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Eukaryotic Comparative Genomics

Barak Cohen

June 2018 GEP Alumni Workshop

Last Update: 12/23/2020

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Detecting Conserved Sequences

Motoo KimuraCharles Darwin

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Evolution of Neutral DNA

A T GC C CGT T GGA A TTT T TT G G GT AA

A T GC C CGT T GGA A TTT T TT G G GT AA

A

G

A G

A

C

AT TT

A

G

AA

G* * * * * * * * * * * * * * * * * * * * * * * * *

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Evolution of Non-Neutral DNA

A

*

AT T T T TGGC G AC CCCA A AA AG GC T TA AC C

A AT T T T TGGC G AC CCCA A AA AG GC T TA AC C*****************************

G

C

CCG

G T

G

A T

T G

T

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Multi-Species Alignment

ATGTGGCGCAGCCTGTGCCAGCTGGACGATCGA

ATGTAGCCTAGCCAGTGCCAGCTGGACGATCGA

GTACATCGATAGCTTAGAATGCTGGACGATCTC

GTACGTCGATAGCATAGAATGCTGGACGATCTC

* * * * ***********

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How to do Comparative Genomics

1. Choose species to analyze2. Align sequences3. Identify streches of highly conserved

nucleotides

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Choose species

closely relatedspecies

distantly relatedspecies

• Closely Related Species– align well– not many changes

• Distantly Related Species– hard to align– lots of changes

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S.cerevisiae

S. paradoxus

S. bayanusS. pastorianus

S. servazziiS. unisporus

S. exiguusS. diarenensis

S. castellii

S. kluyveri

Kluyveromyces lactis

Schizosaccharomyces pombe

S. cariocanus

S. mikataeS. kudriavzevii

~10Mya

~20Mya

~150Mya

>350Mya

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Case Study: Coding vs. Non-Coding

• Coding DNA- codes for protein- triplet code- open reading frame (ORF)- tend to be long (50-500 bp)- highly constrained

• Non-Coding DNA- regulatory functions- short (5-15 bp)- degenerate- variable spacing

ORFATG…. …TAA

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CASE 1:Non-Coding

GAL4ATG… …TAA

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S.cerevisiae

S. paradoxus

S. bayanusS. pastorianus

S. servazziiS. unisporus

S. exiguusS. diarenensis

S. castellii

S. kluyveri

Kluyveromyces lactis

Schizosaccharomyces pombe

S. cariocanus

S. mikataeS. kudriavzevii

~10Mya

~20Mya

~150Mya

>350Mya

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paradoxus TCTTCTGAGACAGCATCACTTCTTCTTNTTTTTTACATAACTTATTCTTCTATAATTTTCcerevisiae TCCTTTGAGACAGCATTCGCCCAGTATTTTTTTTATTCTACA-AACCTTCTATAATTT-C

** * *********** * * ******* ** * ************ *

paradoxus AACGTATTTACATAGTTCTGTATCAGTTTAATCACCATAATATTGTTTTCCCTCAACTAAcerevisiae AAAGTATTTACATAATTCTGTATCAGTTTAATCACCATAATATCGTTTTCT-----TTGT

** *********** **************************** ****** *

paradoxus TGAATGCAATTAGATTTTCTTATTGTTCCCTCGCGGCTTTTTTTTGTTTTATAATCTATTcerevisiae TTAGTGCAATTAATTTTTCCTATTGTTACTTCG-GGCCTTTTTCTGTTTTATGAGCTATT

* * ******** ***** ******* * *** *** ***** ******** * *****

paradoxus TTTTCCGTCATTTCTTCCCCAGATTTCCAACTTCATCTCCAGATTGTGTCTATGTAATGCcerevisiae TTTTCCGTCATC-CTTCCCCAGATTTTCAGCTTCATCTCCAGATTGTGTCTACGTAATGC

*********** ************* ** ********************** *******

paradoxus ATGCTATCATATTGAGAAAAGATAGAGAAACAACCCTCCTGAAAAATGAAGCTACTGTCTcerevisiae ACGCCATCATTTTAAGAGAGGACAGAGAAGCAAGCCTCCTGAAAGATGAAGCTACTGTCT

* ** ***** ** *** * ** ****** *** ********** ***************

Closely-related sequences are uninformative

GAL4ATG…

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S.cerevisiae

S. paradoxus

S. bayanusS. pastorianus

S. servazziiS. unisporus

S. exiguusS. diarenensis

S. castellii

S. kluyveri

Kluyveromyces lactis

Schizosaccharomyces pombe

S. cariocanus

S. mikataeS. kudriavzevii

~10Mya

~20Mya

~150Mya

>350Mya

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Distantly-related sequences do not align

cerevisiae ACTTACCAT-CAAC-CATAGATGGGTAAAC---GGTTAGTAACTAGGAACACGATcastelli AGA-GTCAAACTTTTCGT—ATA--TATATATAATATGTCTGATTGCTGGTT---T

* ** * * * * * * * * *

Noncoding (Promoter)

GAL4ATG…

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S.cerevisiae

S. paradoxus

S. bayanusS. pastorianus

S. servazziiS. unisporus

S. exiguusS. diarenensis

S. castellii

S. kluyveri

Kluyveromyces lactis

Schizosaccharomyces pombe

S. cariocanus

S. mikataeS. kudriavzevii

~10Mya

~20Mya

~150Mya

>350Mya

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Multiple sequence alignments reveal conserved elements

cerevisiae TGAGACAGCAT-CACTTCTT-CTTNTTTTTTACATAACTTATTCTTCTATAATTTTCAACmikatae TGAGACAGCATTCACTTCTTTCTTTTTTTTTACATATCTTATTCTTCTATAATTTTCAACBayanus TGAGACAGCATTCGCCCAGT--ATTTTTTTTAT-TCTACAAACCTTCTATAATTT-CAAAkudriadzevi TGAGACTGCACTCCC--------TCTTCCTTTC------------TCCATAACTT---AC

****** *** * * * ** ** ** **** ** *

paradoxus GTATTTACATAGTTCTGTATCAGTTTAATCACCATAAT------ATTGTTTTCCCTCAACkluyveri GTATTTACATAGTTCTGTATCAGTTTAATCACCATAAT------ATTGTTTTCCCTCAACcerevisiae GTATTTACATAATTCTGTATCAGTTTAATCACCATAAT------ATCGTTTTCTTTGT--bayanus TTATTTACATAGTTTTGTATCAGTTTAATCACCATAATCGTAACACCGTTTTACCTCACC

********** ** *********************** * ***** *

paradoxus TAATGAATGCAATTAGATTTTC-TTATTGTTCCC-TCGCGGCTTTTTTTTGTTTTATAATkluyveri TAATGAATGCAATTAGATTTTCCTTATTGTTCCCCTCGCGGCTTTTTTTTGTTTTATAATcerevisiae ---TTAGTGCAATTAATTTTTC-CTATTGTTACT-TCG-GGCCTTTTTCTGTTTTATGAGbayanus TGATGCGGG--A---ATCCTTC-AGACCGTTCTC-TCGCGC-------------------

* * * *** * *** *** *

paradoxus -CTATTTTTTCCGTCATTTCTTCCCC-AGATTTCCAACTTCAT-CTCCAGATTGTGTCTAkluyveri ACTATTTTTTCCGTCATTTCTTCCCCCAGATTTCCAACTTCATACTCCAGATTGTGTCTAcerevisiae -CTATTTTTTCCGTCATC-CTTCCCC-AGATTTTCAGCTTCAT-CTCCAGATTGTGTCTAbayanus -CTTTTTTTTTCGTCATTTCTTCCCC-AGATCTACAACTTTAA-CTCCAGACGGTGTATA

** ****** ****** ******* **** * ** *** * ******* **** **

paradoxus TGTAATGCATGCTATCATATTGAGAAAAGATAGAGAAACAACCCTCCTGAAAAATGAAGCkluyveri TGTAATGCATGCTATCATATTGAGAAAAGATAGAGAAACAACCCTCCTGAAAAATGAAGCcerevisiae CGTAATGCACGCCATCATTTTAAGAGAGGACAGAGAAGCAAGCCTCCTGAAAGATGAAGCbayanus GGCAGTACAAGCAGTGCTTTTGGGAAGAGGCAAAGCTGCAGACCTCGAGAACAATGAAGC

* * * ** ** * * ** ** * * ** ** **** *** *******

UAS1 UAS2

UES MIG1 MIG1

GAL4ATG…

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CASE 2:Coding

CLN3ATG… …TAA

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S.cerevisiae

S. paradoxus

S. bayanusS. pastorianus

S. servazziiS. unisporus

S. exiguusS. diarenensis

S. castellii

S. kluyveri

Kluyveromyces lactis

Schizosaccharomyces pombe

S. cariocanus

S. mikataeS. kudriavzevii

~10Mya

~20Mya

~150Mya

>350Mya

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Closely-related sequences are uninformative

