Rna sequencing for differential expression genes
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RNA sequencing for differential expression genes . Speaker : tzu-chun lo Advisor : Yao-Ting Haung. Outline . Molecular Central Dogma RNA Sequencing Differential Expression Gene Case–Control Study Negative Binomial Distribution Hypothesis Testing Rice SNP, QTL, Pathway.

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RNA sequencing for differential expression genes

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Rna sequencing for differential expression genes

RNA sequencing for differential expression genes

Speaker : tzu-chun lo

Advisor : Yao-Ting Haung


Outline

Outline

Molecular Central Dogma

RNA Sequencing

Differential Expression Gene

Case–Control Study

Negative Binomial Distribution

Hypothesis Testing

Rice

SNP, QTL, Pathway


Molecular central dogma

Molecular Central Dogma

The central dogma of molecular biology

describes the flow of genetic information

within a biological system.

Forest

Branches

BBQ


Rna sequencing

RNA Sequencing

DNA

RNA

Alignment

Gene 1

exons

Gene 2

mRNA

reads

Spliced alignment

Alignment

Finding differential expression genes

via read counts each gene.

Read counts

DEG process


Differential e xpression g ene

Differential Expression Gene

  • We want to find the cold-resistant genes in rice.

  • Rice genome

  • We should compare with two conditions.

    • Room temperature

    • Low temperature

Gene 1

Gene 3

Gene 2

Gene 1

Gene 3

Gene 2

Cole-resistant differential

expression genes :

Gene 1

Gene 3

Gene 2

13

4

5

7

2

6


Strategy for deg

Strategy for DEG

  • Case–control study

    • Two existing groups differing in outcome are identified and compared on the basis of some supposed causal attribute.

  • Question

    • Is the number adequate to the gene?

    • How to define the gene is differential expression?

 69 v.s 71

Almost the same

?

 86 v.s 56

PossibleDEG

 66 v.s 111

More likely DEG

Gene 4

80

60

 80 v.s 60

How to judge?

It is just one of sample in condition.

Negative binomial distribution

Hypothesis test


Negative binomial d istribution

Negative Binomial Distribution

NB is a count data distribution that can substitute Poisson distribution for better variance.

j

Gene abundance parameter

Smooth function

i

69

i=1~n

j=1~m

Library size parameter

Smooth function is more complex, so let us forget it. 

3


Rna sequencing for differential expression genes

FPKM

An indicator used to represent mRNA expression.

Fragments Per Kilobase of transcript per Million mapper reads.

10 4 reads

Genome

Exon length: 8 10 7 8 9 bases

Gene 1

Gene 2


Rna sequencing for differential expression genes

FPKM

Before hypothesis testing, we have to get FPKM and variance of FPKM.


Hypothesis testing

Hypothesis Testing

Step 1 : You find some observations or clues support

a novel idea.

Step 2 : Assume a against opinion that you want to

fight it.

Step 3 : Go to test it and take a stand.

p-value


T test

T-test

Using t-test to compare the log ratio (log fold-change)

of gene’s expression between condition (a) and (b).


T test1

T-test


Result investigating

Result Investigating

Discussing alpha=0.05 with read counts & p-value.

If alpha=0.04 or 0.03 ?

We don’t know which alpha is the best,

but we can do some subsequent processing.


Rna sequencing for rice

RNA sequencing for Rice

  • Plan

    • Cold-resistant genes

  • Samples

    • Japonica (TN67): room temperature (R), low temperature (L)

    • Indica (IR64): room temperature (R), low temperature (L)

  • Rice

    • 粳稻(TN67) : 米粒闊而短,黏性較大,Q彈,如 : 蓬萊米。

    • 秈稻(IR64) : 米粒細而長,黏性較小,易碎,如 : 在來米。

  • Zone

    • TN67 : High-latitude, or high altitude

    • IR64 : Low-latitude, or low altitude


Strategy for deg1

Strategy for DEG

  • Case–control study

    • Four combinations

      • Different varieties or distinct temperatures

    • Four sets of differential expression genes

      • The DEGs above combination (A,B,C,D)

