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    Sequence Alignments: Felix Sappelt Irina Wagner

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    Pairwise and multiple sequence alignments are important bioinformatics tools. Pairwise alignments can be done with dynamic programming or heuristic methods like BLAST and FASTA. Multiple sequence alignments (MSA) are an NP-complete problem so heuristic methods are used. Popular MSA tools include ClustalW, ProbCons, MAFFT, Muscle, Cobalt, and T-Coffee. MSAs are useful for assessing evolutionary relationships, modeling protein structures, and illustrating evolutionary events. The choice of MSA method depends on factors like number of sequences, sequence identity, and using structural information.

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    Sequence Alignments: Felix Sappelt Irina Wagner

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    Pairwise and multiple sequence alignments are important bioinformatics tools. Pairwise alignments can be done with dynamic programming or heuristic methods like BLAST and FASTA. Multiple sequence alignments (MSA) are an NP-complete problem so heuristic methods are used. Popular MSA tools include ClustalW, ProbCons, MAFFT, Muscle, Cobalt, and T-Coffee. MSAs are useful for assessing evolutionary relationships, modeling protein structures, and illustrating evolutionary events. The choice of MSA method depends on factors like number of sequences, sequence identity, and using structural information.

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    Sequence Alignments

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    Pairwise and multiple sequence alignments are important bioinformatics tools. Pairwise alignments can be done with dynamic programming or heuristic methods like BLAST and FASTA. Multiple sequence alignments (MSA) are an NP-complete problem so heuristic methods are used. Popular MSA tools include ClustalW, ProbCons, MAFFT, Muscle, Cobalt, and T-Coffee. MSAs are useful for assessing evolutionary relationships, modeling protein structures, and illustrating evolutionary events. The choice of MSA method depends on factors like number of sequences, sequence identity, and using structural information.

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    147 views34 pages

    Sequence Alignments: Felix Sappelt Irina Wagner

    Uploaded by

    Felix Spacebook
    Pairwise and multiple sequence alignments are important bioinformatics tools. Pairwise alignments can be done with dynamic programming or heuristic methods like BLAST and FASTA. Multiple sequence alignments (MSA) are an NP-complete problem so heuristic methods are used. Popular MSA tools include ClustalW, ProbCons, MAFFT, Muscle, Cobalt, and T-Coffee. MSAs are useful for assessing evolutionary relationships, modeling protein structures, and illustrating evolutionary events. The choice of MSA method depends on factors like number of sequences, sequence identity, and using structural information.

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    of 34

    Sequence Alignments

    Felix Sappelt
    Irina Wagner
    PAIRWISE SEQUENCE ALIGNMENTS
    Pairwise Sequence Alignment Methods

    • Dynamic Programming
    - Global alignment (Needleman-Wunsch)
    - Local alignment (Smith-Waterman)
    • Heuristic Methods
    - FASTA
    - BLAST
    Heuristic Methods
    • Try only most likely alignments and skip all
    others
    • Much faster than dynamic programming
    methods, but less sensitive
    • For large databases, such as whole genomes,
    speed is extremely important
    • In some cases, heuristic methods are the only
    possibility; exact algorithms take too long.
    FASTA
    • One of the earliest widely used database
    searching tools (Lipman&Pearson in 1985)
    • Heuristic method approximating Smith
    Waterman
    • Search time is proportional to size of DB
    Fasta-Algorithm
    • Find identical substrings
    • Re-Score and keep only
    high-scoring identities
    • Discard substrings that
    cannot be easily joined
    • Optimize using dynamic
    programming around
    diagonal
    Substitution Matrix
    • PAM (Point Accepted Mutation)
    – Created by Margaret Dayhoff in 1970
    – Based on an explicit evolutionary model
    – PAM1 estimate using 1572 changes in 71 groups of protein sequences that
    were at least 85% similar
    – PAM 250 (20% SIMILARITY) obtained by multiplying PAM1 by itself 250 times
    • BLOSUM (Block Substitution Matrix)
    – Deals with sequence changes over long timespans
    – Based on multiple protein alignments
    – Used 500 families of related proteins
    – not based on explicit evolutionary model, but from considering all amino acids
    changes observed in an aligned region from a related family of proteins
    • when the correct scoring matrix is used, alignment statistics are
    meaningful
    PAM250 Matrix
    BLAST
    • BLAST is an improvement over FASTA
    – Greater speed by pre-indexing the database
    – More accurate results
    • BLAST is the centerpiece of many
    bioinformatics assays, because it makes
    genome-scale sequences accessible
    • The original paper was the most cited paper of
    the 1990s (Altschul et al. 1990)
    BLAST
    • Mask low-complexity regions
    Over 50% of genomic DNA is repetitive
    - Retrotransposons
    - Repeats
    - ALU regions
    - Microsatellites
    - UTRs
    BLAST
    • List all k-tuples in the query sequence
    • the lower k-tup value the more
    background you will have
    • the higher the k-tup value the faster
    analysis
    • Find all matching words in the
    database
    • Keep only the high-scoring words
     difference to FASTA
    • Build search tree from remaining
    words
    BLAST
    • Extend the match until the match score decreases or
    the end of the sequence has been reached
    • Extended matches are called High-Scoring Segment
    Pairs
    BLAST
    • List all HSPs in the database whose score is
    high enough to be considered
    • Assess statistical significance via the Gumbel
    Extreme Value Distribution, which describes
    the distribution of Smith-Waterman scores
    • Join HSPs into a longer alignment
    • Output
    BLAST Results
    • The raw score: is calculated by summing the scores for each
    aligned position and the scores for gaps
    • Bit scores: Bit scores are raw scores converted from the log base
    of the scoring matrix that creates the alignment to log base 2, this
    rescaling allows scores to be compared between the alignments
    • E-value: Expected number of chance alignments; the lower the E
    value, the more significant the score. An expect value of 10.0 is
    the default value of statistical significance, but this number can
    be adjusted by the user
    • P-value: The P-value represents the probablity (in the range of 0-
    1) of a given sequence occuring by chance. It is less accurate than
    the E-value
    Other BLAST variants
    BLASTN Nucleotide seqeunece comparison
    BLASTP General protein comparison