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S.cerevisiae

S. paradoxus

S. bayanusS. pastorianus

S. servazziiS. unisporus

S. exiguusS. diarenensis

S. castellii

S. kluyveri

Kluyveromyces lactis

Schizosaccharomyces pombe

S. cariocanus

S. mikataeS. kudriavzevii

~10Mya

~20Mya

~150Mya

>350Mya

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Less distantly related species not informative either

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S.cerevisiae

S. paradoxus

S. bayanusS. pastorianus

S. servazziiS. unisporus

S. exiguusS. diarenensis

S. castellii

S. kluyveri

Kluyveromyces lactis

Schizosaccharomyces pombe

S. cariocanus

S. mikataeS. kudriavzevii

~10Mya

~20Mya

~150Mya

>350Mya

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Distantly-related species reveal functional protein domains

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Identification of Multi-Species Conserved Regions (MCS)

Margulies et al (2003) Gen. Res. 13:2507-18

Human cccattcttttccaagtgtctccg--cctgcagcgattaggttagaaagcatttctctctChimp cccattcttttccaagtgtctccg--cctgcagcgattaggttagaaagcatttctctctMouse ttcagtcgtttcccagtgtctctga-cattcagagactactttagtaagcattt-tctctRat tcagtccttccctggcatctccag-cactcaa-gactactttagtaagcattt-tctctgDog tcaatgactttcccagtctcttctactgggaagagattaggttgcaaatcatttttctct

* * * * * * **

How can we decide if this region is “conserved?”

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Its like flipping coins (really)

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Binomial-Based Method for Detecting Conserved Sequences

p = probability that a site is the same between human and mouse by chance alone (Kimura), q = 1-p

For an alignment N base pairs long with n identities calculate the cumulative binomial probability as:

Margulies et al (2003) Gen. Res. 13:2507-18

Human: AATGGMouse: AATCGStatus: CCCDC

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Large sequencing projects are underway

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species A

species B

species Cspecies D

species E

species F

Star Phylogeny Actual Phylogeny

Tree Topology Influences Power

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Challenges in larger genomes

1) Deciding on the neutral rate of substitution

2) Local differences in neutral rate of substitutions

3) Multiple hypothesis testing

4) Repeat sequences and uneven base composition

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OLIG2

100 kb upstream of OLIG2

PhastCons and the UCSC Genome Browser

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Gene 1 Gene 2 Gene 3 Gene NSpecies 1Species 2Species 3

…

Motif Searching Across Several Multiple Alignments

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Information Content

GAATTCGAATTCGAATTCGAATTCGAATTCGAATTCGAATTC

EcoR1GCCTACACATTCTCATTCCGACTCGAATTCATATCGGAAATG

Random Rap1TGTATGGGTGTGTTCGGATTTGCATGGGTGTGTACAGGTGTGTATGGATGTGTTCGGGTTTGTATGGGTG

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Weight Matrix Model of TATA Box

A: -8 10 -1 2 1 -8

C: -10 -9 -3 -2 -1 -12

G: -7 -9 -1 -1 -4 -9

T: 10 -6 9 0 -1 11

G. Stormo

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A: -8 10 -1 2 1 -8

C: -10 -9 -3 -2 -1 -12

G: -7 -9 -1 -1 -4 -9

T: 10 -6 9 0 -1 11

….A C T A T A A T G T …

Score = -24

G. Stormo

Weight Matrix Model of TATA Box

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A: -8 10 -1 2 1 -8

C: -10 -9 -3 -2 -1 -12

G: -7 -9 -1 -1 -4 -9

T: 10 -6 9 0 -1 11

….A C T A T A A T G T …

Score = 43

G. Stormo

Weight Matrix Model of TATA Box

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N(b,i)

F(b,i)

S(b,i) = log[F(b,i)/P(b)]

G. Stormo

Weight Matrix Model of TATA Box

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Now we can compare motifs to each other

4 -3 5 -6 -2 -52 -1 -2 11 -1 -1

-10 8 2 -4 2 -3-3 2 1 2 -3 15

3 -2 2 1 3 13 -1 -2 7 -2 -1-8 6 3 -2 2 -2-1 1 1 4 -3 9

ACGT

ACGT

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MAGMAunaligned motif finding in multispecies conserved regions

Gene 1 Gene 2 Gene 3

*Ihuegbu, Stormo, & Buhler, JCB 19:139, 2012

Gene NSpecies 1Species 2Species 3

…

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