Refined Annotation of the Arabidopsis Genome by Complete Expressed Sequence Tag Mapping

Abstract

Expressed sequence tags (ESTs) currently encompass more entries in the public databases than any other form of sequence data. Thus, EST data sets provide a vast resource for gene identification and expression profiling. We have mapped the complete set of 176,915 publicly available Arabidopsis EST sequences onto the Arabidopsis genome using GeneSeqer, a spliced alignment program incorporating sequence similarity and splice site scoring. About 96% of the available ESTs could be properly aligned with a genomic locus, with the remaining ESTs deriving from organelle genomes and non-Arabidopsis sources or displaying insufficient sequence quality for alignment. The mapping provides verified sets of EST clusters for evaluation of EST clustering programs. Analysis of the spliced alignments suggests corrections to current gene structure annotation and provides examples of alternative and non-canonical pre-mRNA splicing. All results of this study were parsed into a database and are accessible via a flexible Web interface at http://www.plantgdb.org/AtGDB/.The efforts of an international collaboration to obtain the complete genome sequence of the flowering plant Arabidopsis resulted in the release and annotation of 115.4 Mb of the genome (estimated at 125 Mb) in December of 2000 (Arabidopsis Genome Initiative, 2000). At that time, 25,498 protein-coding genes were identified in the five haploid chromosomes, but only 9% of these genes had been characterized experimentally, and only 69% could be functionally classified by similarity to proteins of known functions. In the interim, sequencing and annotation has progressed. The most current release of the Arabidopsis genome available at GenBank provides 117.3 Mb and 27,288 annotated protein-coding genes (see Data Sets in "Materials and Methods"). Annotation of the Arabidopsis genome and functional characterization of all the genes is an ongoing effort. Initial, high-throughput computational gene structure prediction has likely been successful in identifying most gene locations; however, these methods still suffer from limitations in predicting the precise gene structure for an entire gene, detection of intergenic regions, and identification of non-coding exon sequences (Pavy et al., 1999;Brendel and Zhu, 2002). Recent studies have concentrated on sequencing of full-length cDNAs to improve genome annotation Seki et al., 2002).Expressed sequence tags (ESTs) are single-pass sequencing reads of cDNA clones that have become a widely employed method for gene identification, expression profiling, and polymorphism analysis. Presently, more than 13.4 million EST entries have been deposited into the National Center for Biotechnology Information (NCBI) dbEST public database, including Arabidopsis with 176,915 ESTs and 21 other species with EST sets of more than 100,000 entries (http://www.ncbi.nlm.nih.gov/dbEST/ dbEST_summary.html). In the absence of a wholegenome sequencing project for a particular species, clustering of ESTs into contigs that represent unique genes is one of the most promi...

Evaluation of five ab initio gene prediction programs for the discovery of maize genes

“…In that species the frequencies of the non-canonical GC-AG and AT-AC sites have been estimated to be about 1.0% and 0.06%, respectively. These may, however be over-estimates because ambiguous splice sites were included in this analysis (Zhu et al, 2003). In our data set 2, 99.1% of all sites were canonical GT-AG pairs and non-canonical GC-AG and AT-AC pairs represent 0.822% and 0.103% of all pairs, respectively.…”

“…Analysis of spliced alignments between clustered Arabidopsis EST and genomic sequences also showed that the canonical GT-AG pairs account for the majority of the splice sites in Arabidopsis (Zhu et al, 2003). In that species the frequencies of the non-canonical GC-AG and AT-AC sites have been estimated to be about 1.0% and 0.06%, respectively.…”

Genome-wide survey of Alternative Splicing in Sorghum Bicolor

“…Three other investigations with a lesser assembled of EST/cDNA data briefly described fewer AS events in Arabidopsis (Iida et al 2004;Zhu et al 2003;Haas et al 2003). All these pioneering investigations revealed that reduced parts of genes of 5-10 % are alternatively spliced, with Intron retention the most prevalent AS type in Arabidopsis (Iida et al 2004).…”

Genomewide comparative analysis of alternative splicing in plants

“…They identified 15,214 transcription units (TUs) con- taining at least two sequences each and observed alternative splicing for 11.6% of these TUs (33). Three other studies with a smaller collection of EST͞cDNA data briefly reported fewer AS events in Arabidopsis (9,34,35). All these pioneering studies revealed that a low fraction of genes (5-10%) are alternatively spliced, with IntronR the most prevalent AS type in Arabidopsis.…”

“…Millions of ESTs were used in human AS analyses (9), whereas less than one-10th of that number were available for Arabidopsis (9,(32)(33)(34)(35). The number of publicly available plant cDNA͞EST sequences has increased dramatically since the original studies, and, thus, it seemed likely that more AS events would be identified by using current data.…”