PowerPoint Presentation

Genome-wide association study betweenDSE polymorphism and Poly-A usage inHuman population

Hiren Karathia

Sridhar Hannenhalli

Transcription & Polyadenylation (Poly-A)

http://geneed.nlm.nih.gov/images/transcription_sm.jpg

http://www.mun.ca/biology/scarr/iGen3_05-11b_Figure-L.jpg

Objectives

•Genome-wide estimation of alternate Poly-A (PA) usageon 3’UTR

•Genome-wide Prediction and investigation ofpolymorphisms in DSE (Downstream Sequence Element)motifs

•Population-wide correlation study between the PAusage and DSE polymorphisms

Annotation status of Poly-A sites on 3’UTR ofHuman Genome (hg19 – 2009)

37% - Multiple Poly-A points

Target of theanalysis

RNA-Seq processing for Human Samples

Sample

Fastq files

BWA

Samtools

BAM file

Merged 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

Symbol

Group of Samples

Male

Female

DNA

RNA

British in England and Scotland

Finnish in Finland

Utah residents with Northern and Western European ancestry

Yoruba in Ibadan, Nigeria

Differential Expression of UTR

Cuffdiff tools

Python script

De-novo assembly

Genome-wide estimation of alternate Poly-A(PA) usage on 3’UTR

PA1 Coverage

PA2 Coverage

PA1 Junction

PA2 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

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

Frequency of Poly-A usage in the samples

Correlation of different PA usage in a HumanSample

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\PEARSON_CORR_1_2_USAGE.png$

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\PEARSON_CORR_2_3_USAGE.png$

PA1 – PA2

PA2 – PA3

r = - 0.643; p = 0.0

r = - 0.182; p = 1.06e-33

Correlation of PA usage and correspondingDSE polymorphism

$C:\UMIACS_Work\UTR_Work\UTR_RESULTS\COMPARATIVE_RESULTS\Utah-Male_British-Female_High_Low_DSE_GG.png$

$C:\UMIACS_Work\UTR_Work\UTR_RESULTS\COMPARATIVE_RESULTS\Utah-Male_British-Female_High_Low_GG.png$

$C:\UMIACS_Work\UTR_Work\UTR_RESULTS\COMPARATIVE_RESULTS\Utah-Male_British-Female_High_Low_DSE_GT.png$

$C:\UMIACS_Work\UTR_Work\UTR_RESULTS\COMPARATIVE_RESULTS\Utah-Male_British-Female_High_Low_GT.png$

Correlation of PA usage and corresponding DSEpolymorphism

$C:\UMIACS_Work\UTR_Work\UTR_RESULTS\COMPARATIVE_RESULTS\Finnish-Female_Nigeria-Male_High_Low_GT.png$

$C:\UMIACS_Work\UTR_Work\UTR_RESULTS\COMPARATIVE_RESULTS\Finnish-Female_Nigeria-Male_High_Low_DSE_GT.png$

$C:\UMIACS_Work\UTR_Work\UTR_RESULTS\COMPARATIVE_RESULTS\Finnish-Female_Nigeria-Male_High_Low_GG.png$

$C:\UMIACS_Work\UTR_Work\UTR_RESULTS\COMPARATIVE_RESULTS\Finnish-Female_Nigeria-Male_High_Low_DSE_GG.png$

Functional enrichment of Genes associatedwith Differential PA Usage and Polymorphic forof DSEs in Population

Thank you !!

Differential Expression of complete 3’UTR

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\DE_HG00266_HG00146.png$

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\DE_HG00146_HG00155.png$

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\DE_HG00267_HG00146.png$

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\DE_HG00266_HG00267.png$

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\SAMPLE_VENN.png$

Inter/Intra group correlation of a PA usage

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\PEARSON_CORR_1_USAGE.png$

r = 0.8; p = 0.0

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\PEARSON_CORR_1_USAGE.png$

r = 0.8; p = 0.0

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\PEARSON_CORR_1_USAGE.png$

r = 0.98; p = 0.0

PA1 usage

BR1 – BR2

FN1 – FN2

BR1 – FN1

Statistics of predicted DSE motifs

Sample

PA type

Mean(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

Alternate Poly-A selection mechanism

Complete 3’UTR coverageVSAlternate 3’UTR coverage

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\DE_SAMPLE_2.png$

$U:\Hiren\Poly_A_Samples\Results\Annotation\DE_Expression\DE_HG00266_HG00267.png$

Differential expression of complete 3’UTR usage

Differential expression of PA Usage

Poly Adenylation Usage on 3’UTR

PA1 Coverage

PA2 Coverage

PA1 Junction

PA2 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

Sample

PA type

Mean(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

Locations of annotated multiple PA locations on 3’UTR

PA1 Junction

PA2 Junction

Stop Codon

Cleaved 3’UTR

PA1 Junction

PA2 Junction

Stop Codon

PAs on same exon

PAs on multiple exons

$U:\Hiren\Poly_A_Samples\Results\Annotation\Pearson_Correlation.png$

r = 0.2578

p = 8.44e10-111

Poly-A Location

Length of 3’UTR