1. Experimental Verification Department of Genetic Medicine
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 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
DNA
mRNA
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°

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%...