Multiple Sequence Alignment Clustal W TCoffee Ka Ks

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Multiple Sequence Alignment Clustal. W TCoffee Ka, Ks, and Ka/Ks Anchored alignment 1

Multiple Sequence Alignment Clustal. W TCoffee Ka, Ks, and Ka/Ks Anchored alignment 1

Clustal. W Ø http: //www. ebi. ac. uk/clustalw/ 2

Clustal. W Ø http: //www. ebi. ac. uk/clustalw/ 2

Clustal. W Paste your sequences Multiple sequence Alignment alignment options Submit 3

Clustal. W Paste your sequences Multiple sequence Alignment alignment options Submit 3

Exercise Ø Homolo. Gene is a system for automated detection of homologs among annotated

Exercise Ø Homolo. Gene is a system for automated detection of homologs among annotated genes of several completely sequenced eukaryotic genomes. Ø Download the FASTA sequences of Homolo. Gene: 5276 and align them with Clustal. W 4

Download protein sequences 5

Download protein sequences 5

Result Alignment Guide Tree 6

Result Alignment Guide Tree 6

TCoffee http: //tcoffee. crg. cat/ Tcoffee computes its alignments by combining a collection of

TCoffee http: //tcoffee. crg. cat/ Tcoffee computes its alignments by combining a collection of smaller alignments 7

Alignment at the DNA level based on an alignment at the Protein Level Ø

Alignment at the DNA level based on an alignment at the Protein Level Ø The 18 -k. Da protein plays an important role in fertilization of several abalone species Ø Build a multiple sequence alignment using the following sequences 8

Sequences >gi|604533|gb|AAC 37231. 1| fertilization protein MRSLVLLCVLLMAICAADKKTSVSKENEAAMKVAMMKFLDMKAGVFKEIIEDMGYPITPPQWTTLLYYNR ERLIEFCRSFLALSKKIILLGGNKLNKANFARMGRILGWKSQWAVRQRQWGMVRVSRRHTSTAIAKRIVA MKVADLPCN >gi|604531|gb|AAC 37233. 1| fertilization protein

Sequences >gi|604533|gb|AAC 37231. 1| fertilization protein MRSLVLLCVLLMAICAADKKTSVSKENEAAMKVAMMKFLDMKAGVFKEIIEDMGYPITPPQWTTLLYYNR ERLIEFCRSFLALSKKIILLGGNKLNKANFARMGRILGWKSQWAVRQRQWGMVRVSRRHTSTAIAKRIVA MKVADLPCN >gi|604531|gb|AAC 37233. 1| fertilization protein MRFLLLLCVLMGAVSQAVCRKRPNVWGKIVVKEKNKAAMKIGFMEYLDAKLVKFKRHWLVGANWKLQKFE TDEMRYLAIKRLIKVCHGYTIWSQRLIMLKYRPLNEKYFKKVGRYLAWRNYLIVFRMWIGVLKKNLKRSE ITKPMQKLLDTKDGELPCPVRKIHG >gi|604529|gb|AAC 37232. 1| fertilization protein MRSLVLLCVLMAVGCVAFDDVVVSRQEQSYVQRGMVNFLDEEMHKLVKRFRDMRWNLGPGFVFLLKKVNR ERMMRYCMDYARYSKKILQLKHLPVNKKTLTKMGRFVGYRNYGVIRELYADVFRDVQGFRGPKMTAAMRK YSSKDPGTFPCKNEKRRG >gi|604527|gb|AAC 37230. 1| fertilization protein MRSLVLLCVLLMAICAADKKTTVSKENAAAMKIAMIKFLDARAGKFKKRVENMGYPITPPQWTTLLYYNR QRLMEWCHTYVEFSKKIILMGGNKLNKKNFTRMGRIIGWKNQWVLKRRQWEMVRVMRRYKSTAIAKKIVA MKVADLPCN >gi|604525|gb|AAC 37229. 1| fertilization protein MRSLVLLCVLLMAICAADKKSTVSKENAAAMKVAMIKFLDSRTDRFKKRIEKIGYPITPPQYTTLLYYNR ERLMDWCHNYVEVSKKIILLGGNKLNKKNFARMGRIIGWKNQWILKRRQWHMVRVMRRYKASAIAKKIVA MKVADLPCN 9

Choose TCoffee Regular, paste the sequences in the data box, and press submit 10

Choose TCoffee Regular, paste the sequences in the data box, and press submit 10

Download formats Guide tree 11

Download formats Guide tree 11

Codon Alignment Ø In order to study selection patterns, you will need to have

Codon Alignment Ø In order to study selection patterns, you will need to have the corresponding DNA alignment Ø Using the PROTOGENE (Protein-to. Gene) in Tcoffee, the amino-acid alignment will be transformed into a codon alignment. The actual procedure invloves t. BLASTn. 12

• PROTOGENE (in Tcoffee) is time consuming. Please submit your email address, and

• PROTOGENE (in Tcoffee) is time consuming. Please submit your email address, and the results will be emailed to you. • PROTOGENE may return more that one DNA sequence for any given Protein sequence. For your homework assignment, please choose one sequence for each species. 13

(Result) Codon alignment >gi|604533|gb|AAC 37231. 1|_G_L 36554 _S_ AAC 37231 _DESC_ fertilization protein MATCHES_ON

(Result) Codon alignment >gi|604533|gb|AAC 37231. 1|_G_L 36554 _S_ AAC 37231 _DESC_ fertilization protein MATCHES_ON Haliotis assimilis fertilization protein m. RNA, complete cds ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGCTGATGGCAATATGTGCGGCGGAC------------AAAAAAACCTCGGTCTCGAAGGAAAATGAAGCCGCAATGAAG GTAGCGATGATGAAGTTTTTGGATATGAAGGCGGGTGTATTCAAAGAAATC---ATTGAG GATATGGGATATCCAATAACCCCTCCGCAATGGACAACTCTACTGTACTACAACAGAGAG AGATTGAATTTTGCCGTTCCTTGCATTGTCCAAAAAGATTATATTGCTGGGA GGTAACAAATTAAATAAGGCGAACTTCGCTAGGATGGGTCGAATCCTTGGCTGGAAAAGC CAGTGGGCTGTGAGACAGAGGCAATGGGGGATGGTCAGA-----GTGTCGAGGCGC CATACAAGTACTGCAATAGCTAAAAGGATCGTCGCCATGAAAGTTGCTGACCTACCCTGT AAC---------TAG >gi|604531|gb|AAC 37233. 1|_G_L 36590 _S_ AAC 37233 _DESC_ fertilization protein MATCHES_ON Haliotis corrugata fertilization protein m. RNA, complete cds ATGAGGTTTTTGCTGCTTCTCTGTGTTTTGATGGGGGCAGTATCTCAGGCAGTATGCAGA AAAAGACCTAATGTCTGGGGGAAAATCGTGGTCAAGGAGAAAAATAAAGCCGCAATGAAG ATAGGGTTTATGGAATATTTGGATGCAAAGTTGGTAAAGTTTAAAAGGCACTGGCTTGTT GGAGCCAATTGGAAACTTCAAAAATTTGAAACGGATGAAATGAGATACCTCGCCATAAAG AGACTGATAAAAGTTTGCCATGGATACACTATTTGGTCCCAACGACTAATAATGTTAAAA TATCGACCATTGAATGAGAAATACTTCAAAAAGGTGGGTCGATACCTTGCCTGGCGAAAC TACCTCATAGTTTTTCGGATGTGGATCGGCGTTTTG------AAGAAAAATCTTAAAAGA TCGGAAATAACGAAACCCATGCAAAAACTCCTCGACACAAAGGATGGTGAGTTGCCCTGC CCTGTTAGAAAGATACATGGATAA >gi|604529|gb|AAC 37232. 1|_G_L 36589 _S_ AAC 37232 _DESC_ fertilization protein MATCHES_ON Haliotis fulgens fertilization protein m. RNA, complete cds ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGATGGCGGTAGGATGTGTGGCGTTT------------GATGATGTGGTGGTCTCAAGGCAAGAGCAATCTTATGTGCAG AGAGGGATGGTCAACTTTTTGGATGAAGAAATGCATAAACTGGTTAAACGG---TTTAGA GATATGCGATGGAATTTAGGGCCAGGCTTTGTATTCCTTCTAAAAAAAGTCAACAGAGAG AGAATGATGCGCTACTGCATGGATTACGCCAGATATTCCAAAAAGATTTTACAGCTAAAA CATCTTCCAGTAAATAAGAAGACCCTCACTAAAATGGGTAGATTCGTTGGATATCGAAAC TATGGGGTCATCAGGGAGTTGTACGCCGACGTATTCAGAGACGTTCAAGGATTTAGGGGG CCTAAAATGACTGCAGCCATGAGGAAGTACAGCAGCAAGGATCCTGGTACATTTCCTTGC AAGAACGAGAAACGCCGCGGATGA >gi|604527|gb|AAC 37230. 1|_G_L 36553 _S_ AAC 37230 _DESC_ fertilization protein MATCHES_ON Haliotis sorenseni fertilization protein m. RNA, complete cds ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGCTGATGGCAATATGTGCGGCGGAC------------AAAAAAACCACGGTCTCGAAGGAAAATGCAGCCGCAATGAAG ATAGCTATGATAAAGTTTTTGGATGCGAGGGCGGGTAAATTCAAAAAACGC---GTTGAG AATATGGGATATCCAATAACCCCTCCGCAATGGACAACTCTACTACAACAG AGATTGATGGAATGGTGCCATACCTACGTTGAATTTTCCAAAAAGATTATATTGATGGGA GGTAACAAATTAAATAAGAAGAACTTCACTAGGATGGGTCGAATCATTGGCTGGAAAAAC CAGTGGGTTTTGAAAAGGAGGCAATGGGAGATGGTCAGA-----GTGATGAGGCGC TATAAAAGTACTGCAATAGCTAAAAAGATCGTCGCCATGAAAGTTGCTGACCTACCCTGT AAC---------TAG >gi|604525|gb|AAC 37229. 1|_G_L 36552 _S_ AAC 37229 _DESC_ fertilization protein MATCHES_ON Haliotis rufescens fertilization protein m. RNA, complete cds ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGCTGATGGCAATATGTGCGGCGGAC------------AAAAAATCCACGGTCTCGAAGGAAAATGCAGCCGCAATGAAG GTAGCGATGATAAAGTTTTTGGATTCGAGGACGGATAGATTCAAAAAACGC---ATTGAG AAGATTGGATATCCAATAACCCCTCCGCAATATACAACTCTACTACAACAGAGAG AGATTGATGGATTGGTGCCATAACTACGTTGAAGTATCCAAAAAGATTATATTGTTGGGA GGTAACAAATTAAATAAGAAGAACTTCGCTAGGATGGGTCGAATCATTGGCTGGAAAAAC CAGTGGATTTTGAAAAGGAGGCAATGGCACATGGTCAGA-----GTGATGAGGCGC TATAAAGCTTCTGCAATAGCTAAAAAGATCGTCGCCATGAAAGTTGCTGACCTACCCTGT AAC---------TAG 14

SNAP - Ds/Dn Calculation Tool http: //hcv. lanl. gov/content/sequence/SNAP. html Calculates synonymous and nonsynonymous

SNAP - Ds/Dn Calculation Tool http: //hcv. lanl. gov/content/sequence/SNAP. html Calculates synonymous and nonsynonymous substitution rates based on codon alignments according to Nei and Gojobori (1986) method. 15