  • Negative binomial

    • Inference probability situation by sample

  • Hypothesis test

    • Which is the DEG that we want

  • Subsequent processing

    • SNP, QTL, Pathway

A

TN67R IR64R

TN67L IR64L

D

B

C


Rna sequencing for differential expression genes

SNP

A single-nucleotide polymorphism is a sequence variation occurring when a single nucleotide differs between members of a biological species.

Case

Assembly

SNP

ATGCCCTCGTAA TTACTGCGT

ATGCCCTCGTAA TTACTGCGT

Control

ATGCGCTCGAAA TTACTCCGT

ATGCGCTCGAAA TTACTCCGT


Rna sequencing for differential expression genes

QTL

Quantitative traits refer to phenotypes(characteristics) that vary in degree and can be attributed to polygeniceffects (product of two or more genes)

Quantitative trait loci (QTLs) are stretches of DNA containing or linked to the genes that underlie a quantitative trait.

Ex : QT(cold) Loci : 599~799 (base)

DNA

Cold tolerance (29) & pollen fertility (43)

QTL length : ~million bases

QTL

genes

1

1000


Pathway

Pathway

Pathway is a collection of manually drawn pathway maps representing molecular interaction and reaction networks.

Rice

Gene No.2

Gene No.55

Gene No.99

Cold-resistant


Conclusion

Conclusion

  • Review

    • RNA Sequencing

    • Differential Expression Gene

    • Case–Control Study

    • Negative Binomial Distribution

    • Hypothesis Testing

  • Rice

    • SNP

    • QTL

    • Pathway


Variance of negative binomial

Variance of negative binomial

NB is a count data distribution that can substitute poisson distribution for better variance.


Strategy for deg2

Strategy for DEG

Case-control in the same temperature : A, C

Case-control in the same variety : B, D

Let T is a set of all genes.


Rna sequencing for differential expression genes

QTL

生物的另一類性狀例如人類的身高、體重、高

血壓、糖尿病;水稻株高及產量對疾病的抵抗程度;老鼠的體脂肪百分比;乳牛的乳產量;雞的產卵量,由

於其變異性是連續性的,不易分類,且易受環境影響,故稱為數量性狀(quantitative trait)。數量性狀是由多

個基因所控制,由於每個基因對數量性狀均有影響,所以每一基因的作用便相對地小。這些控制數量性狀的

基因稱為微效基因(polygenes)或又稱為數量性狀基因座(quantitative trait loci,QTL)。

Rice genome size 430Mb


Rna sequencing for differential expression genes

QTL


Negative binomial distribution

Negative binomial distribution

NB is a count data distribution that can inference adequate number by sample.

j

Smooth function

i


Negative binomial distribution1

Negative binomial distribution

NB is a count data distribution that can substitute Poisson distribution for better variance.


Hypothesis test

Hypothesis test

Step 1 : You find some observations or clues support

a novel idea.()

Step 2 : Assume a against opinion that you want to

fight it.

Step 3 : Go to test it and take a stand.

p-value


Case control example

Case-control example

  • Example

  • Question

    • Is the number adequate to the gene?

      • Negative binomial

    • How to define the gene is differential expression?

      • Hypothesis test

 69 v.s 71

Almost the same

 86 v.s 56

PossibleDEG

 66 v.s 111

More likely DEG


Variance of negative binomial1

Variance of negative binomial

NB is a count data distribution that can substitute Poisson distribution for better variance.


Rna sequencing1

RNA sequencing

DNA

RNA

Alignment

Gene 1

exons

Gene 2

mRNA

reads

Spliced alignment

DNA

We should align with regions above blue.


Rna sequencing2

RNA sequencing

  • Spliced alignment

    • TopHat


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