Multiple Sequence Alignment

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Multiple Sequence Alignment

• ClustalW
• TCoffee
• Ka, Ks, and Ka/Ks
• Anchored alignment

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ClustalW

• http//www.ebi.ac.uk/clustalw/

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ClustalW
Paste your sequences
Multiple sequence Alignment alignment options
Submit
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Exercise

• HomoloGene is a system for automated detection of
  homologs among annotated genes of several
  completely sequenced eukaryotic genomes.
• Download the FASTA sequences of HomoloGene5276
  and align them with ClustalW

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Download protein sequences
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Result
Alignment
Guide Tree
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TCoffee

• http//tcoffee.crg.cat/

Tcoffee computes its alignments by combining a
collection of smaller alignments
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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

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Sequences

• gtgi604533gbAAC37231.1 fertilization protein
• MRSLVLLCVLLMAICAADKKTSVSKENEAAMKVAMMKFLDMKAGVFKEII
  EDMGYPITPPQWTTLLYYNR
• ERLIEFCRSFLALSKKIILLGGNKLNKANFARMGRILGWKSQWAVRQRQW
  GMVRVSRRHTSTAIAKRIVA
• MKVADLPCN
• gtgi604531gbAAC37233.1 fertilization protein
• MRFLLLLCVLMGAVSQAVCRKRPNVWGKIVVKEKNKAAMKIGFMEYLDAK
  LVKFKRHWLVGANWKLQKFE
• TDEMRYLAIKRLIKVCHGYTIWSQRLIMLKYRPLNEKYFKKVGRYLAWRN
  YLIVFRMWIGVLKKNLKRSE
• ITKPMQKLLDTKDGELPCPVRKIHG
• gtgi604529gbAAC37232.1 fertilization protein
• MRSLVLLCVLMAVGCVAFDDVVVSRQEQSYVQRGMVNFLDEEMHKLVKRF
  RDMRWNLGPGFVFLLKKVNR
• ERMMRYCMDYARYSKKILQLKHLPVNKKTLTKMGRFVGYRNYGVIRELYA
  DVFRDVQGFRGPKMTAAMRK
• YSSKDPGTFPCKNEKRRG
• gtgi604527gbAAC37230.1 fertilization protein
• MRSLVLLCVLLMAICAADKKTTVSKENAAAMKIAMIKFLDARAGKFKKRV
  ENMGYPITPPQWTTLLYYNR
• QRLMEWCHTYVEFSKKIILMGGNKLNKKNFTRMGRIIGWKNQWVLKRRQW
  EMVRVMRRYKSTAIAKKIVA
• MKVADLPCN

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Choose TCoffee Regular, paste the sequences in
the data box, and press submit
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Download formats
Guide tree
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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.

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• 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.

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(Result) Codon alignment

• gtgi604533gbAAC37231.1_G_L36554 _S_ AAC37231
  _DESC_ fertilization protein MATCHES_ON Haliotis
  assimilis fertilization protein mRNA, complete
  cds
• ATGAGGTCTTTGGTGCTTCTCTGTGTTTTGCTGATGGCAATATGTGCGGC
  GGAC------
• ------------------AAAAAAACCTCGGTCTCGAAGGAAAATGAAGC
  CGCAATGAAG
• GTAGCGATGATGAAGTTTTTGGATATGAAGGCGGGTGTATTCAAAGAAAT
  C---ATTGAG
• GATATGGGATATCCAATAACCCCTCCGCAATGGACAACTCTACTGTACTA
  CAACAGAGAG
• AGATTGATTGAATTTTGCCGTTCCTTCCTTGCATTGTCCAAAAAGATTAT
  ATTGCTGGGA
• GGTAACAAATTAAATAAGGCGAACTTCGCTAGGATGGGTCGAATCCTTGG
  CTGGAAAAGC
• CAGTGGGCTGTGAGACAGAGGCAATGGGGGATGGTCAGA---------GT
  GTCGAGGCGC
• CATACAAGTACTGCAATAGCTAAAAGGATCGTCGCCATGAAAGTTGCTGA
  CCTACCCTGT
• AAC------------------TAG
• gtgi604531gbAAC37233.1_G_L36590 _S_ AAC37233
  _DESC_ fertilization protein MATCHES_ON Haliotis
  corrugata fertilization protein mRNA, complete
  cds
• ATGAGGTTTTTGCTGCTTCTCTGTGTTTTGATGGGGGCAGTATCTCAGGC
  AGTATGCAGA
• AAAAGACCTAATGTCTGGGGGAAAATCGTGGTCAAGGAGAAAAATAAAGC
  CGCAATGAAG
• ATAGGGTTTATGGAATATTTGGATGCAAAGTTGGTAAAGTTTAAAAGGCA
  CTGGCTTGTT
• GGAGCCAATTGGAAACTTCAAAAATTTGAAACGGATGAAATGAGATACCT
  CGCCATAAAG
• AGACTGATAAAAGTTTGCCATGGATACACTATTTGGTCCCAACGACTAAT
  AATGTTAAAA
• TATCGACCATTGAATGAGAAATACTTCAAAAAGGTGGGTCGATACCTTGC
  CTGGCGAAAC
• TACCTCATAGTTTTTCGGATGTGGATCGGCGTTTTG------AAGAAAAA
  TCTTAAAAGA
• TCGGAAATAACGAAACCCATGCAAAAACTCCTCGACACAAAGGATGGTGA
  GTTGCCCTGC

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SNAP - Ds/Dn Calculation Tool

• http//hcv.lanl.gov/content/sequence/SNAP/SNAP.htm
  l
• Calculates synonymous and nonsynonymous
  substitution rates based on codon alignments
  according to Nei and Gojobori (1986) method.

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Input codon alignment
Select output statistics
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SNAP - Ds/Dn Calculation Tool

• Conclusion We detect positive selection in six
  of the comparisons. So did Swanson and Vacquier
  (1998).

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Distmat
http//emboss.bioinformatics.nl/cgi-bin/emboss/dis
tmat
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
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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?

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Distmat
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Distmat
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Anchored multiple-sequence alignment with DIALIGN
http//dialign.gobics.de/anchor/submission.php
User manual
http//dialign.gobics.de/anchor/manual
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• Align the following sequences (use the file
  dalign_sequences.txt)
• gtseq1 WKKNADAPKRAMTSFMKAAY
• gtseq2 WNLDTNSPEEKQAYIQLAKDDRIRYD
• gtseq3 WRMDSNQKNPDSNNPKAAYNKGDANAPK
• 
  

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Results

• DIALIGN makes alignments from fragments

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Results

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

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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

gtseq1 WKKNADAPKRAMTSFMKAAY gtseq2
WNLDTNSPEEKQAYIQLAKDDRIRYD gtseq3
WRMDSNQKNPDSNNPKAAYNKGDANAPK
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• 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

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

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Results

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

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Guidance/HoT
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gtseq1 WKKNADAPKRAMTSFMKAAY gtseq2 WNLDTNSPEEKQAYI
QLAKDDRIRYD gtseq3 WRMDSNQKNPDSNNPKAAYNKGDANAPK gts
eq4 WRMDSNQKNPNNPKAAYNKGDANAPK
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