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splice-acceptor

Influence of SNVs on TA ndem S plice S ites. splice-acceptor. splice-donor. …NAG. NA G/T. GYN…. Consequences of SNVs on tandem splicing. prediction phase. 1000 Genome SNVs. … NAGCGAG. CTCGATGTGTGATT…. …NAG. CGAGCTCGATGTGTGATT…. destruction of ‘usually’ realized AG.

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splice-acceptor

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  1. Influence of SNVs on TAndemSplice Sites splice-acceptor splice-donor …NAG NAG/T GYN…

  2. Consequences of SNVs on tandem splicing predictionphase 1000 Genome SNVs …NAGCGAG CTCGATGTGTGATT… …NAG CGAGCTCGATGTGTGATT… destruction of ‘usually’ realized AG generation of novel AG …NAG CGAXCTCGATGTGAGATT… Do these SNVs actually generate new (or remove) isoforms in individuals carrying the variant?

  3. description of SNVs in splice-junctions sum of reads showing isoform x total number of reads x 1,000,000,000 detected splice-events description start = start of splice event end = end of splice event i.e. M20N30M55 would start at read position + 20 and end at read position + 50 • only consider reads: • MAPQV >= 150 - both tags on the same strand • failedQC = false - insert size <= 100,000 bp • edit distance <= 6 - largest N in cigar string <= 100,000 bp • - normalize number of reads per isoform and sample by total number of reads of the sample: • - calculate p-values for the different isoform combinations using 2x2 contingency tables and Fisher’s exact test

  4. Comparison of every isoform with all possible combinations of the other isoforms isoform 1 isoform 4 isoform 2 isoform 3 example: N = 4 isoforms group 2 2N-2 combinations (= 16-2 = 14) group 1 2(N-1)-1 combinations (= 8-1 = 7) ignore 0000 (all samples in group 2) 1111 (all samples in group 1)

  5. example 1: • good correlation between homozygous reference and heterozygous individuals, despite large N-difference • hom. alt. individuals differ from hom. ref. individuals • number of hom. alt. individuals is low (7) • incomplete penetrance of the SNV (if any) Are the observed differences caused by too few homozygous individuals?

  6. example 2: • good correlation of variant 001 expresssion in all three genotypes • variants 100 and 010 only occur in heterzygous and homozygous alt individuals (they seem rare) Is the number of hom. reference individuals too low?

  7. example 3: expression for all isoforms is lower in the hom. alt. individuals

  8. example 4: increase of one isoform in hom alt. individuals, but decrease of the other isoform

  9. example 4: reverse strand: GC -> GT change • good genotype distribution • much stronger expression of one isoform in hom. alt. individuals • lack of other isoforms in hom. alt. individuals It’s a GY->GY change with large effect?

  10. Problems: • difficult to tell which SNVs actually have an effect on splicing • in general, only few individuals homozygous for SNVs in +/- 30 bp around annotated exon boundaries • expression differences overlay SNV specific splice-effects • (linkage disequilibrium with) other SNVs could falsify the results • the results give no proof that a certain SNV is responsible for the presents or absence of a certain isoform SNV1 SNV2 a causative SNV more than 30 bp inside the intron reflects a significant change in the splice-pattern to an exonic SNV

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