Multiple Sequence Alignment and Molecular Evolution

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Multiple Sequence Alignment and Molecular
Evolution
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Quiz

• What do
• BLAST
• FASTA
• Protein motif searching
• Multiple Sequence alignment
• Have in common?

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Key Concepts

• Appreciate the foundation of sequence alignment
  evolutionary theory
• Appreciate the significance of unique and
  non-unique sequence characters
• Realize potential uses of multiple sequence
  alignments
• Understand the basics of the most popular
  multiple sequence alignment method
• Appreciate importance of accurate multiple
  alignments and the need for manual editing in
  some cases

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18th and 19th centuries The evolution of a theory

• Earth erosion, sediment deposition, strata
  present earth conditions provide keys to the past

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18th and 19th centuries The evolution of a theory

• Discoveries of fossils accumulated
• Remains of unknown but still living species that
  are elsewhere on the planet?
• Cuvier (circa 1800) the deeper the strata, the
  less similar fossils were to existing species

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• Discoveries of fossils accumulated
• Remains of unknown but still living species that
  are elsewhere on the planet?
• Cuvier (circa 1800) the deeper the strata, the
  less similar fossils were to existing species

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Part of Darwins Theory

• The world is not constant, but changing
• All organisms are derived from common ancestors
  by a process of branching.

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Part of Darwins Theory

• This explained
• Fossil record
• Similarities of organisms classified together
  (shared traits inherited from common ancestor)
• Similar species in the same geographic region

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• What is evolution?
• Dynamic changes with selected pressure
• Punctuated equilibrium
• Progressive generational adaptation
• Environmentally imposed mutational success
• E (mutationselective pressure) / time
• Staying fit

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Characters

• Heritable changes in features (morphology,
  DNA sequence etc)
• The more similar characters you have, the more
  related you are
• However.. characters can be unique and non-unique

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Evolution and characters
time
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A Unique Character Hair for Mammals

• Hair evolved only once and is unreversed
• Presence of hair ? strong indication that
  organism is a mammal

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Homoplasy The formation of tails

• Tails evolved independently in the ancestors of
  frogs and humans
• Presence of a tail ? no useful conclusions

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Unique and non-unique characters
Non-unique Unique
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Unique and non-unique characters

• Example Sequence analysis of functionally
  similar transporters
• All share the same deleted sequence region, which
  is not found in any other transporter examined to
  date
• Unique character?
• Further investigate for possible functional
  significance, or use for classification

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Unique and non-unique characters

• Example Sequence analysis of functionally
  similar transporters
• All have isoleucine at the third position in the
  sequence, however some other transporters have
  isoleucine there too, while some other
  transporters have valine at that position
• Non-unique.
• Changes from I ? V ? I are common (see BLOSUM or
  PAM matrices). Not a high priority for further
  analysis of significance and not useful for
  classification.

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Classification according to characters more
characters can be good
Chicken most similar to Tofu?
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Classification according to characters
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Classification according to characters
increasing the number of characters
Chicken most similar to Duck?
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Multiple Sequence Alignment The power of many
many characters
VTISCTGSSSNIGAG-NHVKWYQQLPG
VTISCTGTSSNIGS--ITVNWYQQLPG
LRLSCSSSGFIFSS--YAMYWVRQAPG
LSLTCTVSGTSFDD--YYSTWVRQPPG
PEVTCVVVDVSHEDPQVKFNWYVDG--
ATLVCLISDFYPGA--VTVAWKADS--
AALGCLVKDYFPEP--VTVSWNSG---
VSLTCLVKGFYPSD--IAVEWESNG--
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Evolution and characters the importance of
comparing characters with common origins
(homologous)
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Evolution and characters

• Gaps represent non-homologous positions in the
  sequence.
• They reflect the occurrence of insertions/deletion
  s or other rearrangements during the evolutionary
  process.

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Multiple Sequence Alignment
VTISCTGSSSNIGAG-NHVKWYQQLPG
VTISCTGTSSNIGS--ITVNWYQQLPG
LRLSCSSSGFIFSS--YAMYWVRQAPG
LSLTCTVSGTSFDD--YYSTWVRQPPG
PEVTCVVVDVSHEDPQVKFNWYVDG--
ATLVCLISDFYPGA--VTVAWKADS--
AALGCLVKDYFPEP--VTVSWNSG---
VSLTCLVKGFYPSD--IAVEWESNG--
The sole purpose of multiple sequence alignments
is to place homologous positions of homologous
sequences into the same column.
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On further with Multiple Sequence
AlignmentQuestions?
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Multiple Sequence Alignment - uses

• Powerful tool
• Detect trends/patterns in homologous sequences
  (motifs, domains, indels)
• Indels (insertions and deletions) of evolutionary
  interest, yet not incorporated into some
  phylogenetic tree algorithms
• - ATTYNETCITRTQ -
• - SITYNETCVTITQ -
• - SVTY-----CIVR -

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• Multiple sequence alignments and phylogenetic
  analysis
• First step in any phylogenetic analysis
• Phylogenetic analysis only as good as the
  alignment
• in ?
  out!

