expressed sequence tags ( est ) represent short , unedited , randomly selected single - pass sequence reads derived from cdna libraries , providing a low - cost alternative ( also called poor man 's genome ) to whole genome sequencing ( 1,2 ) and specifically relevant to the transcriptome of an organism at various stages of development or under different experimental conditions . the analysis of est data can enable gene discovery , complement genome annotation , aid gene structure identification , establish the viability of alternative transcripts , guide single nucleotide polymorphism ( snp ) characterization and facilitate proteomic exploration ( 2 ) . ests are highly error prone and require several computational methods for pre - processing , clustering , assembly and annotation to yield biological information . furthermore , it is extremely important to be able to store , organize and annotate ests using a comprehensive analysis pipeline due to their  high - throughput nature . we recently compared ( 2 ) available web resources ( http://biolinfo.org/est/ ) , individual tools and pipelines pertaining to est analysis . we also evaluated currently available methods for each step of analysis , including est clustering , assembly , consensus generation and tools for dna and protein annotation , employing benchmark est datasets . a detailed investigation of different est analysis platforms ( 38 ) revealed that they all terminate prior to functional annotations , such as gene ontologies , motif / pattern analysis and pathway mapping . some platforms terminate at the assembly level , providing contigs and singletons as an output ( 3 ) . other platforms solely run nucleotide - based programs with limited annotation at the protein level ( 5,7,9,10 ) . therefore , we developed estexplorer , a complete est analysis suite which employs programs for both nucleotide- and protein - based annotation . moreover , we have carefully selected the most appropriate combination of programs for each stage of est analysis , based on their ability to accurately reproduce partial gene sequences from ests and annotate them as correctly as possible ( http://estexplorer.biolinfo.org/methodology.html ) . estexplorer comprises a suite of programs with a customizable web interface to manage and analyse est data . optionally , est assembly datasets generated elsewhere , e.g. egassembler ( 3 ) , can be further functionally annotated at the estexplorer website . besides pre - processing and assembly from est sequences , estexplorer annotates input sequences extensively , using gene ontologies ( go ) , domain analysis and pathway mapping . estexplorer has been used extensively for the analysis and annotation of large est datasets from parasitic nematodes generated in our laboratories , and to identify key nematode molecules as potential targets for anti - parasite intervention . estexplorer has been also used for the analysis of differential transcription between adult male and female haemonchus contortus by oligonucleotide microarrays ( unpublished data ) . the estexplorer workflow can be divided into three phases ( shown in supplementary figure 1 ) . phase i is dedicated to est sequence pre - processing and assembly , phase ii carries out dna- level annotation and phase iii provides for protein - level annotation . estexplorer can accept nucleotide sequence input of two types ( figure 1a ; arrows in supplementary figure 1 ) . ests in fasta format can be submitted to phase i for est pre - processing and assembly , followed by analyses in phases ii and iii . alternatively , ests assembled using another program or pipeline into contigs and singletons , may be submitted directly for functional annotation ( phases ii and iii ) . ( a ) est or contig submission with optional parameters for organism and program selection , ( b ) status page to monitor the progress of different programs and ( c ) result download page from where processed data can be downloaded and annotation links accessed . screenshots of the results showing gene ontologies ( accessed via a link for each est / contig ) , interproscan and kegg pathway mapping obtained from processed est data are shown . ( a ) est or contig submission with optional parameters for organism and program selection , ( b ) status page to monitor the progress of different programs and ( c ) result download page from where processed data can be downloaded and annotation links accessed . screenshots of the results showing gene ontologies ( accessed via a link for each est / contig ) , interproscan and kegg pathway mapping obtained from processed est data are shown . phase i comprises three programs run sequentially , to convert input est sequences into high quality ests . seqclean accepts ests in fasta format and performs vector removal ( using ncbi 's univec database ) , polya removal , trimming of low quality segments at the 5 and 3 ends and cleaning of low complexity regions ( using the dust module ) . additionally , all short ests ( < 100  bp ) are eliminated as uninformative . the output from seqclean species - specific repeat masking is done using repeat masker which in turn employs cross_match and up - to - date repeat libraries for different species from repbase . for a novel species , the nearest organism listed in estexplorer , using ncbi taxonomy , may be selected . cap3 ( 12 ) then accepts repeat - masked high quality est sequences and performs clustering and assembly into contigs ( containing multiple ests ) and singletons , based on an overlap percent identity threshold cutoff of 80 . the user can modify this , with the recommendation to provide a value > 65 . phase ii