Characterization of duplicate gene evolution in the recent natural allopolyploid Tragopogon miscellus by next-generation sequencing and Sequenom iPLEX MassARRAY genotyping

The definitive version is available at www.blackwell-synergy.co

High-throughput gene discovery in the rat

The rat is an important animal model for human diseases and is widely used in physiology. In this article we present a new strategy for gene discovery based on the production of ESTs from serially subtracted and normalized cDNA libraries, and we describe its application for the development of a comprehensive nonredundant collection of rat ESTs. Our new strategy appears to yield substantially more EST clusters per ESTs sequenced than do previous approaches that did not use serial subtraction. However, multiple rounds of library subtraction resulted in high frequencies of otherwise rare internally primed cDNAs, defining the limits of this powerful approach. To date, we have generated >200,000 3′ ESTs from >100 cDNA libraries representing a wide range of tissues and developmental stages of the laboratory rat. Most importantly, we have contributed to ∼50,000 rat UniGene clusters. We have identified, arrayed, and derived 5′ ESTs from >30,000 unique rat cDNA clones. Complete information, including radiation hybrid mapping data, is also maintained locally at http://genome.uiowa.edu/clcg.html. All of the sequences described in this article have been submitted to the dbEST division of the NCBI

Generation of a large scale repertoire of Expressed Sequence Tags (ESTs) from normalised rainbow trout cDNA libraries

BACKGROUND: Within the framework of a genomics project on livestock species (AGENAE), we initiated a high-throughput DNA sequencing program of Expressed Sequence Tags (ESTs) in rainbow trout, Oncorhynchus mykiss. RESULTS: We constructed three cDNA libraries including one highly complex pooled-tissue library. These libraries were normalized and subtracted to reduce clone redundancy. ESTs sequences were produced, and 96 472 ESTs corresponding to high quality sequence reads were released on the international database, currently representing 42.5% of the overall sequence knowledge in this species. All these EST sequences and other publicly available ESTs in rainbow trout have been included on a publicly available Website (SIGENAE) and have been clustered into a total of 52 930 clusters of putative transcripts groups, including 24 616 singletons. 57.1% of these 52 930 clusters are represented by at least one Agenae EST and 14 343 clusters (27.1%) are only composed by Agenae ESTs. Sequence analysis also reveals that normalization and especially subtraction were effective in decreasing redundancy, and that the pooled-tissue library was representative of the initial tissue complexity. CONCLUSION: Due to present work on the construction of rainbow trout normalized cDNA libraries and their extensive sequencing, along with other large scale sequencing programs, rainbow trout is now one of the major fish models in term of EST sequences available in a public database, just after Zebrafish, Danio rerio. This information is now used for the selection of a non redundant set of clones for producing DNA micro-arrays in order to examine global gene expression

Analysis of a human brain transcriptome map

BACKGROUND: Genome wide transcriptome maps can provide tools to identify candidate genes that are over-expressed or silenced in certain disease tissue and increase our understanding of the structure and organization of the genome. Expressed Sequence Tags (ESTs) from the public dbEST and proprietary Incyte LifeSeq databases were used to derive a transcript map in conjunction with the working draft assembly of the human genome sequence. RESULTS: Examination of ESTs derived from brain tissues (excluding brain tumor tissues) suggests that these genes are distributed on chromosomes in a non-random fashion. Some regions on the genome are dense with brain-enriched genes while some regions lack brain-enriched genes, suggesting a significant correlation between distribution of genes along the chromosome and tissue type. ESTs from brain tumor tissues have also been mapped to the human genome working draft. We reveal that some regions enriched in brain genes show a significant decrease in gene expression in brain tumors, and, conversely that some regions lacking in brain genes show an increased level of gene expression in brain tumors. CONCLUSIONS: This report demonstrates a novel approach for tissue specific transcriptome mapping using EST-based quantitative assessment

Gene capture prediction and overlap estimation in EST sequencing from one or multiple libraries

BACKGROUND: In expressed sequence tag (EST) sequencing, we are often interested in how many genes we can capture in an EST sample of a targeted size. This information provides insights to sequencing efficiency in experimental design, as well as clues to the diversity of expressed genes in the tissue from which the library was constructed. RESULTS: We propose a compound Poisson process model that can accurately predict the gene capture in a future EST sample based on an initial EST sample. It also allows estimation of the number of expressed genes in one cDNA library or co-expressed in two cDNA libraries. The superior performance of the new prediction method over an existing approach is established by a simulation study. Our analysis of four Arabidopsis thaliana EST sets suggests that the number of expressed genes present in four different cDNA libraries of Arabidopsis thaliana varies from 9155 (root) to 12005 (silique). An observed fraction of co-expressed genes in two different EST sets as low as 25% can correspond to an actual overlap fraction greater than 65%. CONCLUSION: The proposed method provides a convenient tool for gene capture prediction and cDNA library property diagnosis in EST sequencing

Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.)

<p>Abstract</p> <p>Background</p> <p>Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean.</p> <p>Results</p> <p>Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes.</p> <p>Conclusions</p> <p>The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean.</p

Quantitative comparison of EST libraries requires compensation for systematic biases in cDNA generation

BACKGROUND: Publicly accessible EST libraries contain valuable information that can be utilized for studies of tissue-specific gene expression and processing of individual genes. This information is, however, confounded by multiple systematic effects arising from the procedures used to generate these libraries. RESULTS: We used alignment of ESTs against a reference set of transcripts to estimate the size distributions of the cDNA inserts and sampled mRNA transcripts in individual EST libraries and show how these measurements can be used to inform quantitative comparisons of libraries. While significant attention has been paid to the effects of normalization and substraction, we also find significant biases in transcript sampling introduced by the combined procedures of reverse transcription and selection of cDNA clones for sequencing. Using examples drawn from studies of mRNA 3'-processing (cleavage and polyadenylation), we demonstrate effects of the transcript sampling bias, and provide a method for identifying libraries that can be safely compared without bias. All data sets, supplemental data, and software are available at our supplemental web site [1]. CONCLUSION: The biases we characterize in the transcript sampling of EST libraries represent a significant and heretofore under-appreciated source of false positive candidates for tissue-, cell type-, or developmental stage-specific activity or processing of genes. Uncorrected, quantitative comparison of dissimilar EST libraries will likely result in the identification of statistically significant, but biologically meaningless changes