Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii

ABSTRACT: 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.

ORGANISM(S): Athelia rolfsii

SUBMITTER: Jochen Schmid 

PROVIDER: E-GEOD-21040 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress