Expressed sequence tags and their application for plant research

Expressed Sequence Tags (ESTs) are short, usually unedited sequences obtained by single-pass sequencing of cDNA clones from any cDNA library. Analyzing and comparing ESTs can provide information on gene expression, function and evolution. Large-scale EST sequencing has become an attractive alternative to plant genome sequencing. Currently, plant EST collections comprise over 3.8 million sequences from about 200 species. They have proved to be a valuable tool for gene discovery and plant metabolism analysis. Several plant-specific EST databases have been created which provide access to sequence data and bioinformatics-based tools for data mining. Searching EST collections allows pre-selection of genes for preparing cDNA arrays, targeted to bring maximum information on specialized processes, like stress response, symbiotic nitrogen fixation etc. Also, ESt-based molecular markers such as SNP, SSR, and indels are fast developing tools for breeders and researchers.     

EST and transcriptome analysis of cephalochordate amphioxus--past, present and future

The cephalochordates, commonly known as amphioxus or lancelets, are now considered the most basal chordate group, and the studies of these organisms therefore offer important insights into various levels of evolutionary biology. In the past two decades, the investigation of amphioxus developmental biology has provided key knowledge for understanding the basic patterning mechanisms of chordates. Comparative genome studies of vertebrates and amphioxus have uncovered clear evidence supporting the hypothesis of two-round whole-genome duplication thought to have occurred early in vertebrate evolution and have shed light on the evolution of morphological novelties in the complex vertebrate body plan. Complementary to the amphioxus genome-sequencing project, a large collection of expressed sequence tags (ESTs) has been generated for amphioxus in recent years; this valuable collection represents a rich resource for gene discovery, expression profiling and molecular developmental studies in the amphioxus model. Here, we review previous EST analyses and available cDNA resources in amphioxus and discuss their value for use in evolutionary and developmental studies. We also discuss the potential advantages of applying high-throughput, next-generation sequencing (NGS) technologies to the field of amphioxus research.     

HaMStR: Profile hidden markov model based search for orthologs in ESTs

Background  

EST sequencing is a versatile approach for rapidly gathering protein coding sequences. They provide direct access to an organism's gene repertoire bypassing the still error-prone procedure of gene prediction from genomic data. Therefore, ESTs are often the only source for biological sequence data from taxa outside mainstream interest. The widespread use of ESTs in evolutionary studies and particularly in molecular systematics studies is still hindered by the lack of efficient and reliable approaches for automated ortholog predictions in ESTs. Existing methods either depend on a known species tree or cannot cope with redundancy in EST data.     

一种新的EST聚类方法

该研究发展了一种EST（expressed sequence tag）聚类方法（ESTClustering）,用于分析大规模EST测序中所产生的大量数据,以获得高质量,非重复表达序列,该方法在聚类过程中采用MEGABLAST工具对一致序列进行序列同源比较,并用phrap程序对每一EST簇进行拼接检验。这一聚类策略能降低测序错误带来的影响,有效识别基因家族成员,并避免选择性剪接的干扰,与NCB（National Center for Biotechnology Information）的UniGene clustering）方法相比,ESTClustering的聚类结果可以更好地反映表达序列的多样性,用ESTClustering对112256条拟南芥EST聚类测试,产生23581个EST簇,其中13597个EST簇有对应拟南芥基因组编码序列,与该基因组中有EST作为依据的预测基因数目接近。应用该方法对收集的147191条水稻EST序列进行聚类,形成33896个EST簇。     

Efficient clustering of large EST data sets on parallel computers

Clustering expressed sequence tags (ESTs) is a powerful strategy for gene identification, gene expression studies and identifying important genetic variations such as single nucleotide polymorphisms. To enable fast clustering of large-scale EST data, we developed PaCE (for Parallel Clustering of ESTs), a software program for EST clustering on parallel computers. In this paper, we report on the design and development of PaCE and its evaluation using Arabidopsis ESTs. The novel features of our approach include: (i) design of memory efficient algorithms to reduce the memory required to linear in the size of the input, (ii) a combination of algorithmic techniques to reduce the computational work without sacrificing the quality of clustering, and (iii) use of parallel processing to reduce run-time and facilitate clustering of larger data sets. Using a combination of these techniques, we report the clustering of 168 200 Arabidopsis ESTs in 15 min on an IBM xSeries cluster with 30 dual-processor nodes. We also clustered 327 632 rat ESTs in 47 min and 420 694 Triticum aestivum ESTs in 3 h and 15 min. We demonstrate the quality of our software using benchmark Arabidopsis EST data, and by comparing it with CAP3, a software widely used for EST assembly. Our software allows clustering of much larger EST data sets than is possible with current software. Because of its speed, it also facilitates multiple runs with different parameters, providing biologists a tool to better analyze EST sequence data. Using PaCE, we clustered EST data from 23 plant species and the results are available at the PlantGDB website.     

