Sequence alignment

Paul Gardner

March 19, 2015

Paul Gardner Sequence alignment

What is homology?

I A homologous trait is anycharacteristic of organisms that isderived from a common ancestor[1]

Eg. vertebrate forelimbs.

I Contrast this to analagous traits:similarities between organisms thatwere not in the last commonancestor[1] Eg. wings frompterosaurs, bats & birds.

1. Text & image adapted from:http://en.wikipedia.org/wiki/Homology (biology)

Paul Gardner Sequence alignment

How can we measure evolutionary relatedness?

I Is a jellyfish more related to a sponge or the custard apple?

I How can we test this?

Barton et al. (2007) Evolution. Cold Spring Harbor Laboratory Press

Paul Gardner Sequence alignment

The homology search problem

I Given a biologicalsequence, can we identifyhomologues in otherspecies?

Allers & Mevarech (2005) Archaeal genetics the third way. Nature Reviews Genetics

Paul Gardner Sequence alignment

Given gazillions of biomolecular sequences, how can weestimate homology?

I By sequence comparison! Usually by alignment.I NOTE: sequence similarity is used to infer homology.

Homology is binary, either something is or isn’t homologous.Sequences can be 87% similar, they can’t be 87% homologous!

I Alignments are two or more sequences placed on top of eachother, identifying a functional, structural, or evolutionaryrelationships between the sequences.

2

4

5

3

1

>1GCAUCCAUGGCUGAAUGGUUAAAGCGCCCAACUCAUAAUUGGCGAACUCGCGGGUUCAAUUCCUGCUGGAUGCA>2GCAUUGGUGGUUCAGUGGUAGAAUUCUCGCCUGCCACGCGGGAGGCCCGGGUUCGAUUCCCGGCCAAUGCA>3UGGGCUAUGGUGUAAUUGGCAGCACGACUGAUUCUGGUUCAGUUAGUCUAGGUUCGAGUCCUGGUAGCCCAG>4GAAGAUCGUCGUCUCCGGUGAGGCGGCUGGACUUCAAAUCCAGUUGGGGCCGCCAGCGGUCCCGGGCAGGUUCGACUCCUGUGAUCUUCCG>5CUAAAUAUAUUUCAAUGGUUAGCAAAAUACGCUUGUGGUGCGUUAAAUCUAAGUUCGAUUCUUAGUAUUUACC

** * 1 GCAUCCAUGGCUGAAU-GGUU-AAAGCGCCCAACUCAUAAUUGGCGAA-- 2 GCAUUGGUGGUUCAGU-GGU--AGAAUUCUCGCCUGCCACGCGG-GAG-- 3 UGGGCUAUGGUGUAAUUGGC--AGCACGACUGAUUCUGGUUCAG-UUA-- 4 GAAGAUCGUCGUCUCC-GGUG-AGGCGGCUGGACUUCAAAUCCA-GU-UG 5 CUAAAUAUAUUUCAAU-GGUUAGCAAAAUACGCUUGUGGUGCGU-UAA--

**** * ** 1 ------------------CUCGCGGGUUCAAUUCCUGCUGGAUGC-A 2 ------------------G-CCCGGGUUCGAUUCCCGGCCAAUGC-A 3 ------------------G-UCUAGGUUCGAGUCCUGGUAGCCCA-G 4 GGGCCGCCAGCGGUCCCG--GGCAGGUUCGACUCCUGUGAUCUUCCG 5 ------------------A-UCUAAGUUCGAUUCUUAGUAUUUAC-C

SMADMYMURSYUC

AMY-

GGY

u a AV M M M

R MH

CR

MYUSH V R

HK

CV

Rc

KWA--

-- - c c - c c

a-c---

cc

c-V-YS Y R R G U UCR

AYU

CCYRSYMDMYVM

cV

Paul Gardner Sequence alignment

Did you do your homework?

I Play: http://phylo.cs.mcgill.ca

Paul Gardner Sequence alignment

How should a sequence alignment be interpreted & scored?

