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

Mechanism of Poly-Adenylation

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

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 = p = 8.44e Poly-A Location Length of 3’UTR

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

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

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

Frequency of Poly-A usage in the samples

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

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

Differential Expression of complete 3’UTR

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

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

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

DSE statistic SamplePA typeMean(Motif Length)Max(Motif Length)Min(Motif Length)Mean(Distance)Max(Distance)Min(Distance) BR-1 Single Multiple BR-2 Single Multiple FN - 1 Single Multiple

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