Genome-Wide Identification of Transcriptional Start Sites in the Plant Pathogen Pseudomonas syringae pv. tomato str. DC3000

RNA-Seq has provided valuable insights into global gene expression in a wide variety of organisms. Using a modified RNA-Seq approach and Illumina's high-throughput sequencing technology, we globally identified 5′-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5′-ends obtained by this method were consistent with results obtained using global RNA-Seq and 5′RACE. As expected, many 5′-ends were positioned a short distance upstream of annotated genes. We also captured 5′-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, suggesting additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5′-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels

Evaluation of Methods for Sequence Analysis of Highly Repetitive DNA Templates

The DNA Sequencing Research Group (DSRG) of the ABRF conducted a study to assess the ability of DNA sequencing core facilities to successfully sequence a set of well-defined templates containing difficult repeats. The aim of this study was to determine whether repetitive templates could be sequenced accurately by using equipment and chemistries currently utilized in participating sequencing laboratories. The effects of primer and template concentrations, sequencing chemistries, additives, and instrument formats on the ability to successfully sequence repeat elements were examined. The first part of this study was an analysis of the results of 361 chromatograms from participants representing 40 different laboratories who attempted to sequence a panel of difficult-to-sequence templates using their best in-house protocols. The second part of this study was a smaller multi-laboratory evaluation of a single robust protocol with the same panel of templates. This study provides a measure of the potential success of different approaches to sequencing across homopolymer tracts and repetitive elements