>gb|CP001191.1| Rhizobium leguminosarum bv. trifolii WSM2304, complete genome

Length=4537948

Features in this part of subject sequence:

cold-shock DNA-binding domain protein

Score = 57.2 bits (62), Expect = 2e-05

Identities = 78/106 (74%), Gaps = 6/106 (6%)

Strand=Plus/Plus

Query 1 CTTCGTCAGATTTCCTCTCAATATCGATCATACCGGACTGATATTCGTCCGG----GAAC

|| |||||||| ||||||||| |||||| | | | || |||| |||| ||||

Sbjct 828507 CTCCGTCAGATATCCTCTCAACATCGATACGGCTTGTCGGACATTCTTCCGCAGGCGAAC

Query 57 TCTAGCGATTGAAA-GGAAATCGTTATGAACTCAGGCACCGTAAAG

| | || |||||| ||| ||||||||||| |||||| ||| |||

Sbjct 828567 ACAA-CGGTTGAAAAGGAGATCGTTATGAATTCAGGCGTCGTCAAG

Paul Gardner Sequence alignment

Lets play a game of coin toss

I How can we detect cheating or bias?I coin tossI ./coin-toss.pl 100I ./coin-toss2.pl 100

Paul Gardner Sequence alignment

Where do those scores come from?

I Most alignment scores (from BLAST, HMMER, ...) can beinterpreted as log-odds or bit-scores

I What is a log-odds score?I The observed frequency of two independent events is pABI The expected frequency of two independent events is fA ∗ fB

(i.e. what is the likelihood of the event occuring by chance)I The log-odds score is log2( #Observed

#Expected ) = log2( pABfA∗fB )

I What happens when pAB = fA ∗ fB?I What happens when pAB > fA ∗ fB or pAB < fA ∗ fB?

I Often called the bit-score, information theorists like to discusshow many “bits” of information they have, hence “log2”.

Paul Gardner Sequence alignment

Logarithms – reminder

Paul Gardner Sequence alignment

Exercise

I If I was to throw two dice 360 times and rolled double 6’s 80times, what is the log-odds score for that (base 2)?

I What if instead I only rolled 10 double 6’s?

I What if instead I only rolled 5 double 6’s?

Paul Gardner Sequence alignment

Where do the nucleotide scores come from?

TABLE 1. PAM Substitution Scores Based

on the Uniform Mutation Model

PAMDist.

PercentCon-

served

MatchScore(Bits)

M is-m atchScore(Bits)

Match/M is-

m atchScoreRatio

Ave.Inform ation Per

Posi-tion

(Bits)

1 99.0 1.99 -6.24 0.32 1.90

2 98.0 1.97 -5.25 0.38 1.83

5 95.2 1.93 -3.95 0.49 1.64

10 90.6 1.86 -3.00 0.62 1.40

15 86.4 1.79 -2.46 0.73 1.21

20 82.4 1.72 -2.09 0.82 1.05

25 78.7 1.66 -1.82 0.91 0.92

30 75.3 1.59 -1.60 0.99 0.80

35 72.0 1.53 -1.42 1.07 0.70

40 69.0 1.46 -1.27 1.15 0.62

45 66.2 1.40 -1.15 1.22 0.54

50 63.5 1.34 -1.04 1.29 0.47

States, DJ et al. (1991) Improved sensitivity of nucleic acid database searches using application-specific scoringmatrices. Methods Enzymol.

Paul Gardner Sequence alignment

Protein evolution

I What sorts of amino-acid replacements are likely during theevolution of a protein sequence?

