Multiple Multiple Sequence Sequence

Alignment (MSA)Alignment (MSA)andand

Phylogeny Phylogeny

MSA input: multiple sequence MSA input: multiple sequence Fasta fileFasta file

>gi|10835167|ref|NP_000607.1| CD4 antigen precursor [Homo sapiens] >gi|10835167|ref|NP_000607.1| CD4 antigen precursor [Homo sapiens] MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLT MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLT KGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLT KGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLT LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV RCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI RCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI

>gi|57113961|ref|NP_001009043.1| CD4 antigen [Pan troglodytes] >gi|57113961|ref|NP_001009043.1| CD4 antigen [Pan troglodytes] MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQTKILGNQGSFLT MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQTKILGNQGSFLT KGPSKLNDRVDSRRSLWDQGNFTLIIKNLKIEDSDTYICEVGDQKEEVQLLVFGLTANSDTHLLQGQSLT KGPSKLNDRVDSRRSLWDQGNFTLIIKNLKIEDSDTYICEVGDQKEEVQLLVFGLTANSDTHLLQGQSLT LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV RCRHRRRQAQRMSQIKRLLSEKKTCQCPHRFQKTCSPI RCRHRRRQAQRMSQIKRLLSEKKTCQCPHRFQKTCSPI

>gi|50054438|ref|NP_001001908.1| CD4 antigen [Sus scrofa] >gi|50054438|ref|NP_001001908.1| CD4 antigen [Sus scrofa] MDPGTSLRHLFLVLQLAMLPAASGTQEKYLVLGKAGDLAELPCHSSQKKNLPFNWKNSNQTKILGGHGSF MDPGTSLRHLFLVLQLAMLPAASGTQEKYLVLGKAGDLAELPCHSSQKKNLPFNWKNSNQTKILGGHGSF WHTASVTELTSRLDSKKNMWDHGSFPLIIKNLEVTDSGIYICEVEDKRIEVQLLVFRLTASVTRVLLGQS WHTASVTELTSRLDSKKNMWDHGSFPLIIKNLEVTDSGIYICEVEDKRIEVQLLVFRLTASVTRVLLGQS LTLTLEGPSGSHPTVQWKGPGNKSKNDVKSLLLPQVGLEDSGLWTCTVSQDQKTLVFRSNIFVLAFQKVP LTLTLEGPSGSHPTVQWKGPGNKSKNDVKSLLLPQVGLEDSGLWTCTVSQDQKTLVFRSNIFVLAFQKVP STVYVKEGDQVALSFPLTFEAESLSGELMWRQTKGASSPQSWITFSLKDRKVTVQKSLQNLKLRMAEKLP STVYVKEGDQVALSFPLTFEAESLSGELMWRQTKGASSPQSWITFSLKDRKVTVQKSLQNLKLRMAEKLP LQITLLQALPQYAGSGNLTLVLPEGRLHREVNLVVMRATQSKNEVTCEVLGPTPPKVVLSLKLGNQSMKV LQITLLQALPQYAGSGNLTLVLPEGRLHREVNLVVMRATQSKNEVTCEVLGPTPPKVVLSLKLGNQSMKV SDQQKLVTVLDPEAGMWRCLLRDKDKVLLESQVEVLPTAFTRAWPELLASVIGGIIGLLFLAGFCIACVK SDQQKLVTVLDPEAGMWRCLLRDKDKVLLESQVEVLPTAFTRAWPELLASVIGGIIGLLFLAGFCIACVK CWHRRRRAERMSQIKRLLSEKKTCQCAHRQQKNYSLT CWHRRRRAERMSQIKRLLSEKKTCQCAHRQQKNYSLT

