Dataset Information

An optimized protocol for analysis of EST sequences.

ABSTRACT: The vast body of Expressed Sequence Tag (EST) data in the public databases provide an important resource for comparative and functional genomics studies and an invaluable tool for the annotation of genomic sequences. We have developed a rigorous protocol for reconstructing the sequences of transcribed genes from EST and gene sequence fragments. A key element in developing this protocol has been the evaluation of a number of sequence assembly programs to determine which most faithfully reproduce transcript sequences from EST data. The TIGR Gene Indices constructed using this protocol for human, mouse, rat and a variety of other plant and animal models have demonstrated their utility in a variety of applications and are freely available to the scientific research community.

SUBMITTER: Liang F

PROVIDER: S-EPMC110731 | biostudies-literature | 2000 Sep

REPOSITORIES: biostudies-literature

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An optimized protocol for analysis of EST sequences.

Liang F F Holt I I Pertea G G Karamycheva S S Salzberg S L SL Quackenbush J J

Nucleic acids research 20000901 18

The vast body of Expressed Sequence Tag (EST) data in the public databases provide an important resource for comparative and functional genomics studies and an invaluable tool for the annotation of genomic sequences. We have developed a rigorous protocol for reconstructing the sequences of transcribed genes from EST and gene sequence fragments. A key element in developing this protocol has been the evaluation of a number of sequence assembly programs to determine which most faithfully reproduce ...[more]

PMID: 10982889

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Project description:BackgroundOat is an important crop in North America and northern Europe. In Scandinavia, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop such a crop, more knowledge about mechanisms of cold tolerance in oat is required.ResultsFrom an oat cDNA library 9792 single-pass EST sequences were obtained. The library was prepared from pooled RNA samples isolated from leaves of four-week old Avena sativa (oat) plants incubated at +4 degrees C for 4, 8, 16 and 32 hours. Exclusion of sequences shorter than 100 bp resulted in 8508 high-quality ESTs with a mean length of 710.7 bp. Clustering and assembly identified a set of 2800 different transcripts denoted the Avena sativa cold induced UniGene set (AsCIUniGene set). Taking advantage of various tools and databases, putative functions were assigned to 1620 (58%) of these genes. Of the remaining 1180 unclassified sequences, 427 appeared to be oat-specific since they lacked any significant sequence similarity (Blast E values > 10(-10)) to any sequence available in the public databases. Of the 2800 UniGene sequences, 398 displayed significant homology (BlastX E values < or = 10(-10)) to genes previously reported to be involved in cold stress related processes. 107 novel oat transcription factors were also identified, out of which 51 were similar to genes previously shown to be cold induced. The CBF transcription factors have a major role in regulating cold acclimation. Four oat CBF sequences were found, belonging to the monocot cluster of DREB family ERF/AP2 domain proteins. Finally in the total EST sequence data (5.3 Mbp) approximately 400 potential SSRs were found, a frequency similar to what has previously been identified in Arabidopsis ESTs.ConclusionThe AsCIUniGene set will now be used to fabricate an oat biochip, to perform various expression studies with different oat cultivars incubated at varying temperatures, to generate molecular markers and provide tools for various genetic transformation experiments in oat. This will lead to a better understanding of the cellular biology of this important crop and will open up new ways to improve its agronomical properties.

Project description:BACKGROUND: Large scale gene analysis of most organisms is hampered by incomplete genomic sequences. In many organisms, such as soybean, the best source of sequence information is the existence of expressed sequence tag (EST) libraries. Soybean has a large (1115 Mbp) genome that has yet to be fully sequenced. However it does have the 6th largest EST collection comprised of ESTs from a variety of soybean genotypes. Many EST libraries were constructed from RNA extracted from various genetic backgrounds, thus gene identification from these sources is complicated by the existence of both gene and allele sequence differences. We used the ESTminer suite of programs to identify potential soybean gene transcripts from a single genetic background allowing us to observe functional classifications between gene families as well as structural differences between genes and gene paralogs within families. The identification of potential gene sequences (pHaps) from soybean allows us to begin to get a picture of the genomic history of the organism as well as begin to observe the evolutionary fates of gene copies in this highly duplicated genome. RESULTS: We identified approximately 45,000 potential gene sequences (pHaps) from EST sequences of Williams/Williams82, an inbred genotype of soybean (Glycine max L. Merr.) using a redundancy criterion to identify reproducible sequence differences between related genes within gene families. Analysis of these sequences revealed single base substitutions and single base indels are the most frequently observed form of sequence variation between genes within families in the dataset. Genomic sequencing of selected loci indicate that intron-like intervening sequences are numerous and are approximately 220 bp in length. Functional annotation of gene sequences indicate functional classifications are not randomly distributed among gene families containing few or many genes. CONCLUSION: The predominance of single nucleotide insertion/deletions and substitution events between genes within families (individual genes and gene paralogs) is consistent with a model of gene amplification followed by single base random mutational events expected under the classical model of duplicated gene evolution. Molecular functions of small and large gene families appear to be non-randomly distributed possibly indicating a difference in retention of duplicates or local expansion.

Dataset Information

An optimized protocol for analysis of EST sequences.

Publications

An optimized protocol for analysis of EST sequences.

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