芦笋EST资源的SSR信息分析

为了在芦笋中开发EST-SSR功能性标记,对来源于NCBI公共数据库的8590条芦笋(AsparagusofficinalisL.)EST序列进行简单重复序列SSR搜索。剔除冗余序列,得到非冗余序列8377条。在非冗余序列中共挖掘出469个EST-SSR,平均相隔14.80kb出现1个SSR。在所有的重复基序中,二核苷酸重复基序的SSR所占比例最高40.51%(190/469),其次是三核苷酸34.97%(164/469),六核苷酸21.11%(99/469)。在所有基序里,CT/AG出现的频率最高有62次,占全部重复基序的13.22%(62/469)。选取含SSR的EST序列30条,并利用primer5软件设计引物,进行SSR位点的扩增,其中27对引物扩增产物,24对有较清晰可靠的目标扩增条带,占引物数的80%,且所检测出的芦笋等位基因数量较丰富,平均4.93个/对。这些EST-SSR标记的开发将有助于芦笋群体遗传多样性、遗传图谱构建、基因定位、分子标记和系谱分析等方面的研究。     

Species are hypotheses: avoid connectivity assessments based on pillars of sand

Connectivity among populations determines the dynamics and evolution of populations, and its assessment is essential in ecology in general and in conservation biology in particular. The robust basis of any ecological study is the accurate delimitation of evolutionary units, such as populations, metapopulations and species. Yet a disconnect still persists between the work of taxonomists describing species as working hypotheses and the use of species delimitation by molecular ecologists interested in describing patterns of gene flow. This problem is particularly acute in the marine environment where the inventory of biodiversity is relatively delayed, while for the past two decades, molecular studies have shown a high prevalence of cryptic species. In this study, we illustrate, based on marine case studies, how the failure to recognize boundaries of evolutionary‐relevant unit leads to heavily biased estimates of connectivity. We review the conceptual framework within which species delimitation can be formalized as falsifiable hypotheses and show how connectivity studies can feed integrative taxonomic work and vice versa. Finally, we suggest strategies for spatial, temporal and phylogenetic sampling to reduce the probability of inadequately delimiting evolutionary units when engaging in connectivity studies.     

Genomic tools and cDNA derived markers for butterflies

The Lepidoptera have long been used as examples in the study of evolution, but some questions remain difficult to resolve due to a lack of molecular genetic data. However, as technology improves, genomic tools are becoming increasingly available to tackle unanswered evolutionary questions. Here we have used expressed sequence tags (ESTs) to develop genetic markers for two Müllerian mimic species, Heliconius melpomene and Heliconius erato. In total 1363 ESTs were generated, representing 330 gene objects in H. melpomene and 431 in H. erato. User-friendly bioinformatic tools were used to construct a nonredundant database of these putative genes (available at http://www.heliconius.org), and annotate them with blast similarity searches, InterPro matches and Gene Ontology terms. This database will be continually updated with EST sequences for the Papilionideae as they become publicly available, providing a tool for gene finding in the butterflies. Alignments of the Heliconius sequences with putative homologues derived from Bombyx mori or other public data sets were used to identify conserved PCR priming sites, and develop 55 markers that can be amplified from genomic DNA in both H. erato and H. melpomene. These markers will be used for comparative linkage mapping in Heliconius and will have applications in other phylogenetic and genomic studies in the Lepidoptera.     

Gene2EST: a BLAST2 server for searching expressed sequence tag (EST) databases with eukaryotic gene-sized queries

Expressed sequence tags (ESTs) are randomly sequenced cDNA clones. Currently, nearly 3 million human and 2 million mouse ESTs provide valuable resources that enable researchers to investigate the products of gene expression. The EST databases have proven to be useful tools for detecting homologous genes, for exon mapping, revealing differential splicing, etc. With the increasing availability of large amounts of poorly characterised eukaryotic (notably human) genomic sequence, ESTs have now become a vital tool for gene identification, sometimes yielding the only unambiguous evidence for the existence of a gene expression product. However, BLAST-based Web servers available to the general user have not kept pace with these developments and do not provide appropriate tools for querying EST databases with large highly spliced genes, often spanning 50 000-100 000 bases or more. Here we describe Gene2EST (http://woody.embl-heidelberg.de/gene2est/), a server that brings together a set of tools enabling efficient retrieval of ESTs matching large DNA queries and their subsequent analysis. RepeatMasker is used to mask dispersed repetitive sequences (such as Alu elements) in the query, BLAST2 for searching EST databases and Artemis for graphical display of the findings. Gene2EST combines these components into a Web resource targeted at the researcher who wishes to study one or a few genes to a high level of detail.     