>gi|6978631|ref|NP_036837.1| Cd4 molecule [Rattus norvegicus] >gi|6978631|ref|NP_036837.1| Cd4 molecule [Rattus norvegicus] MCRGFSFRHLLPLLLLQLSKLLVVTQGKTVVLGKEGGSAELPCESTSRRSASFAWKSSDQKTILGYKNKL MCRGFSFRHLLPLLLLQLSKLLVVTQGKTVVLGKEGGSAELPCESTSRRSASFAWKSSDQKTILGYKNKL LIKGSLELYSRFDSRKNAWERGSFPLIINKLRMEDSQTYVCELENKKEEVELWVFRVTFNPGTRLLQGQS LIKGSLELYSRFDSRKNAWERGSFPLIINKLRMEDSQTYVCELENKKEEVELWVFRVTFNPGTRLLQGQS LTLILDSNPKVSDPPIECKHKSSNIVKDSKAFSTHSLRIQDSGIWNCTVTLNQKKHSFDMKLSVLGFAST LTLILDSNPKVSDPPIECKHKSSNIVKDSKAFSTHSLRIQDSGIWNCTVTLNQKKHSFDMKLSVLGFAST SITAYKSEGESAEFSFPLNLGEESLQGELRWKAEKAPSSQSWITFSLKNQKVSVQKSTSNPKFQLSETLP SITAYKSEGESAEFSFPLNLGEESLQGELRWKAEKAPSSQSWITFSLKNQKVSVQKSTSNPKFQLSETLP LTLQIPQVSLQFAGSGNLTLTLDRGILYQEVNLVVMKVTQPDSNTLTCEVMGPTSPKMRLILKQENQEAR LTLQIPQVSLQFAGSGNLTLTLDRGILYQEVNLVVMKVTQPDSNTLTCEVMGPTSPKMRLILKQENQEAR VSRQEKVIQVQAPEAGVWQCLLSEGEEVKMDSKIQVLSKGLNQTMFLAVVLGSAFSFLVFTGLCILFCVR VSRQEKVIQVQAPEAGVWQCLLSEGEEVKMDSKIQVLSKGLNQTMFLAVVLGSAFSFLVFTGLCILFCVR CRHQQRQAARMSQIKRLLSEKKTCQCSHRMQKSHNLI CRHQQRQAARMSQIKRLLSEKKTCQCSHRMQKSHNLI

Clustal XClustal X

Step1: Load the sequencesStep1: Load the sequences

Uploaded sequencesUploaded sequences

A little unclear…

Edit Fasta headersEdit Fasta headers…… MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLT MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLT KGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLT KGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLT LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV RCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI RCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI

MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQTKILGNQGSFLT MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQTKILGNQGSFLT KGPSKLNDRVDSRRSLWDQGNFTLIIKNLKIEDSDTYICEVGDQKEEVQLLVFGLTANSDTHLLQGQSLT KGPSKLNDRVDSRRSLWDQGNFTLIIKNLKIEDSDTYICEVGDQKEEVQLLVFGLTANSDTHLLQGQSLT LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV RCRHRRRQAQRMSQIKRLLSEKKTCQCPHRFQKTCSPI RCRHRRRQAQRMSQIKRLLSEKKTCQCPHRFQKTCSPI

MDPGTSLRHLFLVLQLAMLPAASGTQEKYLVLGKAGDLAELPCHSSQKKNLPFNWKNSNQTKILGGHGSF MDPGTSLRHLFLVLQLAMLPAASGTQEKYLVLGKAGDLAELPCHSSQKKNLPFNWKNSNQTKILGGHGSF WHTASVTELTSRLDSKKNMWDHGSFPLIIKNLEVTDSGIYICEVEDKRIEVQLLVFRLTASVTRVLLGQS WHTASVTELTSRLDSKKNMWDHGSFPLIIKNLEVTDSGIYICEVEDKRIEVQLLVFRLTASVTRVLLGQS LTLTLEGPSGSHPTVQWKGPGNKSKNDVKSLLLPQVGLEDSGLWTCTVSQDQKTLVFRSNIFVLAFQKVP LTLTLEGPSGSHPTVQWKGPGNKSKNDVKSLLLPQVGLEDSGLWTCTVSQDQKTLVFRSNIFVLAFQKVP STVYVKEGDQVALSFPLTFEAESLSGELMWRQTKGASSPQSWITFSLKDRKVTVQKSLQNLKLRMAEKLP STVYVKEGDQVALSFPLTFEAESLSGELMWRQTKGASSPQSWITFSLKDRKVTVQKSLQNLKLRMAEKLP LQITLLQALPQYAGSGNLTLVLPEGRLHREVNLVVMRATQSKNEVTCEVLGPTPPKVVLSLKLGNQSMKV LQITLLQALPQYAGSGNLTLVLPEGRLHREVNLVVMRATQSKNEVTCEVLGPTPPKVVLSLKLGNQSMKV SDQQKLVTVLDPEAGMWRCLLRDKDKVLLESQVEVLPTAFTRAWPELLASVIGGIIGLLFLAGFCIACVK SDQQKLVTVLDPEAGMWRCLLRDKDKVLLESQVEVLPTAFTRAWPELLASVIGGIIGLLFLAGFCIACVK CWHRRRRAERMSQIKRLLSEKKTCQCAHRQQKNYSLT CWHRRRRAERMSQIKRLLSEKKTCQCAHRQQKNYSLT

MCRGFSFRHLLPLLLLQLSKLLVVTQGKTVVLGKEGGSAELPCESTSRRSASFAWKSSDQKTILGYKNKL MCRGFSFRHLLPLLLLQLSKLLVVTQGKTVVLGKEGGSAELPCESTSRRSASFAWKSSDQKTILGYKNKL LIKGSLELYSRFDSRKNAWERGSFPLIINKLRMEDSQTYVCELENKKEEVELWVFRVTFNPGTRLLQGQS LIKGSLELYSRFDSRKNAWERGSFPLIINKLRMEDSQTYVCELENKKEEVELWVFRVTFNPGTRLLQGQS LTLILDSNPKVSDPPIECKHKSSNIVKDSKAFSTHSLRIQDSGIWNCTVTLNQKKHSFDMKLSVLGFAST LTLILDSNPKVSDPPIECKHKSSNIVKDSKAFSTHSLRIQDSGIWNCTVTLNQKKHSFDMKLSVLGFAST SITAYKSEGESAEFSFPLNLGEESLQGELRWKAEKAPSSQSWITFSLKNQKVSVQKSTSNPKFQLSETLP SITAYKSEGESAEFSFPLNLGEESLQGELRWKAEKAPSSQSWITFSLKNQKVSVQKSTSNPKFQLSETLP LTLQIPQVSLQFAGSGNLTLTLDRGILYQEVNLVVMKVTQPDSNTLTCEVMGPTSPKMRLILKQENQEAR LTLQIPQVSLQFAGSGNLTLTLDRGILYQEVNLVVMKVTQPDSNTLTCEVMGPTSPKMRLILKQENQEAR VSRQEKVIQVQAPEAGVWQCLLSEGEEVKMDSKIQVLSKGLNQTMFLAVVLGSAFSFLVFTGLCILFCVR VSRQEKVIQVQAPEAGVWQCLLSEGEEVKMDSKIQVLSKGLNQTMFLAVVLGSAFSFLVFTGLCILFCVR CRHQQRQAARMSQIKRLLSEKKTCQCSHRMQKSHNLI CRHQQRQAARMSQIKRLLSEKKTCQCSHRMQKSHNLI

>Homo_sapiens_CD4

>Pan_troglodytes_CD4

>Sus_scrofa_CD4

>Rattus_norvegicus_CD4

>gi|10835167|ref|NP_000607.1| CD4 antigen precursor [Homo sapiens]

>gi|57113961|ref|NP_001009043.1| CD4 antigen [Pan troglodytes]

>gi|50054438|ref|NP_001001908.1| CD4 antigen [Sus scrofa]

>gi|6978631|ref|NP_036837.1| Cd4 molecule [Rattus norvegicus]

Uploaded sequencesUploaded sequences

Much better

Step2: Perform alignmentStep2: Perform alignment

Multiple Sequence Alignment and Multiple Sequence Alignment and conservation viewconservation view

