Multiple sequence alignment msa and phylogeny
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Multiple Sequence Alignment (MSA) and Phylogeny . MSA input: multiple sequence Fasta file.

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Msa input multiple sequence fasta file
MSA input: multiple sequence Fasta file

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

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

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

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




Uploaded sequences
Uploaded sequences

A little unclear…


Edit fasta headers
Edit Fasta headers…

>Homo_sapiens_CD4

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

MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLT KGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLT LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV RCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI

MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQTKILGNQGSFLT KGPSKLNDRVDSRRSLWDQGNFTLIIKNLKIEDSDTYICEVGDQKEEVQLLVFGLTANSDTHLLQGQSLT LTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSI VYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPL HLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCV RCRHRRRQAQRMSQIKRLLSEKKTCQCPHRFQKTCSPI

MDPGTSLRHLFLVLQLAMLPAASGTQEKYLVLGKAGDLAELPCHSSQKKNLPFNWKNSNQTKILGGHGSF WHTASVTELTSRLDSKKNMWDHGSFPLIIKNLEVTDSGIYICEVEDKRIEVQLLVFRLTASVTRVLLGQS LTLTLEGPSGSHPTVQWKGPGNKSKNDVKSLLLPQVGLEDSGLWTCTVSQDQKTLVFRSNIFVLAFQKVP STVYVKEGDQVALSFPLTFEAESLSGELMWRQTKGASSPQSWITFSLKDRKVTVQKSLQNLKLRMAEKLP LQITLLQALPQYAGSGNLTLVLPEGRLHREVNLVVMRATQSKNEVTCEVLGPTPPKVVLSLKLGNQSMKV SDQQKLVTVLDPEAGMWRCLLRDKDKVLLESQVEVLPTAFTRAWPELLASVIGGIIGLLFLAGFCIACVK CWHRRRRAERMSQIKRLLSEKKTCQCAHRQQKNYSLT

MCRGFSFRHLLPLLLLQLSKLLVVTQGKTVVLGKEGGSAELPCESTSRRSASFAWKSSDQKTILGYKNKL LIKGSLELYSRFDSRKNAWERGSFPLIINKLRMEDSQTYVCELENKKEEVELWVFRVTFNPGTRLLQGQS LTLILDSNPKVSDPPIECKHKSSNIVKDSKAFSTHSLRIQDSGIWNCTVTLNQKKHSFDMKLSVLGFAST SITAYKSEGESAEFSFPLNLGEESLQGELRWKAEKAPSSQSWITFSLKNQKVSVQKSTSNPKFQLSETLP LTLQIPQVSLQFAGSGNLTLTLDRGILYQEVNLVVMKVTQPDSNTLTCEVMGPTSPKMRLILKQENQEAR VSRQEKVIQVQAPEAGVWQCLLSEGEEVKMDSKIQVLSKGLNQTMFLAVVLGSAFSFLVFTGLCILFCVR CRHQQRQAARMSQIKRLLSEKKTCQCSHRMQKSHNLI

>Pan_troglodytes_CD4

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

>Sus_scrofa_CD4

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

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

>Rattus_norvegicus_CD4


Uploaded sequences1
Uploaded sequences

Much better





The Newick tree format is used to represent trees as strings

A

C

B

D

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

  • 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 njplot
Step 4: View tree with NJPlot

Note:

unrooted tree



Swapping nodes

Note:

The order inside a split doesn’t matter



Bootstrap

A. Resample (100-1000 time).

12345 100

1 : ATCTG…A

2 : ATCTG…C

3 : ACTTA…C

4 : ACCTA…T

12345 100

1 : AATTT…T

2 : AATTT…G

3 : AACTT…T

4 : AACTT…T

11244 x

12345 100

1 : TTTAT…T

2 : TAACC…G

3 : TAACC…T

4 : TGGGA…T

47789…x

12345 100

1 : AGGTA…T

2 : AGGAC…G

3 : AAAAC…A

4 : AAAGG…C

15578… x


Sp1

Sp2

Sp3

Sp4

Bootstrap

B. Reconstruct a tree from each data set.

12345 100

1 : AATTT…T

2 : AATTT…G

3 : AACTT…T

4 : AACTT…T

11244 x

12345 100

1 : TTTAT…T

2 : TAACC…G

3 : TAACC…T

4 : TGGGA…T

47789…x

12345 100

1 : AGGTA…T

2 : AGGAC…G

3 : AAAAC…A

4 : AAAGG…C

15578… x

Sp1

Sp1

Sp2

Sp2

Sp3

Sp3

Sp4

Sp4


Sp1

Sp2

Sp3

Sp4

Bootstrap

C. We compute the majority rule consensus.

Sp1

Sp1

Sp2

Sp2

Sp3

Sp3

Sp4

Sp4

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

67%

Sp1

100%

Sp2

Sp3

Sp4



Bootstrap values on njplot
Bootstrap values on NJPlot

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


Detecting selection forces using phlogeny conseq consurf selecton

Detecting selection forces using phlogeny (ConSeq, ConSurf, Selecton)


Important”sites evolve

slower

than “unimportant” ones.




Http conseq tau ac il site specific rate computation tool
http://conseq.tau.ac.ilSite-specific rate computation tool





Conservation scores
Conservation scores:

  • The scores are standardized: the average score for all residues is zero, and the standard deviation is one

  • The lowest score represents the most conserved site in the protein

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

  • The highest score represents the most variable site in the protein

    • positive values: rapidly evolving (= fast evolutionary rate), variable sites

  • Scores are relative to the protein. Scores of different proteins are incomparable !!!




Conservation in the structure

Protein core:structurally constrained - usually conserved

Active site: functionally constrained - usually conserved

Surface loops: tolerant to mutations - usually variable

Active site

Surface loops

Hydrophobic core



Http consurf tau ac il
http://consurf.tau.ac.il

Same algorithm as ConSeq, but here the results

are projected onto the 3D structure of the protein





Conseq consurf user intervention advanced options
ConSeq/ConSurf user intervention(advanced options)

  • Choosing the method for calculating the amino-acid conservation scores: (Bayesian/Max Likelihood)

  • Entering your own MSA file

  • Performing the MSA using: (MUSCLE/CLUSTALW)

  • Collecting the homologs from: (SWISS-PROT/UniProt)

  • Max. number of homologs: (50)

  • No. of PSI-BLAST iterations: (1)

  • PSI-BLAST 3-value cutoff: (0.001)

  • Model of substitution for proteins: (JTT/Dayhoff/mtREV/cpREV/WAG)

  • Entering your own PDB file

  • Entering your own TREE file


Solution look at the dna
Solution – look at the DNA

Adaptive evolution = Positive selectionNon-syn > Syn

Purifying selectionSyn > Non-syn

NeutralselectionSyn = Non-syn


Ka/Ks also known as… (or dn/ds, or ω)

Selection score (Ka/Ks) < 1 purifying selection

Selection score (Ka/Ks) > 1 positive selection

Selection score (Ka/Ks) = 1 no selection

Ka

Ks

Non-synonymous mutation rate

Synonymous mutation rate


http://selecton.tau.ac.il


Selecton input
Selecton input

Codon-level sequences !!!

  • The user must provide the sequences – no psi-blast option

  • Coding sequences

  • Only ORF

  • No stop codons

  • If an MSA is provided it must be codon aligned(RevTrans)


Comparing h 0 and h 1 in selecton
Comparing H0 and H1 in Selecton

H1

H0


Solution statistics helps us to compare between hypotheses

> 0.05 (a) accept H0

< 0.05 (a) reject H0

P-value

Solution: statistics helps us to compare between hypotheses

  • H0: There’s no positive selection

  • H1: There is positive selection

  • H0: compute the probability (likelihood) of the data using a model that does not account for positive selection

  • H1: compute the probability (likelihood) of the data using a model that does account for positive selection

  • Perform a statistical test to accept or reject H0

    (likelihood ratio test)


Comparing h 0 and h 1 in selecton1
Comparing H0 and H1 in Selecton


Comparing h 0 and h 1 in selecton2
Comparing H0 and H1 in Selecton



Results
Results

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


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