1. Objectives Genome-wide investigation – to estimate alternate Poly-Adenylation (APA) usage on 3’UTR – to identify polymorphism of Downstream Sequence Elements (DSEs) motifs Correlation of the APA usage and DSE polymorphisms in Human population

2. Mechanism of Poly-Adenylation

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

4. Locations of annotated multiple PA locations on 3’UTR PA1 JunctionPA2 JunctionStop Codon 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

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 Scotland22 FIFinnish in Finland22 UTUtah residents with Northern and Western European ancestry11 YOYoruba in Ibadan, Nigeria11 Differential Expression of UTR Cuffdiff tools Python script De-novo assembly

6. Calculate relative usage on 3’UTR PA1 Coverage PA2 Coverage PA1 JunctionPA2 Junction Complete UTR coverage Coverage (Stop codon – PA1 junction) PA1 Usage = Complete (3’ UTR) Coverage (Stop codon – PA1 junction) PA1 Usage = Complete (3’ UTR) Coverage (PA1 junction – PA2 junction) PA2 Usage = Coverage (3’UTR) Coverage (PA1 junction – PA2 junction) PA2 Usage = Coverage (3’UTR) Stop Codon Cleaved 3’UTR

7. Integrated mode finding and mapping the DSE on Genome Ref genome Sample – 1 RNA-Seq De-novo assembly of downstream RNA fragment Search for DSE motif

8. Frequency of Poly-A usage in the samples

9. 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

10. Correlation of different PA usage PA1 – PA2PA2 – PA3 r = - 0.643; p = 0.0 r = - 0.182; p = 1.06e -33

11. Differential Expression of complete 3’UTR

12. 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

14. Thank you !!

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

16. 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

17. 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

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