ORF-omics Boone –review Vidal – paper
Goal of genomics • To generate a comprehensive and integrated view of cellular function • Develop genome-scale reagents for each model organism. • What do you need to do this? • Good sequence • Gene identification and annotation • Resources (money and people)
What are the genome-scale reagents? • Sequence • Oligo sets or PCR primers for genes (microarrays) • RNAi sets or other knockdown or knockout genome-scale reagents (test phenotype, synthetic lethal, etc.) • Proteomic constructs – tagged proteins, two-hybrid libraries, other genomic libraries, e.g. for overexpression, regulated expression
What is an ORF? • Called hypothetical until protein or RNA product detected • 30 years of genetics – 800 C. elegans genes • Previously, Reboul had PCR’d from a cDNA library – 11,984 ORFs (2,179 actually shown previously) • ORF-sequence tags suggest 50% of the annotations are faulty.
How would you find ORFs? • Sequence analysis • Look for open reading frames, intron/exon boundries • Comparative sequencing • ORFs produce RNAs • How would you detect them using technology you know? • cDNA libraries and tiling
Better clone selection Better cloning system
Results • Increased number of known genes by 35% • Over 3000 ORFs had a structure that differed from that predicted by GeneFinder • Some may be due to splice variants • Assume that most ORFs that weren’t cloned were due to mispredictions of gene structure (1/3 of ORFs) • Suggest 50% of ORFs are misannotated
ORFeome project • Gene prediction tools still need work • Want to identify ORFs • Provide reagents • ORFeome 1.1 11,942 ORFS • 11.4% cloned out of frame because of mispredicted gene boundries • Now making ORFeome v2 – all splice variants • 19,920 predicted genes
What is a phenome? • What is RNAi? • What questions can you address?
Process • Put ORFeome 1.1 clones into vector for E. coli • Makes dsRNA
Using clones for additional Analysis: Two hybrid SDS – MS/MS Isolation of tagged proteins