To assess the accuracy of Rockhopper’s transcript abundance estimates and the specificity of Rockhopper’s novel transcript identifications, we simulated an RNA-Seq experiment in N. gonorrhoeae using the Flux Simulator (http://flux.sammeth.net ), which models RNA-seq experiments in silico. First, using the Flux Simulator, we simulated an artificial expression profile based on the 2002 annotated genes in the N. gonorrhoeae genome (21 (link)). The Flux Simulator chooses gene expression levels at random based on a mixed power and exponential law. We used a mean of 25 for the exponential distribution used to model variations in transcription start sites for each gene, we used random hexamers as primers for first strand synthesis during reverse transcription, and we used uniform random fragmentation during the Flux Simulator’s fragmentation process. The Flux Simulator’s default values were used for PCR amplification during final library preparation. Finally, we used the Flux Simulator to simulate 31 million single-end reads of length 72 nt. Rockhopper aligned to the N.gonorrhoeae genome ∼15 million of the sequencing reads produced by the Flux Simulator. We used these aligned reads together with the simulated expression profiles for the annotated N.gonorrhoeae genes to assess Rockhopper’s accuracy in quantifying transcript abundances and to evaluate Rockhopper’s propensity for false positive identifications of novel transcripts.
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