Download Geuvadis Analysis Meeting

Geuvadis Analysis Meeting
16/02/2012
Micha Sammeth
CNAG – Barcelona
Quantification of Splice-Forms and Variants
- Quantified 615 datasets based on the Gencode v7 annotation
- Sensitivity is a function of sequencing depth
- For every transcript,
normalized RPKM values and
number of deconvoluted reads
Correlation coeff.
0.87 (Pearson and
Spearman)
- Discussion at the end if/what to do before uploading
LoF Definitions
[MacArthur et al. 2012]
LOF = loss of function of a complete transcript
LoF types
SNP that introduces (directly) stop codon
Indels that disrupt/shift reading frame
X
SNP that disrupts splice site
Larger deletions that remove 1st exon or >50% of transcript
LoF scope
“partial” LoF affects just some protein-coding transcripts in
a locus
X
X
“full” LoF affects all protein-coding transcripts annotated
Large deletion
116
Frameshift indel
X
Splice
267
337
565
Stop
across populations
X
Large deletion
24
12
Splice
in a single individual
23
38
Frameshift indel
Stop
LoF Estimates
[MacArthur et al. 2012]
Compare RNA-Seq evidence to LoF predictions
main difference Geuvadis <> 1000 Genomes: RNA-Seq vs. DNA-Seq
Frameshift indel
Large deletion
}
X
X
X
X
directly from mappings / coverage by mappings
X
predicted disruption
of splice site
indirectly called from mappings
Confirmation LoF SNPs in Geuvadis
Stop
- Take phase1 samples where polymorphisms have been found by exome sequencing
- Additionally call SNPs by RNA-Seq (exzessive mappings)
~5000 differences, i.e. on average
>2 out of 1000 calls differ
Example:
(not Geuvadis)
Sufficient
coverage
in DNA
>2 million
genotype calls
possible in both
Experiments
Sufficient
coverage
in RNA
~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?)
Allele-specific RNA Processing
relative abundance distribution 1st form
relative abundance distribution 2nd form
A/A
A/G
G/G
1st 2nd A/A
A/G
G/G
100%
Homozygote
Common Allele
50%
0% or 50%
0% or 100%
[Montgomery
2010 dataset]
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]
(1) classification of AS influences
in LoF based on a certain annotation
5’ frame
2
3’ frame
0
2
1
2
0
(2) extension of an annotation by
RNA-Seq evidence
X
X
?
activation of latent splice sites
(1) classification of AS: AStalavista
1
2
3
4
5
6
7
7
1,2,3,4,5,6,7
^
[
1,2,3,6
-
-
1,2,3,6
^
1,3,5,6
1,2,3,6
1,2,3,4,5,6,7
^
^
6
3,5,6
5
1,2,3,4,5,6,7
bubble
2
1
4
-
3,5,7
-
1,4
^
1,2,3,
4,5,7
-
1,2,3,
4,5,7
]
(2) AS discovery by RNA-Seq
Novel exon junctions
supported by RNA-Seq
add to graph, novel events
7
extend annotated CDSs
1,2,3,4,5,6,7
^
[
1,2,3,6
-
-
1,2,3,6
^
1,3,5,6
1,2,3,6
1,2,3,4,5,6,7
^
-
^
6
3,5,6
5
1,2,3,4,5,6,7
2
1
4
-
3,5,7
-
1,4
^
1,2,3,
4,5,7
-
1,2,3,
4,5,7
]
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?
Acknowledgements
Thasso Griebel (PhD):
Error Models, Pipelining
Paolo Ribeca(PhD),
Santiago Marco:
GEM mapper + conversion
Emanuele Raineri (PhD):
SNP calling