Assessing and improving gene annotation for C. reinhardtii: iterative process integrating gene annotation experiments with metabolic network reconstruction and analysis. Starting with a draft network reconstruction, EC terms associated with model reactions are mapped to corresponding transcripts. Experimentally verified transcripts are used to propose changes in structural annotation, along with functional annotation changes that motivate refinements in the network reconstruction. The reconstructed metabolic network is then used to motivate another round of transcript verification experiments.

Integrating the network model with transcript verification experiments. (a) Comparison of central metabolic EC terms annotated in existing JGI v3.0 and our annotation of JGI v3.1 (Supplementary Note). (b) Applying these two versions of EC annotation to inform the network reconstruction yielded functional differences in core metabolic pathways, as illustrated in acetate uptake pathways inferred from the two sets of annotation. As acetate is the sole carbon source used by wild-type C. reinhardtii in vivo, these pathway differences translate directly to measureable growth phenotypes. (c) Results summary for verification and structural annotation of C. reinhardtii central metabolic transcripts by RT-PCR and RACE. ‘Partially verified’ denotes cases for which the assembled ORF did not completely match the genome sequence or a complete sequence could not be assembled.