expressed sequence tags ( ests ) are obtained from single - pass sequencing of cdna clones prepared from mrnas . they remain a dominant approach for characterizing the active , protein - coding portions of genomes for a variety of species . as of december 2006 , there were 40  227  021 est entries deposited in genbank dbest ( 1 ) . while these data represent by far the most abundant information resource for expressed portions of various genomes , they also present serious challenges for data quality control due to the inherent deficiencies of ests , particularly for those from species without available genome sequences . many public est resources , including ncbi unigene ( 2 ) and tigr gene indices ( 3 ) , have been developed to address the quality limitations , as well as the issues of redundancy and the less - than - full - length nature of ests . unfortunately , the power of these resources relies heavily on the veracity of sequence features that have been submitted to dbest ( 4 ) . many software packages are available for processing raw est traces prior to genbank dbest submission ( 513 ) . before such submission , est sequences are typically screened and trimmed of vector , adapter / linker sequences and insert - flanking restriction endonuclease recognition sites , as well as polya or polyt tails ; however , available sequence processing packages are limited in their ability to detect and trim such sequences correctly ( 13,14 ) . loss of the information in these trimmed sequences can present an obstacle for data validation of error - prone ests , and can impede data mining for certain functional motifs , such as polyadenylation signals , whose detection requires accurate annotation of positional information for polya tails added posttranscriptionally . to alleviate limitations imposed by trimmed sequences , both ncbi trace archive ( www.ncbi.nlm.nih.gov/traces/trace.cgi ? ) and ensembl trace server ( trace.ensembl.org ) were established as public repositories for raw dna sequencer trace files , including those for ests . unfortunately , these databases have so far remained mostly untapped resources for general biologists due to a shortage of freely available , easy - to - use bioinformatics tools with which to identify and highlight sequence features after raw trace processing . to the best of our knowledge , preassemble ( 14 ) is the only freely available sequence processing pipeline service that allows biologists to upload and process raw est traces online without difficult software installation and configuration procedures . like other programs , preassemble creates trimmed sequences , but it produces a quality assessment of sequencing data in color - coded html pages that facilitate online data exploration . like preassemble , webtraceminer 's aim is to be a public sequence processing service that permits biologists to upload their own traces as well as those downloaded from ncbi trace archive . however , webtraceminer differs from preassemble , as well as most other packages , by focusing on est sequence features that characterize the 3 and 5 termini of cdna inserts and by providing robust data visualization and mining capability of these features . webtraceminer is designed to first detect all putative sequence features as single , multiple and/or concatenated occurrence(s ) in each sequence read after basecalling , allowing for both perfect and imperfect ( i.e. mismatch , deletion and insertion ) matching patterns . currently implemented sequence features include vector fragments , adapter / linker sequences , restriction endonuclease recognition sites and polya or polyt runs . based on common canonical models for directional cdna library construction using oligo - dt primers , as shown in figure 1 , webtraceminer then evaluates the number , location , order , fidelity and orientation of the putative features to determine in silico verified features that should in theory occur at the 3 and/or 5 termini of all cdna inserts in a given library . both putative and verified features can easily be explored and compared using sophisticated data querying and visualization web interfaces . all data can be saved locally and selectively , either in fasta , tab - delimited or xml formats , for future exploration if necessary . unambiguous identification and authentication of 3 and 5 termini of cdna inserts through ests provides for the creation of cleaner est clusters , improves annotation of gene position in genomic sequence , facilitates identification of 3 and 5 untranslated regions ( utrs ) , and aids detection of potential functional motifs embedded in abundant ests . figure 1.example of a canonical model for cdna library construction . in some cases , a second adapter / linker sequence ( adapter2 ) might be applied between the polya / t tails and restriction site2 . example of a canonical model for cdna library construction . in some cases , a second adapter / linker sequence ( adapter2 ) might be applied between the polya / t tails and restriction site2 . webtraceminer takes as input raw est trace file(s ) , either a single trace or a zipped file containing multiple traces . as shown in figure 2 panel a , the configuration interface allows a user to define and characterize one trace processing event , which consists of several procedures or steps that are common among existing dna sequence processing packages ( 513 ) . for general users , the only exception is that  adapter and restriction enzyme site configuration requires users to enter the sequences as in the positive cdna strand for enzyme 2 , all of which are mandatory for defining appropriately a canonical model for the user - specific cdna library according to its construction ( see figures 1 and 2 , panel a ) . after a user saves the configuration and finishes uploading raw trace(s ) online , webtraceminer then performs automatic trace processing , produces sequence reads by basecalling and identifies in each sequence read all putative sequence features ( i.e. vector fragments , adapter / linker sequences , restriction sites and polya or