Step 3: Create treeStep 3: Create tree

The Newick tree format is used to represent trees as strings

CA D

In Newick format: ((A,C),(B,D));

B

• Each pair of parenthesis () encloses a clade in the tree • A comma “,” separates the members of the corresponding clade• A semicolon “;” is always the last character

Step 4: View tree with NJPlotStep 4: View tree with NJPlot

Note :unrooted tree

Step 4.5: defining an outgroupStep 4.5: defining an outgroup

Note :The order

inside a split doesn’t matter

Swapping nodesSwapping nodes

Bootstrap

Bootstrap

A. Resample (100-1000 time).

12345 1001 : ATCTG…A 2 : ATCTG…C3 : ACTTA…C 4 : ACCTA…T

12345 1001 : AATTT…T2 : AATTT…G3 : AACTT…T4 : AACTT…T 11244 x

12345 1001 : TTTAT…T2 : TAACC…G3 : TAACC…T4 : TGGGA…T 4 7789…x

12345 1001 : AGGTA…T2 : AGGAC…G3 : AAAAC…A4 : AAAGG…C 15578… x

BootstrapB. Reconstruct a tree from each data set.

12345 1001 : AATTT…T2 : AATTT…G3 : AACTT…T4 : AACTT…T 11244 x

12345 1001 : TTTAT…T2 : TAACC…G3 : TAACC…T4 : TGGGA…T 4 7789…x

12345 1001 : AGGTA…T2 : AGGAC…G3 : AAAAC…A4 : AAAGG…C 15578… x

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3Sp4

C. We compute the majority rule consensus.

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3

Sp4

67%100%

In 67% of the data sets, the split between SP1+SP2 and the rest of the tree was found.

Bootstrap

Step 3.5 - BootstrapStep 3.5 - Bootstrap

Bootstrap values on NJPlotBootstrap values on NJPlot

Note:ClustalX saves trees with .ph extension. Trees with bootstrap are saved with .phb extension

Detecting selection Detecting selection forces using forces using phlogeny phlogeny

(ConSeq, ConSurf, (ConSeq, ConSurf, Selecton)Selecton)

““ImportantImportant”” sites evolvesites evolve

slowerslowerthan “unimportantunimportant” onesones.

Conservation = functional/structural Conservation = functional/structural importanceimportance

Use Phylogenetic informationUse Phylogenetic information 11223344556677

HumanHumanDDMMAAAAHHAAMM

ChimpChimpDDEEAAAAGGGGCC

CowCowDDQQAAAAWWAAPP

FishFishDDLLAAAACCAALL

S. S. cerevisiaecerevisiae

DDDDGGAAFFAAAA

S. pombeS. pombeDDDDGGAALLGGEEAA

GG

AA

AA

AA

GG

AA

AA

AA

AA

GG

GG

http://http://conseqconseq.tau.ac.il.tau.ac.ilSite-specific rate computation toolSite-specific rate computation tool

Using ConSeqUsing ConSeq

ConSeq resultsConSeq results

ConSeq conservation scoresConSeq conservation scores

Conservation scoresConservation scores:: The scores are standardized: the average score for all residues is The scores are standardized: the average score for all residues is

zero, and the standard deviation is one zero, and the standard deviation is one The lowest score represents the most conserved site in the protein The lowest score represents the most conserved site in the protein

negative values: slowly evolving (= low evolutionary rate), negative values: slowly evolving (= low evolutionary rate), conserved sitesconserved sites

The highest score represents the most variable site in the proteinThe highest score represents the most variable site in the protein positive values: rapidly evolving (= fast evolutionary rate), positive values: rapidly evolving (= fast evolutionary rate),

variable sitesvariable sites Scores are relative to the protein. Scores of different proteins Scores are relative to the protein. Scores of different proteins

are incomparable !!!are incomparable !!!

