelic variant and the affected gene. non-programmer biologists have to be autonomous combining themselves different filtering criteria and conduct a personal strategy depending on their assumptions and study design.
 whole exome sequencing has become the strategy of choice to identify a coding allelic variant for a rare human monogenic disorder. this approach is a revolution in medical genetics history, impacting both fundamental research, and diagnostic methods leading to personalized medicine. a plethora of efficient algorithms has been developed to ensure the variant discovery. they generally lead to ~ variations that have to be narrow down to find the potential pathogenic allelic variant and the affected gene. for this purpose, commonly adopted procedures which implicate various filtering strategies have emerged: exclusion of common variations, type of the allelics variants, pathogenicity effect prediction, modes of inheritance and multiple individuals for exome comparison. to deal with the expansion of wes in medical genomics individual laboratories, new convivial and versatile software tools have to implement these filtering steps. non-programmer biologists have to be autonomous combining themselves different filtering criteria and conduct a personal strategy depending on their assumptions and study design.  we describe eva, a user-friendly web-interfaced software dedicated to the filtering strategies for medical wes. thanks to different modules, eva integrates and stores annotated exome variation data as strictly confidential to the project owner, allows to combine the main filters dealing with common variations, molecular types, inheritance mode and multiple samples, offers the browsing of annotated data and filtered results in various interactive tables, graphical visualizations and statistical charts, and finally offers export files and cross-links to external useful databases and softwares for further prioritization of the small subset of sorted candidate variations and genes. we report a demonstrative case study that allowed to identify a new candidate gene related to a rare form of alzheimer disease.  eva is developed to be a user-friendly, versatile, and efficient-filtering assisting software for wes. it constitutes a platform for data storage and for drastic screening of clinical relevant genetics variations by non-programmer geneticists. thereby, it provides a response to new needs at the expanding era of medical genomics investigated by wes for both fundamental research and clinical diagnostics. 12- october nettab workshop on clinical bioinformatics pavia, italy 