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Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33–36, 2015). At its core is the principle of using gene-specific primers (GSPs) to “seed” (or target) the reaction and universal primers to “drive” the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69–74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48–51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.

Background: Cyanobacteria are potential sources of renewable chemicals and biofuels and serve as model organisms for bacterial photosynthesis, nitrogen fixation, and responses to environmental changes. Anabaena (Nostoc) sp. strain PCC 7120 (hereafter Anabaena) is a multicellular filamentous cyanobacterium that can "fix" atmospheric nitrogen into ammonia when grown in the absence of a source of combined nitrogen. Because the nitrogenase enzyme is oxygen sensitive, Anabaena forms specialized cells called heterocysts that create a microoxic environment for nitrogen fixation. We have employed directional RNA-seq to map the Anabaena transcriptome during vegetative cell growth and in response to combined-nitrogen deprivation, which induces filaments to undergo heterocyst development. Our data provide an unprecedented view of transcriptional changes in Anabaena filaments during the induction of heterocyst development and transition to diazotrophic growth.Results: Using the Illumina short read platform and a directional RNA-seq protocol, we obtained deep sequencing data for RNA extracted from filaments at 0, 6, 12, and 21 hours after the removal of combined nitrogen. The RNA-seq data provided information on transcript abundance and boundaries for the entire transcriptome. From these data, we detected novel antisense transcripts within the UTRs (untranslated regions) and coding regions of key genes involved in heterocyst development, suggesting that antisense RNAs may be important regulators of the nitrogen response. In addition, many 5' UTRs were longer than anticipated, sometimes extending into upstream open reading frames (ORFs), and operons often showed complex structure and regulation. Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism.Conclusions: Directional RNA-seq data were obtained that provide comprehensive mapping of transcrip

Background: Cyanobacteria are potential sources of renewable chemicals and biofuels and serve as model organisms for bacterial photosynthesis, nitrogen fixation, and responses to environmental changes. Anabaena (Nostoc) sp. strain PCC 7120 (hereafter Anabaena) is a multicellular filamentous cyanobacterium that can "fix" atmospheric nitrogen into ammonia when grown in the absence of a source of combined nitrogen. Because the nitrogenase enzyme is oxygen sensitive, Anabaena forms specialized cells called heterocysts that create a microoxic environment for nitrogen fixation. We have employed directional RNA-seq to map the Anabaena transcriptome during vegetative cell growth and in response to combined-nitrogen deprivation, which induces filaments to undergo heterocyst development. Our data provide an unprecedented view of transcriptional changes in Anabaena filaments during the induction of heterocyst development and transition to diazotrophic growth.Results: Using the Illumina short read platform and a directional RNA-seq protocol, we obtained deep sequencing data for RNA extracted from filaments at 0, 6, 12, and 21 hours after the removal of combined nitrogen. The RNA-seq data provided information on transcript abundance and boundaries for the entire transcriptome. From these data, we detected novel antisense transcripts within the UTRs (untranslated regions) and coding regions of key genes involved in heterocyst development, suggesting that antisense RNAs may be important regulators of the nitrogen response. In addition, many 5' UTRs were longer than anticipated, sometimes extending into upstream open reading frames (ORFs), and operons often showed complex structure and regulation. Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism.Conclusions: Directional RNA-seq data were obtained that provide comprehensive mapping of transcrip

Background: Cyanobacteria are potential sources of renewable chemicals and biofuels and serve as model organisms for bacterial photosynthesis, nitrogen fixation, and responses to environmental changes. Anabaena (Nostoc) sp. strain PCC 7120 (hereafter Anabaena) is a multicellular filamentous cyanobacterium that can "fix" atmospheric nitrogen into ammonia when grown in the absence of a source of combined nitrogen. Because the nitrogenase enzyme is oxygen sensitive, Anabaena forms specialized cells called heterocysts that create a microoxic environment for nitrogen fixation. We have employed directional RNA-seq to map the Anabaena transcriptome during vegetative cell growth and in response to combined-nitrogen deprivation, which induces filaments to undergo heterocyst development. Our data provide an unprecedented view of transcriptional changes in Anabaena filaments during the induction of heterocyst development and transition to diazotrophic growth.Results: Using the Illumina short read platform and a directional RNA-seq protocol, we obtained deep sequencing data for RNA extracted from filaments at 0, 6, 12, and 21 hours after the removal of combined nitrogen. The RNA-seq data provided information on transcript abundance and boundaries for the entire transcriptome. From these data, we detected novel antisense transcripts within the UTRs (untranslated regions) and coding regions of key genes involved in heterocyst development, suggesting that antisense RNAs may be important regulators of the nitrogen response. In addition, many 5' UTRs were longer than anticipated, sometimes extending into upstream open reading frames (ORFs), and operons often showed complex structure and regulation. Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism.Conclusions: Directional RNA-seq data were obtained that provide comprehensive mapping of transcrip