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Multiple Sequence Alignment

Multiple Sequence Alignment. ClustalW TCoffee Ka, Ks, and Ka/Ks Anchored alignment. ClustalW. http://www.ebi.ac.uk/clustalw/. ClustalW. Paste your sequences. Multiple sequence Alignment alignment options . Submit . Exercise.

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Multiple Sequence Alignment

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

  2. ClustalW • http://www.ebi.ac.uk/clustalw/

  3. ClustalW Paste your sequences Multiple sequence Alignment alignment options Submit

  4. Exercise • HomoloGene is a system for automated detection of homologs among annotated genes of several completely sequenced eukaryotic genomes. • Download the FASTA sequences of HomoloGene:5276 and align them with ClustalW

  5. Download protein sequences

  6. Result Alignment Guide Tree

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

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

  9. Sequences >gi|604533|gb|AAC37231.1| fertilization protein MRSLVLLCVLLMAICAADKKTSVSKENEAAMKVAMMKFLDMKAGVFKEIIEDMGYPITPPQWTTLLYYNR ERLIEFCRSFLALSKKIILLGGNKLNKANFARMGRILGWKSQWAVRQRQWGMVRVSRRHTSTAIAKRIVA MKVADLPCN >gi|604531|gb|AAC37233.1| fertilization protein MRFLLLLCVLMGAVSQAVCRKRPNVWGKIVVKEKNKAAMKIGFMEYLDAKLVKFKRHWLVGANWKLQKFE TDEMRYLAIKRLIKVCHGYTIWSQRLIMLKYRPLNEKYFKKVGRYLAWRNYLIVFRMWIGVLKKNLKRSE ITKPMQKLLDTKDGELPCPVRKIHG >gi|604529|gb|AAC37232.1| fertilization protein MRSLVLLCVLMAVGCVAFDDVVVSRQEQSYVQRGMVNFLDEEMHKLVKRFRDMRWNLGPGFVFLLKKVNR ERMMRYCMDYARYSKKILQLKHLPVNKKTLTKMGRFVGYRNYGVIRELYADVFRDVQGFRGPKMTAAMRK YSSKDPGTFPCKNEKRRG >gi|604527|gb|AAC37230.1| fertilization protein MRSLVLLCVLLMAICAADKKTTVSKENAAAMKIAMIKFLDARAGKFKKRVENMGYPITPPQWTTLLYYNR QRLMEWCHTYVEFSKKIILMGGNKLNKKNFTRMGRIIGWKNQWVLKRRQWEMVRVMRRYKSTAIAKKIVA MKVADLPCN >gi|604525|gb|AAC37229.1| fertilization protein MRSLVLLCVLLMAICAADKKSTVSKENAAAMKVAMIKFLDSRTDRFKKRIEKIGYPITPPQYTTLLYYNR ERLMDWCHNYVEVSKKIILLGGNKLNKKNFARMGRIIGWKNQWILKRRQWHMVRVMRRYKASAIAKKIVA MKVADLPCN

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

  11. Download formats Guide tree

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

  13. 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.

  14. (Result) Codon alignment >gi|604533|gb|AAC37231.1|_G_L36554 _S_ AAC37231 _DESC_ fertilization protein MATCHES_ON Haliotisassimilis fertilization protein mRNA, complete cds ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGCTGATGGCAATATGTGCGGCGGAC------ ------------------AAAAAAACCTCGGTCTCGAAGGAAAATGAAGCCGCAATGAAG GTAGCGATGATGAAGTTTTTGGATATGAAGGCGGGTGTATTCAAAGAAATC---ATTGAG GATATGGGATATCCAATAACCCCTCCGCAATGGACAACTCTACTGTACTACAACAGAGAG AGATTGATTGAATTTTGCCGTTCCTTCCTTGCATTGTCCAAAAAGATTATATTGCTGGGA GGTAACAAATTAAATAAGGCGAACTTCGCTAGGATGGGTCGAATCCTTGGCTGGAAAAGC CAGTGGGCTGTGAGACAGAGGCAATGGGGGATGGTCAGA---------GTGTCGAGGCGC CATACAAGTACTGCAATAGCTAAAAGGATCGTCGCCATGAAAGTTGCTGACCTACCCTGT AAC------------------TAG >gi|604531|gb|AAC37233.1|_G_L36590 _S_ AAC37233 _DESC_ fertilization protein MATCHES_ON Haliotiscorrugata fertilization protein mRNA, complete cds ATGAGGTTTTTGCTGCTTCTCTGTGTTTTGATGGGGGCAGTATCTCAGGCAGTATGCAGA AAAAGACCTAATGTCTGGGGGAAAATCGTGGTCAAGGAGAAAAATAAAGCCGCAATGAAG ATAGGGTTTATGGAATATTTGGATGCAAAGTTGGTAAAGTTTAAAAGGCACTGGCTTGTT GGAGCCAATTGGAAACTTCAAAAATTTGAAACGGATGAAATGAGATACCTCGCCATAAAG AGACTGATAAAAGTTTGCCATGGATACACTATTTGGTCCCAACGACTAATAATGTTAAAA TATCGACCATTGAATGAGAAATACTTCAAAAAGGTGGGTCGATACCTTGCCTGGCGAAAC TACCTCATAGTTTTTCGGATGTGGATCGGCGTTTTG------AAGAAAAATCTTAAAAGA TCGGAAATAACGAAACCCATGCAAAAACTCCTCGACACAAAGGATGGTGAGTTGCCCTGC CCTGTTAGAAAGATACATGGATAA >gi|604529|gb|AAC37232.1|_G_L36589 _S_ AAC37232 _DESC_ fertilization protein MATCHES_ON Haliotisfulgens fertilization protein mRNA, complete cds ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGATGGCGGTAGGATGTGTGGCGTTT------ ------------------GATGATGTGGTGGTCTCAAGGCAAGAGCAATCTTATGTGCAG AGAGGGATGGTCAACTTTTTGGATGAAGAAATGCATAAACTGGTTAAACGG---TTTAGA GATATGCGATGGAATTTAGGGCCAGGCTTTGTATTCCTTCTAAAAAAAGTCAACAGAGAG AGAATGATGCGCTACTGCATGGATTACGCCAGATATTCCAAAAAGATTTTACAGCTAAAA CATCTTCCAGTAAATAAGAAGACCCTCACTAAAATGGGTAGATTCGTTGGATATCGAAAC TATGGGGTCATCAGGGAGTTGTACGCCGACGTATTCAGAGACGTTCAAGGATTTAGGGGG CCTAAAATGACTGCAGCCATGAGGAAGTACAGCAGCAAGGATCCTGGTACATTTCCTTGC AAGAACGAGAAACGCCGCGGATGA >gi|604527|gb|AAC37230.1|_G_L36553 _S_ AAC37230 _DESC_ fertilization protein MATCHES_ON Haliotissorenseni fertilization protein mRNA, complete cds ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGCTGATGGCAATATGTGCGGCGGAC------ ------------------AAAAAAACCACGGTCTCGAAGGAAAATGCAGCCGCAATGAAG ATAGCTATGATAAAGTTTTTGGATGCGAGGGCGGGTAAATTCAAAAAACGC---GTTGAG AATATGGGATATCCAATAACCCCTCCGCAATGGACAACTCTACTATACTACAACAGACAG AGATTGATGGAATGGTGCCATACCTACGTTGAATTTTCCAAAAAGATTATATTGATGGGA GGTAACAAATTAAATAAGAAGAACTTCACTAGGATGGGTCGAATCATTGGCTGGAAAAAC CAGTGGGTTTTGAAAAGGAGGCAATGGGAGATGGTCAGA---------GTGATGAGGCGC TATAAAAGTACTGCAATAGCTAAAAAGATCGTCGCCATGAAAGTTGCTGACCTACCCTGT AAC------------------TAG >gi|604525|gb|AAC37229.1|_G_L36552 _S_ AAC37229 _DESC_ fertilization protein MATCHES_ON Haliotisrufescens fertilization protein mRNA, complete cds ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGCTGATGGCAATATGTGCGGCGGAC------ ------------------AAAAAATCCACGGTCTCGAAGGAAAATGCAGCCGCAATGAAG GTAGCGATGATAAAGTTTTTGGATTCGAGGACGGATAGATTCAAAAAACGC---ATTGAG AAGATTGGATATCCAATAACCCCTCCGCAATATACAACTCTACTATACTACAACAGAGAG AGATTGATGGATTGGTGCCATAACTACGTTGAAGTATCCAAAAAGATTATATTGTTGGGA GGTAACAAATTAAATAAGAAGAACTTCGCTAGGATGGGTCGAATCATTGGCTGGAAAAAC CAGTGGATTTTGAAAAGGAGGCAATGGCACATGGTCAGA---------GTGATGAGGCGC TATAAAGCTTCTGCAATAGCTAAAAAGATCGTCGCCATGAAAGTTGCTGACCTACCCTGT AAC------------------TAG

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

  16. Input codon alignment Select output statistics

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

  18. 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

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

  20. Distmat

  21. Distmat

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

  23. Align the following sequences (use the file dalign_sequences.txt): >seq1 WKKNADAPKRAMTSFMKAAY >seq2 WNLDTNSPEEKQAYIQLAKDDRIRYD >seq3 WRMDSNQKNPDSNNPKAAYNKGDANAPK

  24. Results • DIALIGN makes alignments from fragments

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

  26. Anchored alignment • Now, let us assume that the user has some expert knowledge concerning a certain domain that is present in all the input sequences • The domains marked in red in the three sequences are thought to be homologous to one another >seq1 WKKNADAPKRAMTSFMKAAY >seq2 WNLDTNSPEEKQAYIQLAKDDRIRYD >seq3 WRMDSNQKNPDSNNPKAAYNKGDANAPK

  27. Therefore, the user wants to define this domain as anchor and align the rest of the sequences automatically. • 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

  28. first sequence involved • second sequence involved • start of anchor in first sequence • start of anchor in second sequence • length of anchor

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

  30. Guidance/HoT

  31. >seq1 WKKNADAPKRAMTSFMKAAY >seq2 WNLDTNSPEEKQAYIQLAKDDRIRYD >seq3 WRMDSNQKNPDSNNPKAAYNKGDANAPK >seq4 WRMDSNQKNPNNPKAAYNKGDANAPK

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