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

2. ClustalW

3. ClustalW Paste your sequences Submit
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|AAC | fertilization protein
MRFLLLLCVLMGAVSQAVCRKRPNVWGKIVVKEKNKAAMKIGFMEYLDAKLVKFKRHWLVGANWKLQKFE
TDEMRYLAIKRLIKVCHGYTIWSQRLIMLKYRPLNEKYFKKVGRYLAWRNYLIVFRMWIGVLKKNLKRSE
ITKPMQKLLDTKDGELPCPVRKIHG
>gi|604529|gb|AAC | fertilization protein
MRSLVLLCVLMAVGCVAFDDVVVSRQEQSYVQRGMVNFLDEEMHKLVKRFRDMRWNLGPGFVFLLKKVNR
ERMMRYCMDYARYSKKILQLKHLPVNKKTLTKMGRFVGYRNYGVIRELYADVFRDVQGFRGPKMTAAMRK
YSSKDPGTFPCKNEKRRG
>gi|604527|gb|AAC | fertilization protein
MRSLVLLCVLLMAICAADKKTTVSKENAAAMKIAMIKFLDARAGKFKKRVENMGYPITPPQWTTLLYYNR
QRLMEWCHTYVEFSKKIILMGGNKLNKKNFTRMGRIIGWKNQWVLKRRQWEMVRVMRRYKSTAIAKKIVA
>gi|604525|gb|AAC | fertilization protein
MRSLVLLCVLLMAICAADKKSTVSKENAAAMKVAMIKFLDSRTDRFKKRIEKIGYPITPPQYTTLLYYNR
ERLMDWCHNYVEVSKKIILLGGNKLNKKNFARMGRIIGWKNQWILKRRQWHMVRVMRRYKASAIAKKIVA

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
PROTOGENE (in Tcoffee) is time consuming. Please submit your address, and the results will be ed 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|AAC |_G_L36554 _S_ AAC37231 _DESC_ fertilization protein MATCHES_ON Haliotis assimilis fertilization protein mRNA, complete cds
ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGCTGATGGCAATATGTGCGGCGGAC------
AAAAAAACCTCGGTCTCGAAGGAAAATGAAGCCGCAATGAAG
GTAGCGATGATGAAGTTTTTGGATATGAAGGCGGGTGTATTCAAAGAAATC---ATTGAG
GATATGGGATATCCAATAACCCCTCCGCAATGGACAACTCTACTGTACTACAACAGAGAG
AGATTGATTGAATTTTGCCGTTCCTTCCTTGCATTGTCCAAAAAGATTATATTGCTGGGA
GGTAACAAATTAAATAAGGCGAACTTCGCTAGGATGGGTCGAATCCTTGGCTGGAAAAGC
CAGTGGGCTGTGAGACAGAGGCAATGGGGGATGGTCAGA GTGTCGAGGCGC
CATACAAGTACTGCAATAGCTAAAAGGATCGTCGCCATGAAAGTTGCTGACCTACCCTGT
AAC TAG
>gi|604531|gb|AAC |_G_L36590 _S_ AAC37233 _DESC_ fertilization protein MATCHES_ON Haliotis corrugata fertilization protein mRNA, complete cds
ATGAGGTTTTTGCTGCTTCTCTGTGTTTTGATGGGGGCAGTATCTCAGGCAGTATGCAGA
AAAAGACCTAATGTCTGGGGGAAAATCGTGGTCAAGGAGAAAAATAAAGCCGCAATGAAG
ATAGGGTTTATGGAATATTTGGATGCAAAGTTGGTAAAGTTTAAAAGGCACTGGCTTGTT
GGAGCCAATTGGAAACTTCAAAAATTTGAAACGGATGAAATGAGATACCTCGCCATAAAG
AGACTGATAAAAGTTTGCCATGGATACACTATTTGGTCCCAACGACTAATAATGTTAAAA
TATCGACCATTGAATGAGAAATACTTCAAAAAGGTGGGTCGATACCTTGCCTGGCGAAAC
TACCTCATAGTTTTTCGGATGTGGATCGGCGTTTTG------AAGAAAAATCTTAAAAGA
TCGGAAATAACGAAACCCATGCAAAAACTCCTCGACACAAAGGATGGTGAGTTGCCCTGC
CCTGTTAGAAAGATACATGGATAA
>gi|604529|gb|AAC |_G_L36589 _S_ AAC37232 _DESC_ fertilization protein MATCHES_ON Haliotis fulgens fertilization protein mRNA, complete cds
ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGATGGCGGTAGGATGTGTGGCGTTT------
GATGATGTGGTGGTCTCAAGGCAAGAGCAATCTTATGTGCAG
AGAGGGATGGTCAACTTTTTGGATGAAGAAATGCATAAACTGGTTAAACGG---TTTAGA
GATATGCGATGGAATTTAGGGCCAGGCTTTGTATTCCTTCTAAAAAAAGTCAACAGAGAG
AGAATGATGCGCTACTGCATGGATTACGCCAGATATTCCAAAAAGATTTTACAGCTAAAA
CATCTTCCAGTAAATAAGAAGACCCTCACTAAAATGGGTAGATTCGTTGGATATCGAAAC
TATGGGGTCATCAGGGAGTTGTACGCCGACGTATTCAGAGACGTTCAAGGATTTAGGGGG
CCTAAAATGACTGCAGCCATGAGGAAGTACAGCAGCAAGGATCCTGGTACATTTCCTTGC
AAGAACGAGAAACGCCGCGGATGA
>gi|604527|gb|AAC |_G_L36553 _S_ AAC37230 _DESC_ fertilization protein MATCHES_ON Haliotis sorenseni fertilization protein mRNA, complete cds
AAAAAAACCACGGTCTCGAAGGAAAATGCAGCCGCAATGAAG
ATAGCTATGATAAAGTTTTTGGATGCGAGGGCGGGTAAATTCAAAAAACGC---GTTGAG
AATATGGGATATCCAATAACCCCTCCGCAATGGACAACTCTACTATACTACAACAGACAG
AGATTGATGGAATGGTGCCATACCTACGTTGAATTTTCCAAAAAGATTATATTGATGGGA
GGTAACAAATTAAATAAGAAGAACTTCACTAGGATGGGTCGAATCATTGGCTGGAAAAAC
CAGTGGGTTTTGAAAAGGAGGCAATGGGAGATGGTCAGA GTGATGAGGCGC
TATAAAAGTACTGCAATAGCTAAAAAGATCGTCGCCATGAAAGTTGCTGACCTACCCTGT
>gi|604525|gb|AAC |_G_L36552 _S_ AAC37229 _DESC_ fertilization protein MATCHES_ON Haliotis rufescens fertilization protein mRNA, complete cds
AAAAAATCCACGGTCTCGAAGGAAAATGCAGCCGCAATGAAG
GTAGCGATGATAAAGTTTTTGGATTCGAGGACGGATAGATTCAAAAAACGC---ATTGAG
AAGATTGGATATCCAATAACCCCTCCGCAATATACAACTCTACTATACTACAACAGAGAG
AGATTGATGGATTGGTGCCATAACTACGTTGAAGTATCCAAAAAGATTATATTGTTGGGA
GGTAACAAATTAAATAAGAAGAACTTCGCTAGGATGGGTCGAATCATTGGCTGGAAAAAC
CAGTGGATTTTGAAAAGGAGGCAATGGCACATGGTCAGA GTGATGAGGCGC
TATAAAGCTTCTGCAATAGCTAAAAAGATCGTCGCCATGAAAGTTGCTGACCTACCCTGT

15. SNAP - Ds/Dn Calculation Tool
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
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
User 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