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Abstract
The RNA sequencing (RNA-Seq) analysis pipeline can be complex and variable between transcriptomic studies, and therefore necessitates the need for optimizing methodology to achieve the best results. Further, few transcriptomic methods have been used to analyze the regulation of the Crassulacean acid metabolism (CAM) photosynthesis pathway, much less comparatively against a non-CAM species or in the Orchidaceae. Here we perform a temporal gene expression analysis between CAM species Erycina pusilla and close C3 relative Erycina crista-galli, and establish an optimal methodology to assemble and analyze time series RNA-Seq data for the purpose of studying the evolution and regulation of CAM photosynthesis. Gene clustering for temporal patterns reveals a network of genes associated with canonical CAM gene phosphoenolpyruvate carboxylase (PEPC) present in both E. pusilla and E. crista-galli, suggesting a possible ancestral predisposition for CAM.