定位于染色体9p21-22的一个新基因UBAP1的克隆测序及在鼻咽癌中的表达分析

 在染色体 9p2 1 2 2鼻咽癌杂合性丢失 (lossofheterozygosity,LOH)高频区 ,应用EST介导的定位 侯选克隆策略 ,用RT PCR及Northern杂交检测了 2 2个表达序列标记 (expressedsequencetag ,EST)在鼻咽癌细胞株HNE1和原代培养的正常鼻咽上皮细胞中的表达差异 ,并对其中一个在鼻咽癌细胞株HNE1中表达下调的EST检测了在鼻咽癌活检组织中的表达 .用生物信息学方法获得其全长cDNA序列 ,GenBank登录号AF2 2 2 0 4 3.该基因cDNA全长 2 70 1bp ,其开放阅读框 (openreadingframe ,ORF)编码一个含 50 2个氨基酸、分子量为 55kD的碱性蛋白质 ,在蛋白羧基端含有 2个连续的重要UBA功能域 (ubiquitinassociateddomain) ,属于遍在蛋白相关蛋白家族的一个新成员 ,经国际人类基因命名委员会同意 ,将其命名为UBAP1 (ubiquitinassociatedprotein 1 ) .Northern表达分析显示UBAP1在所检测的人组织中广泛表达 ,但在人的心脏、骨骼肌及肝脏中的表达较强 .UBAP1基因在63 2 % ( 1 2 1 9)的鼻咽癌活检组织中表达下调 .UBAP1基因作为一个遍在蛋白相关蛋白家族的新成员 ,结合其在 9p的重要定位信息 ,有必要进一步研究其表达下调参与鼻咽癌发生发展的可能机制 .     

EST pipeline system: detailed and automated EST data processing and mining

Expressed sequence tags (ESTs) are widely used in gene survey research these years. The EST Pipeline System, software developed by Hangzhou Genomics Institute (HGI), can automatically analyze different scalar EST sequences by suitable methods. All the analysis reports, including those of vector masking, sequence assembly, gene annotation, Gene Ontology classification, and some other analyses, can be browsed and searched as well as downloaded in the Excel format from the web interface, saving research efforts from routine data processing for biological rules embedded in the data.     

Identification and characterization of SSRs from soybean (Glycine max) ESTs

Microsatellites, or simple sequence repeats (SSRs), are very useful molecular markers for a number of plant species. We used a new publicly available module (TROLL) to extract microsatellites from the public database of soybean expressed sequence tag (EST) sequences. A total of 12,833 sequences containing di- to penta-type SSRs were identified from 200,516 non-redundant soybean ESTs. On average, one SSR was found per 7.25?kb of EST sequences, with the tri-nucleotide motifs being the most abundant. Primer sequences flanking the SSR motifs were successfully designed for 9,638 soybean ESTs using the software primer3.0 and only 59 pairs of them were found in earlier studies. We synthesized 124 pairs of the primers to determine the polymorphism and heterozygosity among eight genotypes of soybean cultivars, which represented a wide range of the cultivated soybean cultivars. PCR amplification products with anticipated SSRs were obtained with 81 pairs of primers; 36 PCR products appeared to be homozygous and the remaining 45 PCR products appeared to be heterozygous and displayed polymorphism among the eight cultivars. We further analysed the EST sequences containing 45 polymorphic EST-SSR markers using the programs BLASTN and BLASTX. Sequence alignment showed that 29 ESTs have homologous sequences and 15 ESTs could be classified into a Uni-gene cluster with comparatively convincing protein products. Among these 15 ESTs belonging to a Uni-gene cluster, 9 SSRs were located in 3'-UTR, 4 SSRs were located in the intron region and 2 SSRs were located in the CDS region. None of these SSRs was located in the 5'-UTR. These novel SSRs identified in the ESTs of soybean provide useful information for gene mapping and cloning in future studies.