Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii
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ABSTRACT:
ORGANISM(S): Agroathelia rolfsii
PROVIDER: GSE21040 | GEO | 2010/05/15
SECONDARY ACCESSION(S): PRJNA126417
REPOSITORIES: GEO
Similar Datasets
Project description:Two S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding ~350,000 reads. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Sequence data were used to obtain first insights into the genomics of scleroglucan and oxalate production and to predict putative proteins involved in the synthesis of both metabolites. Using comparative transcriptomics, namely Agilent microarray hybridization and suppression subtractive hybridization, we identified ~ 800 unigenes which are differently expressed under scleroglucan-producing and non-producing conditions. Tailor-made microarrays (44K multiplex chip, Agilent) were designed by imaGenes (Berlin, Germany) using an in-house developed method for empirical selection of best performing probes for each gene (Pre Selection Strategy). Briefly, up to ten probes were designed for each of the 454 unigenes (60 bp long oligomers). The 244K Agilent test array was hybridized with pooled Cy3-labeled cRNAs gained form EPSmax13 and EPSmin17 cultures and (in average) two of the best performing oligos were selected for each unigene. For comparative expression profiling, total RNA was isolated from S. rolfsii, cultured for 37 h in EPSmax13 (biological triplicate) and EPSmin17 media (biological triplicate). RNA quality control, synthesis of Cy3-labeled cRNA including cRNA purification and cRNA quality control, microarray hybridization, scanning and data extraction (Agilent´s feature extraction software) were performed by imaGenes GmbH. Expression data were analyzed by imaGenes GmbH using an in-house developed data analysis pipeline. After quantile normalization, genes were defined as differentially expressed if their expression levels varied at least 2 fold in EPSmax13 samples compared to EPSmin17 samples and if the difference was statistically significant (Student’s t-test, P-value cut-off of 0.05). 454 pyrosequencing cDNA study data was uploaded at SRA and assigned accession number SRP002142.
2010-05-15 | E-GEOD-21040 | biostudies-arrayexpress
Project description:Two S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding ~350,000 reads earlier. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Our first transcriptomic analysis, comparing an early stage of scleroglucan synthesis in high-level and low-level producing media, uncovered that most differentially expressed unigenes belong to the functional categories ‘metabolism’ and ‘transport’ (Schmid et al., 2010). In order to get more insights into transcriptional regulation of scleroglucan production, we extended in this study our transcriptomic analyses. We determined transcriptomic profiles for a later stage of cultivation in both media, thus giving us the opportunity to study temporal phenomena under high- and low-level producing conditions.
2011-03-01 | GSE25254 | GEO
Project description:Seedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored. An integrative strategy combining SSH (suppression subtractive hybridization) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulate Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution showed that the majority of differential genes involved in metabolic process, responded to stimulus and functioned as DNA/RNA binding, catalytic activity and oxidoreductase activity. Expression dynamics of some candidate clones revealed a gene encoding male sterility protein 2 was highly up-regulated, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were specifically down-regulated in the seedless mutant compared with the wild type. Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future.
2012-05-22 | GSE38094 | GEO
Project description:The soil worm Enchytraeus crypticus (oligochaete) is an ecotoxicology model species although without genome or transcriptome sequence information. The present research aimed at studying, via high-throughput pyrosequencing, the transcriptome of Enchytraeus crypticus, sampled from multiple test conditions, and the construction of a high-density microarray for functional genomic studies. A pyrosequencing run retrieved approximately 1.5 million reads representing 645 million bases. After assembly, 27,296 contigs and 87,686 singletons were obtained. from which 44% and 25% were annotated as protein-coding genes. We show that the high amount of orphan genes is not due to poor sequence or assemble quality: 84% of the contig sequences contains an open reading frame with a start codon and E. crypticus homologs were identified for 92% of the core eukaryotic genes. Moreover, 65 and 77% of the unknown singletons and contigs, respectively, showed transcriptional activity. An Agilent 180K microarray platform was designed and validated by hybridizing cDNA from 3 day zinc- exposed E. crypticus to the concentration corresponding to 50% reduction in reproduction (EC50). Overall, 70% of all probes exerted a hybridization signal above background level. More specifically, the probes derived from contigs showed a wider range of average intensities when compared to probes derived from singletons. In total, 522 significantly regulated transcripts were identifying upon zinc exposure. Several significantly regulated genes exerted predicted functions (e.g. zinc efflux, zinc transport) associated with zinc stress. Unexpectedly, the microarray data suggest that zinc exposure alters retrotransposon activity in the E. crypticus genome. In conclusion, characterization of the presented E. crypticus transcriptome and associated microarray platform is a valuable and high quality resource that permits further functional genomics experiments examining gene expression patterns underlying distinct environmental stress conditions. We show that unknown sequences are not the result of technical errors but mostly represent functional genes that are actively transcribed.
