Ying Xu, Manesh Shah, J. Ralph Einstein, Sherri Matis, Xiaojun Guan, Sergey Petrov, Loren Hauser, Richard J. Mural, and Edward C. Uberbacher
Computer Science and Mathematics, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6364. e-mail: GRAILMAIL@ornl.gov
GRAIL is a modular expert system for the analysis and characterization of DNA sequences which facilitates the recognition of gene features and gene modeling. A new version of the system has been created with greater sensitivity for exon prediction (especially in AT rich regions), more accurate splice site prediction, and robust indel error detection capability. GRAIL 1.3 is available to the user in a Motif graphical client-server system (XGRAIL), through WWW-Netscape, by email server, or callable from other analysis programs using Unix sockets.
In addition to the positions of protein coding regions and gene models, the user can view the positions of a number of other features including poly-A addition sites, potential Pol II promoters, CpG islands and both complex and simple repetitive DNA elements using algorithms developed at ORNL. XGRAIL also has a direct link to the genQuest server, allowing characterization of newly obtained sequences by homology-based methods using a number of protein, DNA, and motif databases and comparison methods such as FastA, BLAST, parallel Smith-Waterman, and special algorithms which consider potential frameshifts during sequence comparison.
Following an analysis session, the user can use an annotation tool which is part of the XGRAIL 1.3 system to generate a "feature table" report describing the current sequence and its properties. Links to the GSDB sequence database have been established to record computer-based analysis of sequences during submission to the database or as third party annotation.
Gene Modeling and Client-Server GRAIL: In addition to the current coding region recognition capabilities based on a multiple sensor-neural network and rule base, modules for the recognition of features such as splice junctions, transcription and translation start and stop, and other control regions have been constructed and incorporated into an expert system (GAP III) for reliable computer-based modeling of genes. Heuristic methods and dynamic programming are used to construct first pass gene models which include the potential for modification of initially predicted exons. These actions result in a net improvement in gene characterization, particularly in the recognition of very short coding regions. Translation of gene models and database searches are also supported through access to the genQuest server (described below).
Model Organism Systems: A number of model organism systems have been designed and implemented and can be accessed within the XGRAIL 1.3 client including Escherichia coli, Drosophila melanogaster and Arabidopsis thaliana. The performance of these systems is basically equivalent to the Human GRAIL 1.3 system. Additional model organism systems, including several important microorganisms, are in progress.
Error Detection in Coding Sequences: Single-pass DNA sequencing is becoming a widely used technique for gene identification from both cDNA and genomic DNA sequences. An appreciably higher rate of base insertion and deletion errors (indels) in this type of sequence can cause serious problems in the recognition of coding regions, homology search, and other aspects of sequence interpretation. We have developed two error detection and "correction" strategies and systems which make low-redundancy sequence data more informative for gene identification and characterization purposes. The first algorithm detects sequencing errors by finding changes in the statistically preferred reading frame within a possible coding region and then rectifies the frame at the transition point to make the potential exon candidate frame-consistent. We have incorporated this system in GRAIL 1.3 to provide analysis which is very error tolerant. Currently the system can detect about 70% of the indels with an indel rate of 1%, and GRAIL identifies 89% of the coding nucleotides compared to 69% for the system without error correction. The algorithm uses dynamic programming and runs in time and space linear to the size of the input sequence.
In the second method, a Smith-Waterman type comparison is facilitated in which the frame of DNA translation to protein sequence can change within the sequence. The transition points in the translation frame are determined during the comparison process and a best match to potential protein homologs is obtained with sections of translations from more than one frame. The algorithm can detect homologies with a sensitivity equivalent to Smith-Waterman in the presence of 5% indel errors.
Detection of Regulatory Regions: An initial Polymerase II promoter detection system has been implemented which combines individual detectors for TATA, CAAT, GC, cap, and translation start elements and distance information using a neural network. This system finds about 67% of TATA containing promoters with a false positive rate of one per 35 kilobases. Additionally a systems to detect potential polyA addition sites and CpG islands has been incorporated into GRAIL.
The GenQuest Sequence Comparison Server: The genQuest server is an integrated sequence comparison server which can be accessed via e-mail, using Unix sockets from other applications, Netscape, and through a Motif graphical client-server system. The basic purpose of the server system is to facilitate rapid and sensitive comparison of DNA and protein sequences to existing DNA, protein, and motif databases. Databases accessed by this system include the daily updated GSDB DNA sequence database, SwissProt, the dbEST expressed sequence tag database, protein motif libraries and motif analysis systems (Prosite, BLOCKS), a repetitive DNA library (from J. Jurka), Genpept, and sequences in the PDB protein structural database. These options can also be accessed from the XGRAIL graphical client tool.
The genQuest server supports a variety of sequence query types. For searching protein databases, queries may be sent as amino acid or DNA sequence. DNA sequence can be translated in a user specified frame or in all 6 frames. DNA-DNA searches are also supported. User selectable methods for comparison include the Smith-Waterman dynamic programming algorithm, FastA, versions of BLAST, and the IBM dFLASH protein sequence comparison algorithm. A variety of options for search can be specified including gap penalties and option switches for Smith-Waterman, FastA, and BLAST, the number of alignments and scores to be reported, desired target databases for query, choice of PAM and Blosum matrices, and an option for masking out repetitive elements. Multiple target databases can be accessed within a single query.
Additional Interfaces and Access: Batch GRAIL 1.3 is a new "batch" GRAIL client allows users to analyze groups of short (300-400 bp) sequences for coding character and automates a wide choice of database searches for homology and motifs. A Command Line Sockets Client has been constructed which allows remote programs to call all the basic analysis services provided by the GRAIL-genQuest system without the need to use the XGRAIL interface. This allows convenient integration of selected GRAIL analyses into automated analysis pipelines being constructed at some genome centers. An XGRAIL Motif Graphical Client for the GRAIL release 1.3 has been constructed using Motif with versions for a wide variety of UNIX platforms including Sun, Dec, and SGI. The e-mail version of GRAIL can be accessed at firstname.lastname@example.org and the e-mail version of genQuest can be accessed at Q@ornl.gov. Instructions can be obtained by sending the word "help" to either address. The Motif or Sun versions of XGRAIL, batch GRAIL, and XgenQuest client software are available by anonymous ftp from arthur.epm.ornl.gov (220.127.116.11). Both GRAIL and genQuest are accessible over the World Wide Web (URL http://avalon.epm.ornl.gov/). Communications with the GRAIL staff should be addressed to GRAILMAIL@ornl.gov.
(Supported by the Office of Health and Environmental Research, United States Department of Energy, under contract DE-AC05-840R21400 with Lockheed Martin Energy Systems, Inc.)
 Biology Division
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