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
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ClustalW
Paste your sequences
Run
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Results
Press Start Jalview for interactive view of the
alignment
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ClustalW output format
Guide Tree Cladogram
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Exercise

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

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Result
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TCoffee

• http//www.tcoffee.org/

Tcoffee computes its alignments by combining a
collection of smaller alignments
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Main features

• Multiple Sequence Alignment
• Structure based Multiple Sequence Alignment
• Combining the output of several multiple sequence
  alignment packages
• Combining two (or more) multiple sequence
  alignments into a single one
• Turning amino acid alignments into CDS nucleotide
  alignments

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Exercise

• 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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Estimating the rate of evolution

• In order to study selection patterns, you will
  need to have the corresponding DNA alignment
• By using the PROTOGENE (Protein-to-Gene) in
  Tcoffee, the amino-acid alignment will be
  transformed into the corresponding DNA
  alignment. The actual procedure is tBLASTn.

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Results
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In case it takes too long

• 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 non-synonymous
  substitution rates for codon-aligned nucleotide
  sequences according to Nei and Gojobori (1986)
  method.
• This program will only yield valid results if the
  input alignment is codon-aligned

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SNAP - Ds/Dn Calculation tool

• Using the alignment we obtained previously.
• Averages of all pairwise comparisons
• ds 0.3510, dn 0.3535, ds/dn 0.8241
• The positive selection in sperm protein genes
  from abalone (genus Haliotis) is assumed to be
  the result of species-specific interaction with
  egg surface proteins during fertilization
  (Swanson and Vacquier 1998).

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Distmat
http//sbcr.bii.a-star.edu.sg/cgi-bin/emboss/menu/
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
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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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http//dialign.gobics.de/anchor/submission.php
User manual
http//dialign.gobics.de/anchor/manual
Align the following sequences (use the file
dalign_sequences.txt) gtseq1 WKKNADAPKRAMTSFMKAA
Y gtseq2 WNLDTNSPEEKQAYIQLAKDDRIRYD gtseq3
WRMDSNQKNPDSNNPKAAYNKGDANAPK
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Results

• DIALIGN composes alignments from fragments
• Lower-case letters denote residues not belonging
  to any of these selected fragments. They are not
  considered to be aligned.

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Results

• Numbers below the alignment roughly reflect the
  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