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• Multiple alignments not just sequence
• insertions and deletions in sequences

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• Automated Analysis or Manual Intervention?
•  
• Automated more explicit or objective than manual
• Leads to false sense of security
• Aligns residues that are likely similar only by
  chance
• ILPITSPSKEGYESGKAPDEFSSGG
• ILPEH--IKDDGELGAAPHSFSTAG
• VLPLD-----S--AGRPADSFSAAG
• VLPVDR-------DGQARDEYTKVG
• VLPVDN-------KGEARDEYTKVG
• LLPYDD-------QGRPQDDYSRAG
• GIVSRSG---SNFDGEPKDSYGKVG

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• Clustal
• Thompson, J.D., Higgins, D.G. and Gibson,
  T.J. (1994)
• CLUSTAL W improving the sensitivity of
  progressive multiple sequence alignment through
  sequence weighting, positions-specific gap
  penalties and weight matrix choice. Nucleic
  Acids Research, 224673-4680.

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• Clustal Incorporation of phylogenetic criterion
  into multiple sequence alignment algorithms
• 1. Pairwise alignments calculate a distance
  matrix
• 2. Guide tree constructed
• 3. Sequences progressively aligned according to
  guide tree hierarchy

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Clustal Incorporating Biology into Sequence
Alignment Algorithms

• Matrices varied at different alignment stages
  according to the divergence of the sequences
• For proteins, gap penalties differ for
  hydrophilic (water-loving) sequence regions to
  encourage new gaps in potential loop regions on
  the protein surface (which is usually exposed to
  water)
•  
• Gapped positions in early alignments have reduced
  gap penalties to encourage the opening up of new
  gaps at these positions
• (gaps not penalized as much at the end of
  proteins)
• gh

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ClustalX

• Subset of sequences in alignment can be selected
  and realigned. Useful when trying to align very
  divergent sequences.
• A range of the sequence alignment can be selected
  for realignment. Guide tree built based only on
  the residue range selected.

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Differences between Clustal and BLAST?Clustal
has full length (global) alignmentGap penalty
differencesInput differences selection of
sequencesClustal vary gap penalties and
matricesmany to many Speed Clustal
slowerAlign pro-pro or nuc-nucSimilarities?Iden
tifies conserved domainsUse the same matrices
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Algorithms in Molecular Biology http//www.math.t
au.ac.il/rshamir/algmb/00/algmb00.html
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ClustalX features

• 'Alignment Quality Score' below the alignment.

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MACAW - a program for semi-manual local multiple
alignment of DNA and protein sequences.

• User delimits the sequences and regions in which
  to search for blocks or specify blocks
• Decides which to keep and significance of each
  block is given statistical value.

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Genedoc - for editing and flexible display of
alignments

• view your alignment with different forms of
  shading that you customize
• edit your alignment (add or remove gaps) or the
  sequence order or the sequences themselves
• print directly, or export a graphic of your
  alignment

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Genedoc - for editing and flexible display of
alignments
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• Statistics Report
• 1 residues identical
• 2 residues gt zero score (similar residues)
• 3 residues lined up with a gap
• human rat rabbit turtle
• human 1870 97 96 22
• 0 98 96 28
• 0 0 2 61
• rat 1830 1874 94 22
• 1846 0 95 28
• 18 0 2 61
• rabbit 1818 1793 1863 22
• 1828 1815 0 28
• 45 53 0 61

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Standard multiple sequence alignment approach

• Be as sure as possible that the sequences
  included are homologous
• Know as much as possible about the gene/protein
  in question before trying to create an alignment
  (secondary structure, domains etc..)
• Start with an automated alignment preferably one
  that utilizes some evolutionary theory such as
  Clustal

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• Examine alignment
• Are you confident that aligned residues/bases
  evolved from a common ancestor?
• Are domains of the proteins/predicted secondary
  structures, etc. aligning correctly?
• ? No? May need to edit sequences and redo
• _______________________________
• _________________ ___ __ ____ _
• ? Yes? Move on!
• Note indels (insertions and deletions)
• Possible insights into functionally important
  regions

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• Use in subsequent analyses (identify consensus
  or other pattern recognition, for HMM
  construction, phylogenetic analysis, etc..)
• For phylogenetic analysis Remove unreliably
  aligned regions
• ILPITSPSKEGYESGKAPDEFSSGG
• ILPEH--IKDDGELGAAPHSFSTAG
• VLPLD-----S--AGRPADSFSAAG
• VLPVDR-------DGQARDEYT-VG
• VLPVDN-------KGEARDEYT-VG
• LLPYDD-------QGRPQDDYSRAG
• GIVSRSG---SNFDGEPKDSYGKVG

Delete?
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• If aligning DNA sequence for phylogenetic
  analysis may remove every third codon position

MMET GLY SER GLYMET GLY SER GLY MET ARG
CYS ARG AATG GGA AGT GGA ATG GGG AGC GGGATG
AGG TGC AGG
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Key Concepts

• Appreciate the foundation of sequence alignment
  evolutionary theory
• Appreciate the significance of unique and
  non-unique sequence characters
• Realize potential uses of multiple sequence
  alignments
• Understand the basics of the most popular
  multiple sequence alignment method
• Appreciate importance of accurate multiple
  alignments and the need for manual editing in
  some cases

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• Questions?

M