ORF verification by RT-PCR followed by multiplex sequencing. RNA isolated from C. reinhardtii grown under a permissive condition (continuous illumination and acetate as a source of carbon) was reverse transcribed, then used as template for PCR in which ORF-specific primers were used to amplify the JGI annotated ORFs. The amplicons were then sequenced directly using the 454FLX platform, or cloned, then sequenced by 454. (A) Amplification of representative metabolic ORFs are shown after electrophoresis (192 amplicons analyzed in two 96 well E-gels). (B) Percent coverage of 1,427 enzymatic ORF reference sequences by the obtained reads from 454 sequencing. The 454 reads were aligned to the JGI ORF reference sequences and percent coverage of the length of each reference sequence was determined (100% denotes all bases of the reference sequences could be covered by one or more 454 read). The entire lengths of 699 ORFs were 100% verified.
Ghamsari et al. BMC Genomics 2011 12(Suppl 1):S4 doi:10.1186/1471-2164-12-S1-S4