carries out annotation at the nucleotide level , of assembled est contigs and singletons from phase i or directly uploaded by the user , using the blastx ( 13 ) program and ncbi 's non - redundant protein database , followed by the assignment of functionality via gene ontologies ( 14 ) using blast2go ( 15 ) . blast2go extracts go terms for each blast hit obtained by mapping to extant annotation associations , using a default cutoff of e-03 , which the user can modify . additionally , blast2go provides a data file which can be used to reconstruct go relationships and perform statistical analysis on gene function information . estexplorer , in turn , retrieves gene ontologies from blast2go and links each go identifier to its ontology tree , displayed by the amigo browser . estscan ( 16 ) accepts contigs and singletons from cap3 and provides conceptual translations , using the genetic code from the nearest organism , in a two - step process . in the first step , coding regions or open reading frames ( orfs ) are detected and extracted , while correcting for frame shift errors . in the second step , these orfs are translated into putative peptides . estexplorer currently implements the genetic codes ( smat files generated from mrna sequences ) for the ten organisms : human , mouse , rat , rice , zebrafish , chicken , fly , dog , thale cress ( arabidopsis thaliana ) and roundworm ( caenorhabditis elegans ) provided by the authors of estscan . for a novel species , the nearest organism listed in estexplorer , using ncbi taxonomy , the peptide sequences from estscan are simultaneously passed on to interproscan ( 17 ) and kobas ( 18 ) for processing . interproscan matches protein sequences against interpro , an integrated resource for protein families , domains and functional sites from member databases such as prosite , prints , pfam , prodom and smart . estexplorer runs interproscan in the backend and provides an html output that users can download and analyse , with details of domain / motif architecture for each sequence . kobas ( kegg orthology - based annotation system ) maps protein sequences to pathways based on kegg ( 19 ) . kobas uses controlled vocabularies ( ko ) to annotate a set of sequences and assigns pathways to individual proteins , using a two - step process . in the first step , it takes a set of sequences and assigns kegg orthology terms based on a blastp similarity search against kegg genes or direct cross - sequence identifier mapping . in the second step , estexplorer provides an html output for the mapped pathways through which the user can directly access the pathways at the kegg website . proteins that are mapped from the processed est dataset are highlighted and coloured differently for easy identification . once an est or contig dataset has been submitted to estexplorer , a status page is accessible ( figure 1b ) , for monitoring the progress of the analysis , at the program level . as each selected program is completed , the status page is updated and the output from that program becomes available immediately . estexplorer provides an integrated workflow approach to est analysis , by combining assembly with traditional and well - established resources , such as blast2go and interpro . while some components are available separately as web servers , estexplorer has extended functionality over these as well as added additional features , interfaced seamlessly together . additionally , we have also provided the ability to use quality values during the assembly process . phase ii involves dna - level orthologue mapping , directly from the phase i output . when there are several contigs and singletons after phase i , the user does not have to submit each one to ncbi to run blast . additionally , we can process each of the contigs and singletons from the phase i for protein - level annotation , via phase iii where the complete interproscan , go mapping and kegg pathway mapping are carried out . recently , pavy and coworkers ( 20 ) have used go and pfam matches for annotating their ests at a functional level . estexplorer provides these along with the additional advantage of kegg and the complete interproscan currently comprising 12 modules in addition to pfam , for protein and domain analysis ( details available from our website ) . the outcome for each run is summarized , with links to output files from each selected program . an email with the url of the results will be sent to the user after the completion of the entire run . users can either download output files from the download page for each step or as a single zipped file for each phase of the analysis ( figure 1c ) . all of the programs in phase ii and iii , excepting estscan , are optional . we update the backend databases ( non - redundant protein and univec databases from ncbi , repeat database from repbase , gene ontologies , interproscan and kegg ) every month using automated scripts . a detailed tutorial and faq ( http://estexplorer.biolinfo.org/tutorial.html ) are available for running sample est datasets and understanding the different analysis programs . it is usually difficult to collate the analysis results at the final output stage when a large dataset is analysed using a workflow containing several phases and multiple programs . to address this issue , estexplorer tracks each assembled sequence ( contig / singleton ) which has been functionally annotated ( more details are available from the example section ) . estexplorer has been developed using open source technologies ; zope ( v2.8.1 ) , python ( v2.4.3 ) and mysql ( v4.1.10a ) , for est data management and analysis . estexplorer runs on a 16-node