Lecture 2: Character Homology with particular attention to sequence alignment

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Lecture 2Character Homology(with particular
attention to sequence alignment)
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DNA sequences

• Strings of characters
• Each with one of 4 possible states
• 4 nucleic acids
• adenine, cytosine, guanine, thymine

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DNA sequences

• Protein-coding genes
• Other structural genes
• Ribosomal RNA
• Transfer RNA
• Other DNA - "non-coding"
• Introns
• Repetitive DNA

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Protein Coding Sequences
Structure and function determined by sequence of
amino acids

• Myoglobin
• 3D model

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Protein-Coding Genes
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Possible Changes in DNA sequences

• Substitutions
• Inversions
• Insertions and Deletions
• indels

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Protein-Coding Genes

• 3rd position of codon generally redundant
• 1st position changes more common than 2nd
  position

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Purines
Pyrimidines
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Substitutions

• Transitions
• purine gt purine
• pyrimidine gt pyrimidine
• Transversions
• purine gt pyrimidine
• pyrimidine gt purine

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Substitutions

• Transitions
• purine gt purine
• pyrimidine gt pyrimidine

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Substitutions

• Transitions
• purine gt purine
• pyrimidine gt pyrimidine
• Transversions
• purine gt pyrimidine
• pyrimidine gt purine

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Homology in gene sequences
t c t c c a g g t g c a c g t c t t c t
a g t c t c c a g g t g c a c g t c t t
???
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Homology in gene sequences
t c t c c a g g t g c a c g t c t t c t
a g t c t c c a g g t g c a c g t c t t
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Homology in gene sequences
t c t c c a g g t g c a c g t c t t c t
a g t c t c c a g g t g c a c g t c t t
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Homology in gene sequences
t c t c c a g g t g c a c g t c t t c t
a g t c t c c a g g t g c a c g t c t t
a g t c c c c a g g t g c a c g t c t t
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Introns and exons
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Insertions and deletions

• indels
• generally occur in multiples of three in exons of
  protein-coding genes
• May be any length in introns

1 a g t c t c c a g g t g c a c g t c t t
2 a g t c c c c a g g t g c a c g t c t t
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Insertions and deletions

• indels
• generally occur in multiples of three in exons of
  protein-coding genes
• May be any length in introns

Frame-Shift Mutation
1 a g t c t c c a g g t g c a c g t c t t
2 a g t c c c c a g g t g c a c g t c t t
3 a g t t c c c c a g g t g c a c g t c t t
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Gene families arise from gene duplications
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• Paralogous genes two or more different gene
  loci in the same organism that originated by gene
  duplication

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• Paralogous genes two or more different gene
  loci in the same organism that originated by gene
  duplication
• Orthologous genes same gene in two different
  organisms, homologous due to presence in common
  ancestor

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Gene Copy

• May be more than one paralogous copy of a gene in
  the genome
• Copies may be functional
• e.g. EF1? gene occurs in two copies in insects
• Non-functional copies are called pseudogenes
• Insertions and deletions of any length
• May contain stop codons (TGA, TAG, TAA)

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BLAST(Basic Local Alignment Search Tool)

• National Center for Biotechnology Information
• Algorithms to match query sequence with sequences
  in database (Genbank)
• If sequences are the same, there should be short
  stretches of complete identity (seeds, or
  "words")
• Should be able to find seeds even if sequence has
  insertions and deletions
• Finds best matches and computes overall
  similarity, probability of match this close, etc.

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(No Transcript)
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Ribosomes - the workbench for protein synthesis
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Ribosomes - the workbench for protein synthesis
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Ribosomes

• Consist of ribosomal RNA and proteins
• Protein manufacturing machinery
• rRNA synthesized in nucleolus
• The rRNA self-assembles into two folded
  structures, the large and small subunits

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(image of ribosome by Harry Noller, U.C. Santa
Cruz, Venki Ramakrishnan at Cambridge, and Thomas
Steitz at Yale)
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Eukaryotic rDNA

• NTS - non-transcribed spacer regions
• ITS - internal transcribed spacers
• In thousands of tandem repeats
• Identical due to concerted evolution

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(No Transcript)
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28s rRNA
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Ribosomal DNA

• Insertions and deletions are common
• Alignment of sequences necessary to establish
  positional homologies of nucleotides

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Sequence Alignment
X g a c g t t a g a g c t a a t c
Y g a c a g c t c g t c g a
Z g a c g c c c a t c g a g
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Sequence Alignment
X g a c g t t a g a g c t a a t c
Y 1 g a c - - - a g c t c g t c g a
Y g a c a g c t c g t c g a
Z g a c g c c c a t c g a g
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Sequence Alignment
X g a c g t t a g a g c t a a t c
Y 1 g a c - - - a g c t c g t c g a
Y 2 g a c - - - - - a g c t c g t c g a
Y g a c a g c t c g t c g a
Z g a c g c c c a t c g a g
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Sequence Alignment
X g a c g t t a g a g c t a a t c
Y 1 g a c - - - a g c t c g t c g a
Y 2 g a c - - - - - a g c t c g t c g a
Y g a c a g c t c g t c g a
Z g a c g c c c a t c g a g
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Sequence Alignment
X g a c g t t a g a g c t a a t c
Z 1 g a c - - - - - - g c c c a t c g a g
Y 1 g a c - - - a g c t c g t c g a
Y 2 g a c - - - - - a g c t c g t c g a
Y g a c a g c t c g t c g a
Z g a c g c c c a t c g a g
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Sequence alignment

• How many gaps should we insert?
• Assign cost to whole gaps or each space in a gap
  separately?
• Less cost for expanding an existing gap?
• Should gaps be treated as character states?
• Assign different costs for transitions and
  transversions that result from an alignment?
• What order to align the sequences?
• Programs such as CLUSTAL, MALIGN, POY and others
  attempt to optimize functions with all of these,
  and more, parameters

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CLUSTAL X

• Multiple Alignment Program
• First, produce guide tree
• Compute distances between sequences
• Cluster most similar sequences together
• Use tree as template for order of sequential
  pairwise alignments
• Conduct sequential pairwise alignments

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Clustal X Alignment ParametersSlow accurate
alignments

• Penalty for opening first gap
• Penalty for changing size of existing gap
• Penalty for each inferred transition
• Penalty for each inferred transversion

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Optimization AlignmentWard Wheeler (AMNH)

• Completely different approach
• Strategy of multiple sequence alignment followed
  by phylogenetic analysis is fundamentally flawed
• Better approach is to integrate process of
  sequence alignment with phylogenetic analysis in
  an iterative, recursive analysis
• POY program
• Attempts to optimize both phylogenetic analysis
  and alignment simultaneously
• VERY CPU intensive

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Optimization Alignment

• Minimize both insertion/deletion events and
  substitutions
• Alignments are dynamic and uniquely tailored to
  each topology
• Alignments chosen to minimize tree length
• Homoplasy and alignment cost functions minimized
  simultaneously

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Sensitivity Analysis

• Explore different regions of alignment parameter
  space by trying different combinations of
  parameters
• Determine how sensitive results are to changes in
  different parameters
• Determine optimal combinations of parameters
• Criterion for optimality is often congruence
  (concordance) with other data sets after analysis

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28s rRNA
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Structural Alignment

• Another approach to alignment of molecules for
  which we have models of secondary structure
• ribosomal sequences, tRNA, etc.

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28s rRNA - D2 region
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28s rRNA - D2 region
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Structural Alignment(with thanks to Joe
Gillespie and Matt Yoder)
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Structural Alignment(with thanks to Joe
Gillespie and Matt Yoder)
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Structural Alignment
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Structural Alignment
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Structural Alignment
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Structural Alignment
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Structural Alignment
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Structural Alignment
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Structural Alignment
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Structural Alignment
Regions of unambiguous homology
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Some recent reviews

• Morrison, D. A. (2006). Multiple sequence
  alignment for phylogenetic purposes. Australian
  Systematic Botany (19) 479-539.
• Ogden Rosenberg. 2006. Multiple Sequence
  Alignment Accuracy and Phylogenetic Inference.
  Syst. Biol. 55(2)314328
• Höhl Ragan. 2007. Is Multiple-Sequence
  Alignment Required for Accurate Inference of
  Phylogeny? Syst. Biol. 56(2)206221
• Parmentier et al. 2006. Large scale multiple
  sequence alignment with simultaneous phylogeny
  inference. J. Parallel Distrib. Comput. 66 (2006)
  15341545
• Phillips, A. (2000). Multiple Sequence Alignment
  in Phylogenetic Analysis. Molecular Phylogenetics
  and Evolution 16(3) 317-330.

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Lutzoni et al. (2000) Systematic Biology 49(4)
628-651

• Method to integrate ambiguously aligned regions
  into analysis
• Retain positional homologies
• Calculate minimum distances necessary to
  transform one fragment into another

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Structural Alignment
Regions of unambiguous homology
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Fixed State Optimization

• Treat contiguous strings of nucleotides as
  character states
• Calculate minimum pairwise distances between such
  states
• Attempt to find topology that minimizes these
  distances

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Alignment methods(Wheeler, Cladistics, 2001)
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Saturation of gene sequences
t c t c c a g g t g c a c g t c t t c t
1 a g t c t c c a g g t g c a c g t c t t
2 a g t c c c c a g g t g c a c g t c t t
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Saturation of gene sequences
t c t c c a g g t g c a c g t c t t c t
1 a g t c t c c a g g t g c a c g t c t t
2 a g t c c c c a g g t g c a c g t c t t
3 a g t c t c c a g g t g c a c g t c t t
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Saturation of gene sequences
t c t c c a g g t g c a c g t c t t c t
1 a g t c t c c a g g t g c a c g t c t t
2 a g t c c c c a g g t g c a c g t c t t
3 a g t c t c c a g g t g c a c g t c t t
4 a g t c a c c a g g t g c a c g t c t t