ConSeq resultsConSeq results

Color-coded resultsColor-coded results

Protein core: structurally constrained - usually conserved

Active site: functionally constrained - usually conserved

Surface loops: tolerant to mutations - usually variable

Hydrophobic core

Surface loops

Conservation in the structureConservation in the structure

Active site

Color-coded resultsColor-coded results

http://http://consurfconsurf.tau.ac.il.tau.ac.ilSame algorithm as ConSeq, but here the resultsSame algorithm as ConSeq, but here the results are projected onto the 3D structure of the proteinare projected onto the 3D structure of the protein

Using ConSurfUsing ConSurf

ConSurf resultsConSurf results

First Glance in Jmol visual presentationFirst Glance in Jmol visual presentation

ConSeqConSeq//ConSurfConSurf user intervention user intervention(advanced options)(advanced options)

1.1. Choosing the method for calculating the amino-acid Choosing the method for calculating the amino-acid conservation scores: (conservation scores: (BayesianBayesian/Max Likelihood)/Max Likelihood)

2.2. Entering your own MSA fileEntering your own MSA file3.3. Performing the MSA using: (Performing the MSA using: (MUSCLEMUSCLE/CLUSTALW)/CLUSTALW)4.4. Collecting the homologs from: (Collecting the homologs from: (SWISS-PROTSWISS-PROT/UniProt)/UniProt)5.5. Max. number of homologs: (Max. number of homologs: (5050))6.6. No. of PSI-BLAST iterations: (No. of PSI-BLAST iterations: (11))7.7. PSI-BLAST 3-value cutoff: (PSI-BLAST 3-value cutoff: (0.0010.001))8.8. Model of substitution for proteins: Model of substitution for proteins:

((JTTJTT/Dayhoff/mtREV/cpREV/WAG)/Dayhoff/mtREV/cpREV/WAG)9.9. Entering your own PDB fileEntering your own PDB file10.10. Entering your own TREE fileEntering your own TREE file

Solution – look at the DNASolution – look at the DNA

Purifying selectionSyn > Non-syn

Adaptive evolution = Positive selectionNon-syn > Syn

NeutralselectionSyn = Non-syn

Selection score Selection score ((Ka/KsKa/Ks) < 1) < 1 purifying selectionpurifying selection

Selection score Selection score ((Ka/KsKa/Ks) > 1) > 1 positive selectionpositive selection

Selection score Selection score ((Ka/KsKa/Ks) = 1) = 1 no selectionno selection

Ka/Ks also known as… Ka/Ks also known as… (or dn/ds, or (or dn/ds, or ωω))

Non-synonymous mutation rate

Synonymous mutation rate

http://selecton.tau.ac.il

Selecton inputSelecton input

The user must provide the sequences – no psi-blast option The user must provide the sequences – no psi-blast option Coding sequencesCoding sequences Only ORFOnly ORF No stop codonsNo stop codons If an MSA is provided it must be If an MSA is provided it must be codon alignedcodon aligned ((RevTransRevTrans))

Codon-level sequences !!!

Comparing HComparing H00 and H and H11 in Selecton in Selecton

H0

H1

Solution: Solution: statisticsstatistics helps us to helps us to compare between hypothesescompare between hypotheses

HH00: There’s no positive selection: There’s no positive selection

HH11: There is positive selection: There is positive selection

2~)))0(|(

))1(|(ln(2

HMDataL

HMDataL

HH00: compute the probability: compute the probability (likelihood) (likelihood) of the data using of the data using

a model that a model that does does not not account for positive selectionaccount for positive selection HH11: compute the probability: compute the probability (likelihood) (likelihood) of the data using of the data using

a model that a model that does account for positive selectiondoes account for positive selection Perform a statistical test to accept or reject HPerform a statistical test to accept or reject H00

(likelihood ratio test)(likelihood ratio test)

P-value

> 0.05 (aa) accept H0

> 0.05 (aa) reject H0

Comparing HComparing H00 and H and H11 in Selecton in Selecton

Comparing HComparing H00 and H and H11 in Selecton in Selecton

Selecton resultsSelecton results::

ResultsResults

Human rhesus swaps at sites 332, 335-340 confer human resistance to HIV and rhesus resistance to SIV