1. Genome-wide association study between DSE polymorphism and Poly-A usage in Human population Hiren Karathia Sridhar Hannenhalli

2. Transcription & Polyadenylation (Poly-A)

3. Objectives Genome-wide estimation of alternate Poly-A (PA) usage on 3’UTR Genome-wide Prediction and investigation of polymorphisms in DSE (Downstream Sequence Element) motifs Population-wide correlation study between the PA usage and DSE polymorphisms

4. Annotation status of Poly-A sites on 3’UTR of Human Genome (hg19 – 2009) 37% - Multiple Poly-A points Target of the analysis

5. RNA-Seq processing for Human Samples Sample Fastq files BWA Samtools BAM fileMerged BAM file Samtools Sorted BAM file De-duplicated file Picard tool Indexing the BAM Samtools SAM file Calculate Coverage Bed tools Calculate Relative usage of PAs Python script SymbolGroup of SamplesMaleFemaleDNARNA BRBritish in England and Scotland11 FIFinnish in Finland11 UTUtah residents with Northern and Western European ancestry11 YOYoruba in Ibadan, Nigeria11 Differential Expression of UTR Cuffdiff tools Python script De-novo assembly

6. Genome-wide estimation of alternate Poly-A (PA) usage on 3’UTR PA1 Coverage PA2 Coverage PA1 JunctionPA2 Junction Complete UTR coverage Coverage (Stop codon – PA1 junction) / Distance PA1 Usage = Complete (complete 3’ UTR) / Distance Coverage (Stop codon – PA1 junction) / Distance PA1 Usage = Complete (complete 3’ UTR) / Distance Coverage (Stop codon - PA2 junction) / Distance PA2 Usage = Coverage (complete 3’UTR) / Distance Coverage (Stop codon - PA2 junction) / Distance PA2 Usage = Coverage (complete 3’UTR) / Distance Stop Codon Cleaved 3’UTR

7. Prediction of DSE Coding Strand of DNA Sample A  RNA-Seq Sample A  DNA-Seq De-novo assembled 3’UTR fragment Prediction of DSE motif Template Strand of DNA

8. Frequency of Poly-A usage in the samples

9. Correlation of different PA usage in a Human Sample PA1 – PA2PA2 – PA3 r = - 0.643; p = 0.0 r = - 0.182; p = 1.06e -33

10. Correlation of PA usage and corresponding DSE polymorphism

13. Functional enrichment of Genes associated with Differential PA Usage and Polymorphic for of DSEs in Population

14. Thank you !!

16. Differential Expression of complete 3’UTR

17. Inter/Intra group correlation of a PA usage r = 0.8; p = 0.0 r = 0.98; p = 0.0 PA1 usage BR1 – BR2FN1 – FN2 BR1 – FN1

18. Statistics of predicted DSE motifs SamplePA typeMean(Motif Length)Max(Motif Length)Min(Motif Length)Mean(Distance)Max(Distance)Min(Distance) BR-1 Single 1279930891 Multiple 1252934891 BR-2 Single1262931891 Multiple1252934891 FN - 1 Single1290935891 Multiple1254939891 Find Polymorphism in the DSEs Find Correlation between the PA-usage and DSE polymorphism Pending

20. Alternate Poly-A selection mechanism

21. Complete 3’UTR coverage VS Alternate 3’UTR coverage Differential expression of complete 3’UTR usageDifferential expression of PA Usage

22. Poly Adenylation Usage on 3’UTR PA1 CoveragePA2 Coverage PA1 JunctionPA2 Junction Complete UTR coverage PA1 Coverage Relative PA1 Usage = Longest UTR Coverage PA1 Coverage Relative PA1 Usage = Longest UTR Coverage PA2 Coverage Relative PA2 Usage = Longest UTR Coverage PA2 Coverage Relative PA2 Usage = Longest UTR Coverage Stop Codon Intron Cleaved 3’UTR

23. DSE statistic SamplePA typeMean(Motif Length)Max(Motif Length)Min(Motif Length)Mean(Distance)Max(Distance)Min(Distance) BR-1 Single 1279930891 Multiple 1252934891 BR-2 Single1262931891 Multiple1252934891 FN - 1 Single1290935891 Multiple1254939891

24. + strand - strand Gene Strand Template Strand + Read - Read RNA Strand DNA Strand

25. Locations of annotated multiple PA locations on 3’UTR PA1 JunctionPA2 JunctionStop Codon Cleaved 3’UTR PA1 Junction PA2 Junction Stop Codon PAs on same exon PAs on multiple exons r = 0.2578 p = 8.44e10 -111 Poly-A Location Length of 3’UTR