Identification and analysis of expressed sequence tags present in xylem tissues of kelampayan (Neolamarckia cadamba (Roxb.) Bosser)

The large-scale genomic resource for kelampayan was generated from a developing xylem cDNA library. A total of 6,622 high quality expressed sequence tags (ESTs) were generated through high-throughput 5’ EST sequencing of cDNA clones. The ESTs were analyzed and assembled to generate 4,728 xylogenesis unigenes distributed in 2,100 contigs and 2,628 singletons. About 59.3 % of the ESTs were assigned with putative identifications whereas 40.7 % of the sequences showed no significant similarity to any sequences in GenBank. Interestingly, most genes involved in lignin biosynthesis and several other cell wall biosynthesis genes were identified in the kelampayan EST database. The identified genes in this study will be candidates for functional genomics and association genetic studies in kelampayan aiming at the production of high value forests.     

Analysis of expressed sequence tags from the fungus Aspergillus oryzae cultured under different conditions.

We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories.     

Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis

Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.     

Analysis of expressed sequence tags from oil palm (Elaeis guineensis)

This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non-redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array-based expression analysis in oil palm.     

Comparative gene expression in the symbiotic and aposymbiotic Aiptasia pulchella by expressed sequence tag analysis

Intracellular symbiotic relationships are prevalent between cnidarians, such as corals and sea anemones, and the photosynthetic dinoflagellate symbionts. However, there is little understanding about how the genes express when the symbiotic relationship is set up. To characterize genes involved in this association, the endosymbiosis between sea anemone, Aiptasia pulchella, and dinoflagellate zooxanthellae, Symbiodinium spp., was employed as a model. Two complementary DNA (cDNA) libraries were constructed from RNA isolated from symbiotic and aposymbiotic A. pulchella. Using single-pass sequencing of cDNA clones, a total of 870 expressed sequence tags (ESTs) clones were generated from the two libraries: 474 from symbiotic animal and 396 from aposymbiotic animal. The initial ESTs consisted of 143 clusters and 231 singletons. A BLASTX search revealed that 147 unique genes had similarities with protein sequences available from databases; 120 of these clones were categorized according to their putative function. However, many ESTs could not assign functionally. The putative roles of some of the identified genes relative to endosymbiosis were discussed. This is the first report of the use of EST analysis to examine the gene expression in symbiotic and aposymbiotic states of the cnidarians. The systematic analysis of EST from this study provides a useful database for future investigations of the molecular mechanisms involved in algal-cnidarian symbiosis.     

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.     

Transcriptome generation and analysis from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758) through normalized cDNA library

Catfishes are commercially important fish for both the fisheries and aquaculture industry. Clarias batrachus, an Indian catfish species is economically important owing to its high demand. A normalized cDNA library was constructed from spleen of the Indian catfish to identify genes associated with immune function. One thousand nine hundred thirty seven ESTs were submitted to the GenBank with an average read length of approximately 700 bp. Clustering analysis of ESTs yielded 1,698 unique sequences, including 184 contigs and 1,514 singletons. Significant homology to known genes was found by homology searches against data in GenBank in 576 (34 %) ESTs, including similarity to functionally annotated unigenes for 158 ESTs. Additionally, 433 ESTs revealed similarity to unigenes and ESTs in the dbEST but the remaining 658 EST sequences (39 %) did not match any sequence in GenBank. Of a total of 1,698 ESTs generated, 65 ESTs were found to be associated with immune functions. Gene Ontology and KEGG pathway analyses of C. batrachus ESTs collectively revealed a preponderance of immune relevant pathways apart from the presence of pathways involved in protein processing, localization, folding and protein degradation. This study constitutes first EST analysis of lymphoid organ in aquaculturally important Indian catfish species and could pave the way for further research of immune-related genes and functional genomics in this catfish.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.     

Using mtDNA Sequences to Estimate SNP Parameters in ESTs

Discovery of single nucleotide polymorphisms (SNPs) requires analysis of redundant sequences such as those available in large public databases. The ability to detect SNPs, especially those of low frequency, is dependent on the depth and scale of the discovery effort. Large numbers of SNPs have been identified by mining large-scale EST surveys and whole genome sequencing projects. These surveys however are subject to ascertainment bias and the inherent errors in large-scale single pass sequencing efforts. For example, the number of steps involved in the construction and sequencing of cDNA libraries make ESTs highly error prone, resulting in an increased frequency of nonvalid SNPs obtained in these surveys. Sequences of mtDNA genes are often incorporated into cDNA libraries as an artifact of the library construction process and are typically either subtracted from cDNA libraries or are considered superfluous when evaluating the information content of EST datasets. Sequences of mtDNA genes provide a unique resource for the analysis of SNP parameters in EST projects. This study uses sequences from four turkey muscle cDNA libraries to demonstrate how mtDNA sequences gleaned from collections of ESTs can be used to estimate SNP parameters and thus help predict the validity of SNPs.     

Using mtDNA sequences to estimate SNP parameters in ESTs

Discovery of single nucleotide polymorphisms (SNPs) requires analysis of redundant sequences such as those available in large public databases. The ability to detect SNPs, especially those of low frequency, is dependent on the depth and scale of the discovery effort. Large numbers of SNPs have been identified by mining large-scale EST surveys and whole genome sequencing projects. These surveys however are subject to ascertainment bias and the inherent errors in large-scale single pass sequencing efforts. For example, the number of steps involved in the construction and sequencing of cDNA libraries make ESTs highly error prone, resulting in an increased frequency of nonvalid SNPs obtained in these surveys. Sequences of mtDNA genes are often incorporated into cDNA libraries as an artifact of the library construction process and are typically either subtracted from cDNA libraries or are considered superfluous when evaluating the information content of EST datasets. Sequences of mtDNA genes provide a unique resource for the analysis of SNP parameters in EST projects. This study uses sequences from four turkey muscle cDNA libraries to demonstrate how mtDNA sequences gleaned from collections of ESTs can be used to estimate SNP parameters and thus help predict the validity of SNPs.     

Development of Expressed Sequence Tags from the Bay Scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value ≤ 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.     

Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination

In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 × 10⁵ cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination.