Genome-wide association study between DSE polymorphism and Poly-A

usage in Human population

Hiren KarathiaSridhar Hannenhalli

Transcription & Polyadenylation (Poly-A)

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

Annotation status of Poly-A sites on 3’UTR of

Human Genome (hg19 – 2009)

1 2 3 4 5 6 7 8 9 10 11

Poly-A usage transcripts 12224 4784 1567 588 189 107 19 19 3 2 1

3.16227766016838

31.6227766016838

316.227766016838

3162.27766016838

31622.7766016838

Frequency of Transcripts Vs

Cleavage Points

log1

0 (T

rans

crip

t fre

quen

cy)

1 2 3 4 5 6 7 8 9 10 11

Poly-A usage transcripts 12224 4784 1567 588 189 107 19 19 3 2 1

3.16227766016838

31.6227766016838

316.227766016838

3162.27766016838

31622.7766016838

Frequency of Transcripts Vs

Cleavage Points

log1

0 (T

rans

crip

t fre

quen

cy)

37% - Multiple Poly-A points

Target of the analysis

RNA-Seq processing for Human Samples

SampleFastq files

BWA SamtoolsBAM file Merged BAM file

Samtools

Samtools

Sorted BAM fileDe-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 RNABR British in England and Scotland 1 1 FI Finnish in Finland 1 1 UT Utah residents with Northern and Western European ancestry 1 1 YO Yoruba in Ibadan, Nigeria 1 1

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) / DistancePA1 Usage = Complete (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

BR - 1 (F) BR - 2 (M) FN - 1 (F) FN - 2 (M) UT - 1 (F) UT - 1 (M)

Not Expressed 5499 5833 5211 5677 5849 5514

Single PA Usage 9185 8913 9302 9037 8852 9012

Multiple PA Usage 4819 4757 4988 4787 4802 4975

500

1500

2500

3500

4500

5500

6500

7500

8500

9500

Freq

uenc

y of

Tra

nscr

ipt

Correlation of different PA usage in a Human Sample

PA1 – PA2 PA2 – PA3

r = - 0.643; p = 0.0 r = - 0.182; p = 1.06e-33

Correlation of PA usage and corresponding DSE polymorphism

Utah-Fe

male<=

>Briti

sh-M

ale

Utah-Fe

male<=

>Briti

sh-Fe

male

Nigeria

-Male

<=>U

tah-M

ale

Nigeria

-Male

<=>F

innish-M

ale

British

-Male

<=>B

ritish

-Female

Finnish

-Male

<=>B

ritish

-Male

Nigeria

-Male

<=>B

ritish

-Female

British

-Female

<=>U

tah-Fe

male

Finnish

-Male

<=>U

tah-M

ale

Nigeria

-Male

<=>U

tah-Fe

male

Utah-M

ale<=

>Briti

sh-M

ale

Finnish

-Female

<=>F

innish-M

ale

Nigeria

-Male

<=>B

ritish

-Male

0

1

2

3

4

5

6

TT composition in DSE motifs

-LO

G (P

)

Finnish

-Male

<=>B

ritish

-Male

Nigeria

-Male

<=>F

innish-M

ale

Finnish

-Female

<=>B

ritish

-Male

British

-Female

<=>U

tah-Fe

male

Nigeria

-Male

<=>B

ritish

-Male

Finnish

-Male

<=>U

tah-M

ale

Finnish

-Female

<=>B

ritish

-Female

British

-Female

<=>B

ritish

-Male

Utah-Fe

male<=

>Briti

sh-Fe

male

Nigeria

-Male

<=>B

ritish

-Female

Nigeria

-Male

<=>B

ritish

-Female

British

-Female

<=>B

ritish

-Female

Utah-Fe

male<=

>Briti

sh-M

ale

Nigeria

-Male

<=>U

tah-Fe

male0

0.51

1.52

2.53

3.5

GT composition in DSE motifs

-LO

G (P

)

British

-Female

<=>B

ritish

-Female

Finnish

-Female

<=>B

ritish

-Male

Utah-Fe

male<=

>Briti

sh-M

ale

Finnish

-Male

<=>U

tah-M

ale

Nigeria

-Male

<=>B

ritish

-Male

Utah-Fe

male<=

>Briti

sh-Fe

male

Nigeria

-Male

<=>B

ritish

-Female

Nigeria

-Male

<=>B

ritish

-Female

00.5

11.5

22.5

33.5

GG composition in DSE motifs

- LO

G (P

)

Finnish

-Male

<=>U

tah-M

ale

Nigeria

-Male

<=>F

innish-M

ale

Nigeria

-Male

<=>B

ritish

-Male

Nigeria

-Male

<=>U

tah-Fe

male

Utah-Fe

male<=

>Briti

sh-M

ale

Finnish

-Female

<=>U

tah-M

ale

Nigeria

-Male

<=>B

ritish

-Female

Nigeria

-Male

<=>U

tah-M

ale

British

-Female

<=>B

ritish

-Male

Utah-Fe

male<=

>Briti

sh-Fe

male0

1

2

3

4

5

6

Length of DSE motifs

-LO

G (P

)

Correlation of PA usage and corresponding DSE polymorphism

Functional enrichment of Genes associated with Differential PA Usage and

Polymorphic for of DSEs in Population

Thank you !!

Differential Expression of complete 3’UTR

Inter/Intra group correlation of a PA usage

r = 0.8; p = 0.0 r = 0.8; p = 0.0

r = 0.98; p = 0.0

PA1 usageBR1 – 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-1Single 12 79 9 30 89 1

Multiple 12 52 9 34 89 1

BR-2 Single 12 62 9 31 89 1

Multiple 12 52 9 34 89 1

FN - 1 Single 12 90 9 35 89 1

Multiple 12 54 9 39 89 1

Find Polymorphism in the DSEs

Find Correlation between the PA-usage and DSE polymorphism

Pending

Alternate Poly-A selection mechanism

Complete 3’UTR coverage VS

Alternate 3’UTR coverage

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 CoverageRelative PA1 Usage = Longest UTR Coverage

PA2 CoverageRelative PA2 Usage = Longest UTR Coverage

Stop Codon

Intron

Cleaved 3’UTR

DSE statisticSample PA type Mean(Motif Length) Max(Motif Length) Min(Motif Length) Mean(Distance) Max(Distance) Min(Distance)

BR-1

Single 12 79 9 30 89 1

Multiple 12 52 9 34 89 1

BR-2

Single 12 62 9 31 89 1

Multiple 12 52 9 34 89 1

FN - 1

Single 12 90 9 35 89 1

Multiple 12 54 9 39 89 1

+ strand

- strand

Gene Strand

Template Strand

+ Read

+ Read

+ Read

- Read

- Read

RNA Strand DNA Strand

Locations of annotated multiple PA locations on 3’UTR

PA1 Junction PA2 JunctionStop CodonCleaved 3’UTR

PA1 Junction PA2 JunctionStop Codon

PAs on same exon

PAs on multiple exonsr = 0.2578p = 8.44e10-111

Poly-A Location

Leng

th o

f 3’

UTR