linux cluster ( 1.3  ghz , itanium 2 rev , 5 processors , 16  gb ram ) running on red hat enterprise linux as release 3 . the workflow architecture has been designed based on a  distributed control approach. the user request from the central zope controller is diverted to one of the data - processing machines after appropriate load balancing . browser and platform independent java scripts have been used for data validation , in order to enhance the flexibility of query and output pages . the server refreshes the intermediate result page every 30s and updates the user with the status of processing in the individual programs in the pipeline . a final output page provides the user with detailed output files for viewing and for downloading the results . from dbest ( 21 ) , we provide a small dataset of 372 ests ( input option 1 in supplementary figure 1 ) for the plant capsicum chinense and the complete analysis results from estexplorer . additionally , assembled sequences ( contigs / singletons ) from these ests have been provided as an example for input option 2 ( supplementary figure 1 ) . detailed sequence - wise annotation summaries are provided to facilitate rapid functional analysis of est datasets ( http://estexplorer.biolinfo.org/example_capsicum/summary_table.html ) . the detailed summary of the analysis of contig 9 shows the contributing ests , protein domains , gene ontologies and mapped pathway ( shown in supplementary figure 2 ) . estexplorer has allowed the rapid and accurate analysis of ests by providing robust annotation at the gene and protein levels , matching evidence from multiple sources . using estexplorer to analyse 873 ests from a parasitic nematode oesophagostomum dentatum ( 22 ) yielded 133 contigs and 314 singletons , compared with 128 contigs and 388 singletons reported by cottee et al . overall , 29 entries were annotated with gene ontology data , 44 sequences had protein domain information and 246 sequences were mapped to kegg pathways . this rapid and comprehensive analysis together with additional analyses of specific molecules enabled the identification of novel genes and molecules predicted , based on comparisons with extensive data in wormbase ( 23 ) , to be involved in biological pathways critical for development , reproduction and survival . with estexplorer , the analysis was systematic and additional information on domain and pathway mapping made it easier to validate functional annotation with low scoring hits . this dataset is provided as the second example dataset ( input data 1 ) on the server ( http://estexplorer.biolinfo.org/examples.html ) . a moderate dataset of 10  651 ests for ancylostoma ceylanicum , downloaded from dbest ( 21 ) , is also available , as ests in fasta format ( input option 1 ) and assembled ests ( input option 2 ) . additionally , we have also applied estexplorer for the analysis of a number of est datasets ranging from 717 ests from a related parasitic nematode trichostrongylus vitrinus ( 24 ) to 21  967 ests from haemonchus contortus for subsequent analysis of differential transcription between adult male and female worms by oligonucleotide microarrays . we used two types of data that were annotated using estexplorer : the first comprised unprocessed 21  967 ests and the second contained 1885 contigs . by annotating both the ests as well as these contigs , we have been able to get better representation of biologically relevant genes for oligonucleotide design and subsequent microarray analysis ( unpublished data ) . estexplorer has been used extensively for the annotation of transcript and protein sequence data for the aspergillus niger and mycosphaerella graminicola fungal genomes , a collaborative effort of our group ( n.d . and s.r . ) with doe joint genome institute ( jgi ) , usa . estexplorer currently supports organism - based repeat masking and conceptual translation for ten commonly researched model organisms per se . our goal is to extend this capability to several newly sequenced organisms . in this direction users will also be able to upload their own data files during pre - processing ( vectors , adaptors , organism - specific repeats ) and their own databases for similarity searches , for the targeted analysis of est sequences .
the analysis of expressed sequence tag ( est ) datasets offers a rapid and cost - effective approach to elucidate the transcriptome of an organism , but requiring several computational methods for assembly and annotation . 
 estexplorer is a comprehensive workflow system for est data management and analysis . 
 the pipeline uses a  distributed control approach in which the most appropriate bioinformatics tools are implemented over different dedicated processors . 
 species - specific repeat masking and conceptual translation are in - built . 
 estexplorer accepts a set of ests in fasta format which can be analysed using programs selected by the user . 
 after pre - processing and assembly , the dataset is annotated at the nucleotide and protein levels , following conceptual translation . 
 users may optionally provide estexplorer with assembled contigs for annotation purposes . 
 functionally annotated contigs / ests can be analysed individually . 
 the overall outputs are gene ontologies , protein functional identifications in terms of mapping to protein domains and metabolic pathways . 
 estexplorer has been applied successfully to annotate large est datasets from parasitic nematodes and to identify novel genes as potential targets for parasite intervention . 
 estexplorer runs on a linux cluster and is freely available for the academic community at http://estexplorer.biolinfo.org .