Input codon alignment Select output statistics 16

Input codon alignment Select output statistics 16

SNAP - Ds/Dn Calculation Tool Conclusion: We detect positive selection in six of the

SNAP - Ds/Dn Calculation Tool Conclusion: We detect positive selection in six of the comparisons. So did Swanson and Vacquier (1998). 17

Distmat http: //emboss. bioinformatics. nl/cgi-bin/emboss/distmat Distmat calculates the evolutionary distances between every pair of

Distmat http: //emboss. bioinformatics. nl/cgi-bin/emboss/distmat Distmat calculates the evolutionary distances between every pair of sequences in a multiple alignment. The distances are expressed in terms of the number per 100 nucleotides or number of replacements per 100 amino acids 18

Distmat Ø Feed the DNA alignment of 18 -k. Da protein into distmat. Ø

Distmat Ø Feed the DNA alignment of 18 -k. Da protein into distmat. Ø Calculate separately the distances between the sequences for codon positions 1 and 2, and for codon position 3. Ø Are the results in agreement with those from the dn/ds analysis? 19

Distmat

Distmat

Distmat

Distmat

Anchored multiple-sequence alignment with DIALIGN http: //dialign. gobics. de/anchor/submission. php User manual: http: //dialign.

Anchored multiple-sequence alignment with DIALIGN http: //dialign. gobics. de/anchor/submission. php User manual: http: //dialign. gobics. de/anchor/manual 22

Align the following sequences (use the file dalign_sequences. txt): >seq 1 WKKNADAPKRAMTSFMKAAY >seq 2

Align the following sequences (use the file dalign_sequences. txt): >seq 1 WKKNADAPKRAMTSFMKAAY >seq 2 WNLDTNSPEEKQAYIQLAKDDRIRYD >seq 3 WRMDSNQKNPDSNNPKAAYNKGDANAPK 23

Results Ø DIALIGN makes alignments from fragments 24

Results Ø DIALIGN makes alignments from fragments 24

Results Ø Numbers below the alignment reflect some rough degree of local similarity among

Results Ø Numbers below the alignment reflect some rough degree of local similarity among the sequences 25

Anchored alignment Ø Now, let us assume that the user has some expert knowledge

Anchored alignment Ø Now, let us assume that the user has some expert knowledge concerning a certain domain that is present in all the input sequences Ø The domains marked in red in the three sequences are thought to be homologous to one another >seq 1 WKKNADAPKRAMTSFMKAAY >seq 2 WNLDTNSPEEKQAYIQLAKDDRIRYD >seq 3 WRMDSNQKNPDSNNPKAAYNKGDANAPK 26

Ø Therefore, the user wants to define this domain as anchor and align the

Ø Therefore, the user wants to define this domain as anchor and align the rest of the sequences automatically. Ø To specify a set of anchor points, each anchor point corresponds to a equal-length segment pair involving two of the input sequences should be defined 27

Ø first sequence involved Ø second sequence involved Ø start of anchor in first

Ø first sequence involved Ø second sequence involved Ø start of anchor in first sequence Ø start of anchor in second sequence Ø length of anchor 28

Results Ø The specified domain is aligned and the remainder of the sequences is

Results Ø The specified domain is aligned and the remainder of the sequences is aligned automatically respecting the constraints given by the anchor points: 29

Guidance/Ho. T

Guidance/Ho. T

>seq 1 WKKNADAPKRAMTSFMKAAY >seq 2 WNLDTNSPEEKQAYIQLAKDDRIRYD >seq 3 WRMDSNQKNPDSNNPKAAYNKGDANAPK >seq 4 WRMDSNQKNPNNPKAAYNKGDANAPK

>seq 1 WKKNADAPKRAMTSFMKAAY >seq 2 WNLDTNSPEEKQAYIQLAKDDRIRYD >seq 3 WRMDSNQKNPDSNNPKAAYNKGDANAPK >seq 4 WRMDSNQKNPNNPKAAYNKGDANAPK

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