Experimental Verification Alexandre Reymond Department of Genetic Medicine

1. Experimental Verification Alexandre Reymond Department of Genetic Medicine

2. Gene predictions outside of VEGA • RT-PCR all pairs of exons predicted outside of VEGA objects • Goal: • Confirm experimentally new gene models • Question(s): • How good are the predictions outside of the mainstream? • Is there any coding gene out there?

3. Gene predictions outside of VEGA • RT-PCR all pairs of exons predicted outside of VEGA objects in 13 of the 44 ENCODE regions • Oligos are designed with Primer 3 and default parameters • Individually on 24 human polyA RNA

4. Gene predictions outside of VEGA • 1255 pairs of exons predicted by nine different programs outside of VEGA objects (13 ENCODE regions)

5. Gene predictions outside of VEGA • Tested 1215 pairs for a total of 29160 reactions • 2.6% of success rate (32/1215) • 1.9% with perfectly predicted exon boundaries (23/1215) • 0.7% with displaced exon boundaries (9/1215)

6. Gene predictions outside of VEGA • RT-PCR all pairs of exons predicted outside of VEGA objects • Goal: • Confirm experimentally new gene models • Question(s): • How good are the predictions outside of the mainstream? • Is there any coding gene out there?

7. RT-PCR VEGA objects • RT-PCR of all the VEGA objects belonging to the « novel transcripts », the « putative transcripts» and the « non-canonical splicing » class. • Goal: • Confirm experimentally new genes • Question: • Is the VEGA annotation team doing a good job?

8. 5’ RACE • 5’ RACE of all the VEGA objects belonging to the « known » and the « novel  protein» class • Goal: • Find new 5’ end exons and/or new 5’ UTRs due to alternative splicing • Question: • Should a 5’RACE effort be pursued for the entire genome (ENCODE consolidation phase)?

9. 5’ RACE • Primers are designed with “Primer 3” just 3’ of the ATG in an exon common to most (all) described transcripts of this gene • Parameters (25 nts, 68°<Tm<72°, best 70°, 50-70% GC) • cDNA is prepared with “BD SMART RACE” kit • Touchdown 72°, 70° and 68° DNA mRNA

10. 5’ RACE results • 5’RACE in 12 tissues on 426 genes • Pick tissue with a single large bands • 52% success rate (220/426) • Pick a second tissue with a single large band • Add 6% and get to 58% rate success (245/426) • Clone and sequence band from tissues with multiple hits • Add 5-15%...