GA

A

A

A

A

G

G

G

G

C

C

C

C

U

U

U

U

UC AG UCAGUCAGUCAGUCAGU

CAG

UCAGUCAGUCAGUCAGU

CA

GUCAGUCAGUCAG

UCAG UCAG

P

S

U

nG

nG

oG

oG

oG

G

P

P

P

P

P

nM

nM

M

M

nM

nM

nM

Phenylalanine

Phe

Leucine

Leu

Leucine

Leu

Proline

Pro

Histidine

His

Glutamine

Gln

Isoleucine

Ile

Methionine

Met

Threonine

Thr

Asparagine

Asn

Lysine

Lys

Arginine

Arg

Arginine

Arg

Valine

Val

Alanine

Ala

Glutamic acid

Glu

Aspartic acid

Asp

Glycine

Gly

Serine

Ser

Serine

Ser

Tyrosine

Tyr

Cysteine

Cys

Tryptophan

Trp

Stops

Stop

E G F LS

S

Y

C

WL

P

H

R

R

QIM

TN

K

V

A

D89.09

75.07

174.20

174.20

146.19

165.19

133.11

117.15

147.13

146.15

155.16

115.13

105.09

105.09

131.18

132.12

MW

= 1

49.2

1 Da

131.18

119.12

204.23

131.18

181.19

121.16

HN

NH2

NH

H2N

OH

O

H2N

CH3 OH

O

H2N

O

H2N

OH

O

O

HO

H2N

OH

O

HS

H2N

OH

O

H2N

O

NH2

OH

O

O

OH

H2N

OH

OH2N

OH

O

NH

H2N

OH

O

N

CH3 CH3

H2N

OH

O

CH3

CH3

H2N

OH

O

CH3

CH3

H2N

OH

O

H2N

H2N

OH

O

CH3 S

H2N

OH

O

H2N

OH

O

NH

OH

O

H2N

HO OH

O

H2N

HO OH

O

H2N

HO

CH3

OH

O

NH

H2N

OH

O

HO

H2N

OH

O

H2N

CH3

CH3

OH

O

BasicAcidicPolarNonpolar(hydrophobic)

S -M - P - U - nM -oG - nG -

SumoMethyl

PhosphoUbiquitinN-Methyl

O-glycosylN-glycosyl

Modification

am

ino a

cid

2nd1st position 3rdUC

Image source: http://upload.wikimedia.org/wikipedia/en/d/d6/GeneticCode21-version-2.svg

Paul Gardner Sequence alignment

Where do the protein score matrices come from?

I The BLOSUM62 matrix

Henikoff & Henikoff (1992) Amino acid substitution matrices from protein blocks. PNAS.Image source: http://www.mathgon.com/Cours/TP/TP1/Alignements.html

Paul Gardner Sequence alignment

Multiple and pairwise sequence alignment

I Score matrices are used to evaluate how “good” an alignmentis...

I Does the alignment explain the likely evolutionary history ofthese sequences?

I Is the biochemical function likely to have been preserved?I The highest-scoring pairwise sequence alignment can be found

using dynamic programmingI The highest-scoring multiple sequence alignment cannot be

found easily

H.pylori ------GVDA NALHRPKRFF GAARNIEEGG SLTIIATALI ETGSRMDEVI ------FEEFD.radiodurans ------GVDS TALYPPKRFL GAARNIEEGG SLTIIATAMV ETGSTGDTVI ------FEEFM.tuberculosis ------GVDS TALYPPKRFL GAARNIEEGG SLTIIATAMV ETGSTGDTVI ------FEEFC.pneumoniae ------GVDA SALHKPKRFF GAARNIEGGG SLTILATALI DTGSRMDEVI ------FEEFF.nucleatum ------GIDP TALYHPKNFF GAARNIKDGG SLTIIATILV DTGSKMDEVI ------YEEFS.enterica ------GVDA NALHRPKRFF GAARNVEEGG SLTIIATALI DTGSKMDEVI ------YEEFB.thetaiotaomicron ------GVDA NALHKPKRFF GAARNIENGG SLTIIATALI DTGSKMDEVI ------FEEFL.interrogans ------GVDS NALHKPKRFF GAARNIEEGG SLTIIATALI DTGSKMDEVI ------FEEFP.marinus GYQPTLGTDV GELQER---- -ITSTLE--G SITSIQAVYV PADDLTDPAP ------ATTFU.parvum GYQSTLESDV THIQNR---- -LFRNKN--G SITSFQTIFL PMDDLSDPSA ------VAVFB.subtilis ------GIDP AAFHRPKRFF GAARNIEEGG SLTILATALV DTGSRMDDVI ------YEEFC.difficile ------GIDP GALHGPKKFF GAARNIRQGG SLTILGTALV ETGSRMDDVI ------FEEFS.griseus ------GVDS TALYPPKRFF GAARNIEDGG SLTILATALV ETGSRMDEVI ------FEEFF.nodosum ------GVDP AALYKPKHFF GAARNTREGG SLTIIATALI ETGSKMDEVI ------FEEFM.infernorum ------GVDA NALQKPRRFF ATARNLEEGG SVTIIATALI DTGSKMDDVI ------FEEFT.yellowstonii ------GLEA TALQKPKRFF GTARNIEEGG SLTIIATALV ETGSRMDDVI ------FEEFE.coli ------GVDA NALHRPKRFF GAARNVEEGG SLTIIATALI DTGSKMDEVI ------YEEFH.volcanii GYPAYLAARL SEFYERAGYF TTVNGEE--G SVSVIGAVSP PGGDFSEPVT QNTLRIVKTF

