1. Geuvadis Analysis Meeting 16/02/2012 Micha Sammeth CNAG – Barcelona

2. Quantification of Splice-Forms and Variants - Quantified 615 datasets based on the Gencode v7 annotation -For every transcript, normalized RPKM values and number of deconvoluted reads - Sensitivity is a function of sequencing depth Correlation coeff. 0.87 (Pearson and Spearman) - Discussion at the end if/what to do before uploading

3. LoF Definitions LOF = loss of function of a complete transcript X SNP that introduces (directly) stop codon Indels that disrupt/shift reading frame SNP that disrupts splice site Larger deletions that remove 1 st exon or >50% of transcript X X “partial” LoF affects just some protein-coding transcripts in a locus “full” LoF affects all protein-coding transcripts annotated LoF scope LoF types [MacArthur et al. 2012]

4. X Splice Large deletion Stop Frameshift indel LoF Estimates [MacArthur et al. 2012] X Splice Large deletion Stop Frameshift indel in a single individual across populations

5. Compare RNA-Seq evidence to LoF predictions X X X X predicted disruption of splice site X Large deletion Frameshift indel main difference Geuvadis <> 1000 Genomes: RNA-Seq vs. DNA-Seq } directly from mappings / coverage by mappings indirectly called from mappings

6. Confirmation LoF SNPs in Geuvadis - Take phase1 samples where polymorphisms have been found by exome sequencing - Additionally call SNPs by RNA-Seq (exzessive mappings) Sufficient coverage in DNA Sufficient coverage in RNA >2 million genotype calls possible in both Experiments Example: (not Geuvadis) ~5000 differences, i.e. on average >2 out of 1000 calls differ ~1000 cases where RNA is homozygous and DNA not could be explainable by allele-specific expression ~4000 cases where DNA is homozygous and RNA not (!!!) remove FPs from computational or experimental artifacts (PCR artifacts?) Stop

7. Homozygote Common Allele Heterozygote Homozygote Minority Allele 100% 0% or 100% 0% or 50% 50% relative abundance distribution 1st form relative abundance distribution 2nd form A/AA/GG/G A/AA/GG/G 1 st 2 nd Allele-specific RNA Processing [Montgomery 2010 dataset]

8. LoF and Alternative Splicing (AS) “28.7% LoF events in a single individual affect only a subset of the known transcripts from the affected gene, Emphasizing the need to consider alternative splicing” [MacArthur et al. 2012] X X (1)classification of AS influences in LoF based on a certain annotation (2) extension of an annotation by RNA-Seq evidence 5’ frame 2 2 2 0 1 0 3’ frame ? activation of latent splice sites

9. - - ^ ^ 1 2 3 4 5 6 7 1,2,3,4,5,6,7 1,2,3,6 [ ^ 1,2,3,4,5,6,7 - - ^ - ] 1,2,3, 4,5,7 1,2,3, 4,5,7 1,4 - 6 3,5,7 3,5,6 7 2 5 1 4 1,3,5,6 ^ 1,2,3,6 bubble (1) classification of AS: AStalavista

10. (2) AS discovery by RNA-Seq - - ^ ^ 1,2,3,4,5,6,7 1,2,3,6 [ ^ 1,2,3,4,5,6,7 - - ^ - ] 1,2,3, 4,5,7 1,2,3, 4,5,7 1,4 - 6 3,5,7 3,5,6 7 2 5 1 4 1,3,5,6 ^ 1,2,3,6 Novel exon junctions supported by RNA-Seq add to graph, novel events extend annotated CDSs

11. My Points Quantifications: do you want a normalization before uploading or is this in the responsibility of the analyzing group? Quantifications: Timeline for studies—main paper Oct-end of the year. Separate publications possible if there is sufficient material for a separate story? What would be the constraints for a separate publication on Geuvadis data?

12. Acknowledgements Thasso Griebel (PhD): Error Models, Pipelining Paolo Ribeca(PhD), Santiago Marco: GEM mapper + conversion Emanuele Raineri (PhD): SNP calling