polyt runs ) . xml view ,  fasta view and  color - coded view for both normal and reverse complement sequences , as shown in figure 2 panel b. using tabulated view ( see figure 2 , panel c ) , a user can obtain a quick overview of sequencing performance and various putative sequence features in perfect and/or imperfect matching patterns . sequencing performance can be determined easily by the relative proportion of passed ( i.e. qtag = 1 ) versus failed ( i.e. qtag = 0 ) sequence reads according to whether or not the sequence reads meet the minimum length threshold of the good quality region for each sequence read after quality trimming . the default threshold is 100 bases and can be customized in terms of users requirement in the  quality trimming configuration ( see figure 2 , panel a ) . the tabulated results in tabulated view can be sorted in either descending or ascending order by clicking individual column heads in a toggle manner , and they also can be filtered and searched using the expandable  sequence filter displayed above the tabulated results ( see figure 2 , panel c ) .  xml view presents all processed data in the xml format to enhance data exchangeability and interoperability . using fasta view , a user can retrieve either whole raw sequence reads or trimmed high - quality portions of sequence reads in the fasta format . fasta view ,  color - coded view also furnishes fasta - format results for all raw sequence reads in a html page , but with color - coded bases to indicate putative sequence features , as well as regions have high or low phred ( 15 ) quality scores . panel ( a ) : the configuration interface allows users to save customized information for how to process est traces . for general users , the only required information is entered into the  adapter and restriction enzyme site section to define a specific canonical model for directional cdna library construction , such as that illustrated in figure 1 . panel ( b ) : after processing , resultant data are available as four viewstabulated view ,  xml view ,  fasta view and  color - coded view. with satisfactory processing , users can proceed with optional database integration by clicking the database icon . panel ( c ) : tabulated results are available from  tabulated view. the results can be sorted by clicking relevant column heads . they can also be searched and filtered using the expandable  sequence filter on the top . by clicking a sequence name link , a new panel , such as that shown in panel ( d ) , will pop up automatically . panel ( d ) : an est sequence comprising a complete cdna insert with in silico authenticated 3 and 5 termini . both the svg graph and the color - coded sequence read can be redrawn by applying different parameters available in the  sequence color view control panel. webtraceminer web interfaces . panel ( a ) : the configuration interface allows users to save customized information for how to process est traces . for general users , the only required information is entered into the  adapter and restriction enzyme site section to define a specific canonical model for directional cdna library construction , such as that illustrated in figure 1 . panel ( b ) : after processing , resultant data are available as four viewstabulated view ,  xml view ,  fasta view and  color - coded view. with satisfactory processing , users can proceed with optional database integration by clicking the database icon . panel ( c ) : tabulated results are available from  tabulated view. the results can be sorted by clicking relevant column heads . they can also be searched and filtered using the expandable  sequence filter on the top . by clicking a sequence name link , a new panel , such as that shown in panel ( d ) , will pop up automatically . panel ( d ) : an est sequence comprising a complete cdna insert with in silico authenticated 3 and 5 termini . both the svg graph and the color - coded sequence read can be redrawn by applying different parameters available in the  sequence color view control panel. after trace processing and preliminary characterization , a user may choose to proceed with optional database integration by clicking the icon for  dumpling xml data into mysql database ( see figure 2 , panel b ) . subsequently , all putative sequence features , with raw sequence reads and corresponding phred quality values , are populated into the database . based on the number , location , order , fidelity and orientation of all putative features , sequence features that fit the canonical model of cdna library construction are then determined for each sequence read and inserted into the database automatically . fasta view and  color - coded view will be then available for biologists to query and visualize both putative and in silico verified features . because of the database integration , these newly created views provide far more advanced functionalities than views based on resultant data files ( see figure 2 , panel b ) . for example , when a user clicks a sequence name link displayed in tabulated view , an html page containing the corresponding color - coded sequence read with a scalable vector graphics ( svg ) graph is created on - the - fly ( see figure 2 , panel d ) . this page allows users to inspect individual nucleotides and their associated quality values for putative and/or verified sequence features . the svg graph can be redrawn with different zooming scales from 25 , 50 , 75 to 100% , while the color - coded sequence read can also be recreated with or without space separators , to facilitate searching and text capture for other tools , such as blast ( 16 ) . as shown in figure 2 , panel d , the  sequence color view control panel furnishes several integrated data options that users can use to explore various data . within the detailed data link , the menu items  all putative features and  verified features allow users to toggle between web pages for all putative sequence features and for verified sequence features . within the pages for putative sequence features , users can selectively view each type of