Paul Gardner Sequence alignment

Multiple sequence alignment

Different approaches & issuesI A NP-hard problem (Wang & Jiang (1994))

I Which means there is no mathematically optimal solution formultiple sequence alignment that will run on a moderncomputer in reasonable time

I Which means we have to identify heuristic approaches that runquickly and produce reasonable solutions

H.pylori ------GVDA NALHRPKRFF GAARNIEEGG SLTIIATALI ETGSRMDEVI ------FEEFD.radiodurans ------GVDS TALYPPKRFL GAARNIEEGG SLTIIATAMV ETGSTGDTVI ------FEEFM.tuberculosis ------GVDS TALYPPKRFL GAARNIEEGG SLTIIATAMV ETGSTGDTVI ------FEEFC.pneumoniae ------GVDA SALHKPKRFF GAARNIEGGG SLTILATALI DTGSRMDEVI ------FEEFF.nucleatum ------GIDP TALYHPKNFF GAARNIKDGG SLTIIATILV DTGSKMDEVI ------YEEFS.enterica ------GVDA NALHRPKRFF GAARNVEEGG SLTIIATALI DTGSKMDEVI ------YEEFB.thetaiotaomicron ------GVDA NALHKPKRFF GAARNIENGG SLTIIATALI DTGSKMDEVI ------FEEFL.interrogans ------GVDS NALHKPKRFF GAARNIEEGG SLTIIATALI DTGSKMDEVI ------FEEFP.marinus GYQPTLGTDV GELQER---- -ITSTLE--G SITSIQAVYV PADDLTDPAP ------ATTFU.parvum GYQSTLESDV THIQNR---- -LFRNKN--G SITSFQTIFL PMDDLSDPSA ------VAVFB.subtilis ------GIDP AAFHRPKRFF GAARNIEEGG SLTILATALV DTGSRMDDVI ------YEEFC.difficile ------GIDP GALHGPKKFF GAARNIRQGG SLTILGTALV ETGSRMDDVI ------FEEFS.griseus ------GVDS TALYPPKRFF GAARNIEDGG SLTILATALV ETGSRMDEVI ------FEEFF.nodosum ------GVDP AALYKPKHFF GAARNTREGG SLTIIATALI ETGSKMDEVI ------FEEFM.infernorum ------GVDA NALQKPRRFF ATARNLEEGG SVTIIATALI DTGSKMDDVI ------FEEFT.yellowstonii ------GLEA TALQKPKRFF GTARNIEEGG SLTIIATALV ETGSRMDDVI ------FEEFE.coli ------GVDA NALHRPKRFF GAARNVEEGG SLTIIATALI DTGSKMDEVI ------YEEFH.volcanii GYPAYLAARL SEFYERAGYF TTVNGEE--G SVSVIGAVSP PGGDFSEPVT QNTLRIVKTF

Paul Gardner Sequence alignment

Introducing ClustalW

Paul Gardner Sequence alignment

ClustalW

I Clustering Alignment

I A progressive method

I Very fast

I Widely used

I Very good for rather similar sequences

I Advanced settings, fine tuned

I ClustalW (for weighting)

I ClustalX: Graphical version

Credit: text lifted verbatim from Stinus Lindgreen’s slides. Image source: www.wikimedia.org

Paul Gardner Sequence alignment

ClustalW

I Three steps:

1 Do all-against-all pairwise alignments

2 Build guide tree T

3 Perform multiple alignment along TI sequence-sequence, sequence-profile

& profile-profile

Basic idea: Align most similar sequencesfirst → add more divergent sequences later

Credit: text lifted verbatim from Stinus Lindgreen’s slides. Image source: www.wikimedia.org

Paul Gardner Sequence alignment

Discussion

I What do we use multiple sequence alignments for?

Paul Gardner Sequence alignment

Relevant reading

I Reviews:I Eddy SR (2004) Where did the BLOSUM62 alignment score

matrix come from? Nature Biotechnology

I Methods:I Thompson J et al. (1994) CLUSTAL W: improving the

sensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weightmatrix choice. Nucleic acids research.

I Altschul SF et al. (1997) Gapped BLAST and PSI-BLAST: anew generation of protein database search programs. Nucleicacids research.

Paul Gardner Sequence alignment

The End

Paul Gardner Sequence alignment