System workflow
PLAN adopts project-based data organization. A project contains a set of logically associated data, such as a sequence library to be searched against several target databases, multiple sequence libraries to be annotated using the same criteria, or two sets of sequences to be aligned to each other. A user may have multiple projects. There is no limitation on the size of the project.
In each project, PLAN manages two types of data from users, i.e. sequence files in FASTA format and BLAST result files in NCBI plain text format. The system workflow (Figure 1) generally consists of two stages, pre-BLAST and post-BLAST. In the pre-BLAST stage, PLAN helps the user to manage sequences, customize BLAST search options and conduct BLAST searches. In the post-BLAST stage, PLAN retrieves search results from the BLAST server, imports optional custom BLAST results, manages and presents these results in different ways and performs various downstream analyses.
Query sequences are initially uploaded by users and archived in the database. Sequence ID duplication is checked and various options are provided to deal with such duplication – ignore, discard, replace, etc. BLAST searches are automated with reference to pre-defined search templates. A search template saves all necessary BLAST settings, such as alignment method (BLASTN, BLASTP, BLASTX, TBLASTX, etc.), alignment parameters (e.g. scoring matrix, score and e-value thresholds) and target database. The use of templates ensures that the user applies the same set of customized search criteria in different search sessions. The system administrator has a full control of the utilization of templates over all users, according to user grouping. For example, an institute may separate its PLAN users into public and internal groups, which can perform BLAST searches against their published or private (unpublished) sequence databases, respectively.
PLAN monitors the search process in the background and displays it online upon user request. Once the search is complete, the results, in NCBI plain text format by default, are automatically downloaded from the BLAST server and further imported into PLAN's database. The original search result files are archived for the user to download and review. A user may also upload custom search result files generated by other systems. There is no limit on the number of BLAST searches or uploaded search results in each project, and this enables the user to align multiple query sequence sets against multiple target databases for comprehensive studies.
PLAN converts text-style BLAST results into well-organized, rational database records. It indexes and hierarchically organizes the results into three levels, namely: (1) global parameters of the search session, such as search algorithm, version, database name etc.; (2) sequence level mapping between all search queries and the returned targets (hits), including bit scores and e-values (or p-values if applicable); and (3) alignment details of all high scoring pairs (HSPs). PLAN offers a wide range of high-level database manipulation functions for various post-BLAST analytical tasks. Some major post-BLAST functions are introduced in detail below.
Multi-angle presentation of results
One of the most direct uses of PLAN is to review the BLAST results and find results of interest. PLAN adopts an intuitive web interface that simulates Microsoft Outlook. The presentation of results generally follows the three-level data hierarchy mentioned above. In brief, search session statistics are summarized in spreadsheets; sequence-level query-target mappings are displayed in spreadsheets and graphs; and various tables, texts and hyperlinks are utilized to present various alignment details. Following this schema, PLAN organizes and represents the BLAST results from several angles, summarized as follows.
First, and most intuitively, the results may be viewed according to unique query IDs. When the results are presented from this angle, PLAN summarizes and lists each query's multiple targets/hits returned from all search sessions. This facilitates comprehensive annotation of unknown sequences. Figure 2 depicts a spreadsheet for the identification of an in-house Medicago truncatula (Mt) insertion sequence NF0013-INSERTION-4 through searches against five major databases, namely IMGAG predicted Mt genes, Mt BAC clone genome sequences, and the NCBI protein (NR), NCBI nucleotide (NT) and TIGR Mt EST databases. These results not only identify the insertion site (on the basis of the result against the BAC clone database), but also suggest that the insertion potentially affects a predicted gene (implied by the result against the IMGAG database), the EST of which is included in the TIGR EST database.
Similarly, and more importantly, PLAN provides a novel, global view of BLAST results by grouping them according to unique target IDs. In other words, it helps the user to investigate which multiple queries hit the same target and how they do so. This provides useful information for many purposes. Examples include, but are not limited to, checking the redundancy of a library, estimating EST expressions in-silico, visualizing sequence assembly results, and investigating insertion/deletion mutation sites on the genome.
Figure 3 illustrates how the mutation sites of five Mt insertion sequences are visualized on the same BAC clone sequence. These insertion sites conform to a generally random distribution. In addition, when a graphical bar (corresponding to a specific HSP) is clicked, PLAN displays the alignment details of a specific insertion sequence (Figure 4).
Besides the above intuitive data hierarchy inherited from typical BLAST results, PLAN provides a customizable plug-in mechanism, which encodes a set of translation rules provided by administrators and/or users, enabling the source of in-house sequences to be identified in terms of sequencing plate ID and gene line ID, and allowing the statistics and results to be viewed further on the basis of these in-house IDs. Such statistics are of great value in various in-house sequencing studies. Examples include, but are not limited to, checking the sequencing quality according to the percentage of "good read" sequences, validating an experiment according to data from biological replicates, and identifying unique insertion/deletion mutants in different gene lines.
Lastly, PLAN can be customized by the administrator to extract and translate certain terms in specific patterns during the presentation of results. For example, the current setup of the PLAN system on our server automatically highlights all Gene Ontology (GO) [10, 11] and Plant Ontology (PO) [12, 13] terms. When a GO or PO term is clicked, a window will pop up to show the corresponding ontology tree for a more in-depth study.
Searching, filtering and organization of data of interest
PLAN provides a wide range of search options for identifying records of interest efficiently from large-scale BLAST results. It allows users to search for one or many query/target IDs, with different matching options (e.g. partial or full). It also provides a full-text search engine for matching any search term in the query/target IDs, names and descriptions. This is particularly valuable for functional analytical tasks, such as finding query sequences with hits to a specific protein family, or categorizing sequences according to functional descriptions.
In addition to these search functions, PLAN enables users to filter results on the basis of bit scores and e-value thresholds on the fly. This facilitates the review of results at different homology levels – higher bit scores and lower e-values lead to fewer results at higher homology. In addition, on each query, PLAN allows users to view a specific number of "top hits" (such as the best hit as indicated by the lowest e-value), further reducing the number of returned results for simpler yet more significant investigation. Furthermore, queries with no target (hit) may be hidden to obtain a cleaner workspace.
It is important to have a mechanism that archives, organizes and manages records of interest for further studies. PLAN fully integrates a hierarchical "favorites" management system. A user has full control over the favorite categories (e.g. creation, edition and deletion) as well as records in each category. While the user browses any portion of the project data (such as some search results), he/she may add the data of interest to either a new or an existing favorite category. Subsequently, records in each favorite category may be browsed, removed from the category, or copied/moved to another category. Our current users consider this a very useful function. Figure 5 illustrates how PLAN assists functional genomics studies by organizing a number of sequences into different user-customized functional categories.
Functional annotation and data export
One of the most routine post-BLAST tasks is the functional annotation of the query sequences. PLAN provides comprehensive options for this purpose. The sequences to be annotated may be all the queries in a project, or any sequence subsets from the following sources: (1) an uploaded FASTA file, (2) a BLAST search session, (3) a favorite category, (4) an in-house gene line or plate, or (5) any user-selected sequences from any PLAN-displayed spreadsheets. A user may annotate the sequences with either of the following: (1) the top-hit in terms of either lowest e-value or highest score, or (2) all targets (hits) that passed the project filters and are visible to the user. If the query sequences have been searched against multiple databases, the user may further customize the annotation to use any one of the databases, or all of them. The output format of the annotation may be either natural language (such as "Similar to ..." or "Weakly similar to..."), which follows the format of NCBI GenBank, or tab-delimited format for downstream analysis using other software such as Microsoft Excel. PLAN may also save the user-uploaded original query sequences in well-annotated FASTA format for submission to NCBI GenBank. Figure 6 depicts the annotations of three sequences, with top hits from multiple databases, saved in tab-delimited format and viewed in Microsoft Excel. Figure 7 gives an example of a sequence being annotated with the NCBI protein database (NR) and saved in FASTA format.
In addition to these flexible annotation functions, PLAN provides multiple means of data export. Any subset of sequences and intermediate BLAST results can be exported for further analysis. For example, PLAN may export a well-formatted summary of the overlap between two libraries for Venn diagram analysis. It may also export query sequences with no hit in one search session for further searches. More information on data export may be found at the project web site.
Control of data publication
On each project, PLAN provides three levels of data publication, summarized as follows. (1) At the most private and secure level, all data are hidden from the public. A personal password is required to access the protected data. This security level is most suitable for private, unpublished data. (2) At the medium level, data are published for read-only access. General site visitors may browse and search the project data but may not make changes. With a personal password, the project owner has a full access and all modification rights to the data. This level is best for data presentation and demonstration. (3) At the most open level, all the project data are fully accessible to all site visitors. All visitors may read and modify these data. This level is best for team work, with local deployment of the PLAN system on an institutional network. At the latter two levels, a direct hyperlink of the published project to a specific record (such as file, favorite category, gene line, plate, query or target) can be made from outside the project scope without visiting the PLAN home page. This extends PLAN as a sequence identification reference web site for knowledge sharing.
The project level is editable by the project owner at any time. For example, a user may work on his/her EST library as a private project. Once the EST library is accepted for publication, he/she may further publish the project as a read-only public project. It is worth mentioning that PLAN also provides a special category of "public projects" to generic users who want to utilize its analytical functions, without caring about data privacy or retention period. Manipulating a public project does not require user registration.
System architecture and software implementation
PLAN is implemented in PHP and Perl languages. It follows a typical three-tier software architecture, comprising a presentation layer, a processing layer and a data layer (Figure 8). The presentation layer, which interacts with users, consists of a versatile web interface written in PHP. The processing layer consists of various data manipulation and analysis modules written in PHP and Perl. Some BioPHP [14] and BioPerl [15] functions are utilized for data processing. The data layer consists of a set of file handling, BLAST server communication and database-abstraction modules written in PHP and Perl. ADOdb [16] is adopted for database abstraction, which makes the system independent of the database server. The system supports, by default, the Decypher hardware-based BLAST server provided by Active Motif Inc. Software BLAST solutions may be integrated with no difficulty.
The three tiers are clearly defined while seamlessly interconnected. The platform-independent design makes PLAN capable of working on various mainstream web servers (Apache, IIS, etc.) and operation systems (Unix, Linux, Windows, etc.), and of supporting a large variety of database servers (MySQL, PostgreSQL, Oracle, Microsoft SQL, etc.). In practice, we released the system for public use on an Apache 2 web server running under the Fedora Core 5 Linux operation system, using the MySQL database and a Decypher BLAST server that contains two accelerator cards.