sequence features in perfect , imperfect or both matching patterns . in particular , the  sequence feature table menu item provides users detailed information about each sequence feature , either putative or verified , including start and stop positions , length , identity percentage ( i.e. 100 stands for perfect matches ) and matching orientation ( i.e. d indicates normal direction , p represents palindrome or reverse complement and 0 is indeterminate ) . by applying the menu items  sequence quality or detailed data link , users can obtain individual phred quality scores for all bases or retrieve relevant reverse complement sequences . all anonymous input and output data are stored in our server with a unique identifier . the data files , as well as relevant data entries in our database , are maintained on the server for 7 days . no user data is disclosed , either internally or externally . in the current release ( 1.0 ) , however , we can not necessarily guarantee complete confidentiality of any users data . in the future , webtraceminer consists of three components : an object - oriented perl pipeline , a mysql 5.0 relational database and a php web application that communicates with both the pipeline and database . the core of webtraceminer is a perl pipeline , which contains a few classes that have been designed as extensions of bioperl modules ( 17 ) . it is utilized as a parameter by almost all other classes , except some utility classes . config.pm parses a xml configuration file saved by the user and acts as a driving force for all processing procedures . trace.pm represents individual traces as targets for processing , whereas tracegroup.pm stands for a grouping of trace(s ) from any given processing event . in webtraceminer , trace files are processed using phred ( 15 ) or other basecallers to conduct basecalling and create sequence reads . in addition , webtraceminer provides several options for vector screening , including cross_match ( bozeman.mbt.washington.edu/phrap.docs/phrap.html ) and vmatch ( www.vmatch.de ) . correspondingly , webtraceminer possesses some wrapper classes , including phred.pm , crossmatch.pm and vmatch.pm , which help with executions of the above - mentioned thirty - party software . qualitytrim.pm is a class that characterizes the high - quality region determination for individual sequence reads . homopolymerdetect.pm is a class representing the detection process of homopolymers , such as polya / t . seqread.pm defines each sequence read obtained from basecalling procedures . as the counterpart of tracegroup.pm , seqreadgroup.pm represents the grouping of individual sequence reads , facilitating procedures performed on a group of sequence reads ( e.g. writing tab - delimited text and xml output files ) . the design and implementation of the perl pipeline provides for easy modification and expansion in the future . webtraceminer adopts open - source mysql 5.0 ( www.mysql.com ) as its relational database component . with five tables in its current database schema , sequence table stores all processed results for a group of traces using a specific configuration , while seq_base table houses relevant sequence reads and corresponding quality values . feature table holds all properties ( i.e. start and stop positions , length , identity percentage and matching orientation ) for putative features , whereas sequence_ver houses information for verified sequence features . the web application of webtraceminer was implemented using php ( php : hypertext preprocessor , 5.0 ) . we adopted the smarty template engine ( smarty.php.net ) to speed development cycles , enhance software usability and simplify maintenance . the smarty template engine is a php template engine that separates php , as a business logic , from html , as a presentation logic , to facilitate cleaner coding and more flexible modification of codes . in particular smarty tags within documents and then pre - compiles these codes for faster runtime execution . while est analysis remain a dominant approach for characterizing the protein - coding portions of various genomes , freely available and easy - to - use bioinformatics tools for processing est traces without the hassles of software installation and configuration are rare . like preassemble ( 14 ) , webtraceminer provides a valuable public sequence processing service that allows biologists to process their own est traces or traces downloaded from ncbi trace archive . webtraceminer , however , provides greater flexibility in trace processing and sophisticated functionality of data querying and visualization , which are not available in preassemble . although general users are only required to furnish sequence information for the canonical model of directional cdna library construction , advanced users have many different options for configuration to customize their trace processing events . for example , different criteria have been adopted in other software for the detection of polya / t tails in ests ( 8,10,13,18,19 ) . to investigate the impact of these different criteria , a user might want to compare polya / t detection using two different criteria , e.g. , a minimum length of 12 bases with 2 base maximum internal errors versus a minimum length of 8 continuous bases without any error base . using two different web sessions , a user can save these two criteria separately in two configuration files , each of which stands for an independent trace processing event , and then upload the same set of traces independently to do comparative trace processing . with robust functionalities in data sorting , filtration , search and visualization , a user can easily appreciate the differences resulting from use of these two criteria . clearly , webtraceminer provides advanced users a handy  play - and - try research tool that can be customized in terms of individual research needs . expanding at an exponential rate , ncbi dbest ( 1 ) currently houses 40  227  021 entries of ests . however , est data quality is highly variable and suffers from deficiencies inherent to both sequencing ( e.g. partial , single - pass sequencing of variable quality ) and cdna library construction procedures ( e.g. concatenated adapters / linkers , chimeric genes or inversely inserted cdnas ) . the value of these est sequences can be enhanced tremendously by removing redundancy and grouping them into clusters , as has been done in creating the ncbi unigene ( 2 ) and tigr gene indices ( 3 ) resources . however , data quality of individual sequence reads is critical for correct est clustering and , thus , impacts results from applications using the resultant clusters . unfortunately , to our best knowledge , there has been until now no public est resource that allows biologists to validate individual sequence reads by highlighting features in the 3 and/or 5 ends of cdna inserts . before genbank dbest submission , ests are typically trimmed of vector and adapter / linker sequences , as well as polya or polyt tails . unfortunately , existing packages are also limited in their ability to cleanly detect and trim such sequences . for example , preassemble , which utilizes pregap4 of the open - source staden package ( 5 ) , proves to be problematic sometimes in detecting polya / t tails shorter than 10 bases ( 14 ) . for adapter / linker detection , magic - spp ( 13 ) does a better job than other existing packages , including tigr lucy ( 6 ) , but has problems with inserts having more than two concatenated adapters . adopting a different approach , webtraceminer first detects in an unbiased fashion all vector fragments , restriction sites , adapter / linker sequences and polya or polyt runs in each sequence read as putative features . the putative features can be identified in single / multiple occurrence(s ) , in independent / concatenated status and with perfect / imperfect ( i.e. mismatch , insertion or deletion ) matching patterns . based on the canonical model for directional cdna library construction , webtraceminer then examines the number , location , order , fidelity and orientation of the putative features and identifies in silico verified sequence features to characterize 3 and/or 5 termini of cdna inserts . currently , authenticated polya / t tails are defined as those immediately adjacent to an appropriate restriction site in correct orientation ( e.g. either polya + xhoi ( ctcgag ) or xhoi ( ctcgag ) + polyt ) , with an allowance for a minimal number of low - quality bases between the polya / t tails and the restriction site ( see figure 1 ) . meanwhile , adjacency between a restriction site and an adapter sequence , both of which must also be in correct orientation ( e.g. either ecori(gaattc )  + adapter1(ggcacgagg ) or adapter1(cctcgtgcc ) + ecori(gaattc ) ) with the same allowance for low - quality bases between restriction sites and adapters , is mandatory for annotating the other terminus of the cdna insert ( see figure 1 ) . although in silico verified 3 and 5 termini might not be all true in every case because of imperfections in molecular biology manipulations ( e.g. oligo - dt primer misprimes from a stretch of adenosyl residues internal to the mrna transcript ) , they enable biologists to better create cleaner est clusters , annotate gene positions , explore 3 and 5 utrs , and detect functional motifs , among other things . webtraceminer represents a unique service that provides great flexibility in est trace processing and robust functionality in visualizing and mining both putative and verified sequence features . through easy - to - use web interfaces , biologists can inspect and validate their data and identify artifacts , such as chimeric cdnas and adapter concatemers . clearly , webtraceminer can fill the gaps between ncbi dbest and trace archive , and alleviate the limitation in est data mining imposed by trimmed sequences in dbest . focusing on sequence features that characterize the 3 and 5 termini of cdna inserts , webtraceminer provides a unique yet complimentary tool to facilitate data validation and data mining of enormous , error - prone ests . for example , where base calls and associated quality values obtained from applying abi or kb basecaller ( docs.appliedbiosystems.com/pebiodocs/04362968.pdf ) are embedded in traces , sequence reads will be retrievable via the staden package modules ( 5 ) , as an alternative to using phred ( 15 ) . in a near future release , webtraceminer will also provide users an option of utilizing blast ( 16 ) to screen vector and other contaminants , as well as to annotate their sequences . in particular , we are now in the process of improving our classifier to more accurately identify verified features from putative sequence features .
expressed sequence tags ( ests ) remain a dominant approach for characterizing the protein - encoding portions of various genomes . due to inherent deficiencies , they also present serious challenges for data quality control . before genbank submission , 
 est sequences are typically screened and trimmed of vector and adapter / linker sequences , as well as polya / t tails . 
 removal of these sequences presents an obstacle for data validation of error - prone ests and impedes data mining of certain functional motifs , whose detection relies on accurate annotation of positional information for polya tails added posttranscriptionally . as raw dna sequence information is made increasingly available from public repositories , such as ncbi trace archive , new tools will be necessary to reanalyze and mine this data for new information . 
 webtraceminer ( www.conifergdb.org/software/wtm ) was designed as a public sequence processing service for raw est traces , with a focus on detection and mining of sequence features that help characterize 3 and 5 termini of cdna inserts , including vector fragments , adapter / linker sequences , insert - flanking restriction endonuclease recognition sites and polya or polyt tails . 
 webtraceminer complements other public est resources and should prove to be a unique tool to facilitate data validation and mining of error - prone ests ( e.g. discovery of new functional motifs ) .