2014-05-01 | GSE51838 | GEO
Project description:Transcriptome from high throughput sequencing-by-synthesis is a good resource of molecular markers. In this study, we present utility of massively parallel sequencing by synthesis for profiling the transcriptome of red pepper (Capsicum annuum L. TF68) by 454 GS-FLX pyrosequencing. Through the generation of approximately 30.63 megabases (Mb) of Expressed Sequence Tags (ESTs) data with the average length of 375 base pairs (bp), 9,818 contigs and 23,712 singletons were obtained by assembly. Using BLAST alignment against NCBI non-redundant and a UniProt protein database, 30% of the tentative consensus sequences were assigned to specific function annotation, while 24% returned alignments of unknown function, leaving up to 46% with no alignment. Functional classification using FunCat revealed that sequences with putative known function were distributed cross 18 categories. Furthermore, over 200 high quality single nucleotide discrepancies were discovered using the Bukang cDNA collection as a reference database. Moreover, 758 simple sequence repeat (SSR) motif loci were mined from over 600 contigs, from which 572 primer sets were designed. The SSR motifs corresponded to di- and tri- nucleotide motifs (27.03 and 61.92%, respectively). These molecular markers may be of great value for application in linkage mapping and association mapping research.
2011-09-08 | GSE29215 | GEO
Project description:A method was developed to isolate RNA from 1 dpa fiber. ESTs derived from this and other cotton mRNAs were sequenced and assembled into contigs. Contigs composed of EST unique to libraries of interest and unique singletons were represented on a microarray. Microarrays were hybridized with labed nucleic acids derived from whole 1 dpa ovules and 1 dpa fiber to identify genes differentially regulated during fiber initiation. Genes with known expression in fiber were included to validate the microarray. Analysis of the gene ontology (GO) of differentially expressed genes suported previously reported aspects of fiber initiation such as an increase in protein biosynthesis. An increase in the "membrane" was indicated by the GO analysis ans supported by increased staining of fiber initial ER. Analysis of individule genes differentially regulated in fiber initials indicated calcium may play a role in fiber deveopment which was also supported by an increase in calcium in fiber initials. Keywords: comparative hybridization
2007-06-26 | GSE6851 | GEO
Project description:Transcriptome from high throughput sequencing-by-synthesis is a good resource of molecular markers. In this study, we present utility of massively parallel sequencing by synthesis for profiling the transcriptome of red pepper (Capsicum annuum L. TF68) by 454 GS-FLX pyrosequencing. Through the generation of approximately 30.63 megabases (Mb) of Expressed Sequence Tags (ESTs) data with the average length of 375 base pairs (bp), 9,818 contigs and 23,712 singletons were obtained by assembly. Using BLAST alignment against NCBI non-redundant and a UniProt protein database, 30% of the tentative consensus sequences were assigned to specific function annotation, while 24% returned alignments of unknown function, leaving up to 46% with no alignment. Functional classification using FunCat revealed that sequences with putative known function were distributed cross 18 categories. Furthermore, over 200 high quality single nucleotide discrepancies were discovered using the Bukang cDNA collection as a reference database. Moreover, 758 simple sequence repeat (SSR) motif loci were mined from over 600 contigs, from which 572 primer sets were designed. The SSR motifs corresponded to di- and tri- nucleotide motifs (27.03 and 61.92%, respectively). These molecular markers may be of great value for application in linkage mapping and association mapping research. 1 sample TF68 accession examined
2011-09-08 | E-GEOD-29215 | biostudies-arrayexpress
Project description:Seedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored. An integrative strategy combining SSH (suppression subtractive hybridization) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulate Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution showed that the majority of differential genes involved in metabolic process, responded to stimulus and functioned as DNA/RNA binding, catalytic activity and oxidoreductase activity. Expression dynamics of some candidate clones revealed a gene encoding male sterility protein 2 was highly up-regulated, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were specifically down-regulated in the seedless mutant compared with the wild type. Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future. Flower buds from the seedless citrus mutant and its wild type were collected at four developmental stages: quaring stage (SF, about 20 DBF), medium bud stage (MF, about 10 DBF), flowers at full bloom stage (BF) and young ovaries of 2-3 days after flowering (OV). Total RNA extracted from the mutant and the wild type was hybridized to the array. The hybridization was performed in duplicate by dye swap.
2012-05-21 | E-GEOD-38094 | biostudies-arrayexpress
Project description:Gene expression analysis of stomach ECL cells and mixed gastric fundic epithelial cells Keywords: Gastric ECL cells, Subtractive Hybridization, Comparative genomic hybridization
2006-01-26 | GSE3518 | GEO
Project description:Transcriptional profiling of gastric tumors compared to paired adjacent non-tumoral gastric tissue by two strategies. The first strategy is a conventional microarray assay, while the second strategy involves a previous Suppression Subtractive Hybridization (SSH) followed by a conventional microarray assay. We applied subtractive hybridization followed by microarray analysis to identify tumor-specific low-abundance transcripts. The goal was to determine differences in gene expression profile in tumor samples by both strategies.
2012-06-27 | GSE38932 | GEO