    TBLASTN compares a protein sequence to a translated


    DNA DB
    Use if homolog not found in protein DB
    TBLASTN, TBLASTX compares a translated DNA sequence to a
    translated DNA DB
    Identify new orthologs in closely related
    species
    BLASTX Compares a translated nucleotide query to a
    protein DB
    Other BLAST variants
    PSI-BLAST PHI-BLAST
    • Position Specific Iterative BLAST • Pattern Hit Initiated BLAST
    • is used to find distant relatives • Uses protein motifs to
    of a protein increase the chance of
    • Easy to use version of a „profile“ finding biologically
    search
    significant matches
    • Uses an iterative alignment
    procedur to develop position
    specific scoring matrices which
    increases its capability to detect
    weak pattern matches
    HHSearch
    • Represents query and database by profile Hidden
    Markov Models
    • Database profiles derived from multiple
    sequence alignments
    • Before searching the HMM database, a MSA of
    related sequences is compiled using CSI-Blast
    • From this MSA, a profile is calculated
    • Search is being done with this profile as the
    query
    MULTIPLE SEQUENCE ALIGNMENTS
    The MSA problem
    • Correctly align more than two sequences
    • NP-complete problem
    – For k sequences of length n, complexity is O(nk)
    – For 10 sequences of length 50, nk is about 1017
    – For 50 sequences of length 500, nk is about 10136
    • World‘s biggest supercomputer: 2.5 TFLOPS (1012)
    • Since Planet Earth will be around for just 6 billion
    years, all current approaches are heuristic.
    What are MSAs good for?
    • Assess evolutionary history and sequence
    homology of a set of sequences
    • Useful for ...
    – Homology modelling
    – Phylogenic research
    – Illustrating mutation events and evolutionary
    processes
    MSA Workflow
    Methods
    • ClustalW: Basic Tree-Based approach (1994)
    • ProbCons: Probabilistic approach (2005)
    • MAFFT: Fast Fourier Transformation (2002)
    • Muscle: K-Substring counting, Profiles (2004)
    • Cobalt: Proteins, user input (2007)
    • T-Coffee: Library-Based (2000)

    … and many more.


    ClustalW
    • Published in 1994
    • How it works:
    – First, do all possible pairwise
    alignments
    – Build a guide tree
    • Neighbor Joining Method
    – Progressively align according to
    branching order in guide tree
    • Starting from leafs, build
    pairwise alignments towards
    root
    ClustalW
    • Pros:
    – It‘s fast
    – Results are good for highly similar sequences
    – Position-specific gaps protect hydrophobic core
    • Cons:
    – Simple approach
    – Errors in pairwise alignment stage propagate,
    cannot be corrected
    Probcons
    • Probabilistic
    • Idea of consistency
    – Prevents misalignments due to „faulty“ pairwise
    alignments
    – Sequences x, y, z:
    x i
    if xi aligns with yj,
    and yj aligns with zk, y j

    then xi aligns with zk. z k


    Probcons
    • Compute posterior probability
    matrix using HMM
    • Construct pairwise alignments
    that maximize „expected accuracy“
    • Probabilistic consistency
    transformation of posterior matrix
    – Incorporate similarity to other sequences into pairwise
    comparisons
    • Build guide tree
    • Progressive alignment
    MAFFT
    • Uses Fast Fourier Transformation to identify
    homologous regions
    • Uses polarity and volume information for amino
    acids
    • Can run in progressive and iterative mode
    • Extremely fast
    • Very accurate
    Muscle
    • Iterative method
    • K-mer counting
    – Approximate distance
    between two sequences
    by number of common
    k-substrings
    – Very fast
    • Log expectation
    – Profile function used to
    iteratively improve alignments
    Cobalt
    • Specializes in Proteins
    • Designed to exploit three strategies:
    – Using biological information by deriving
    constraints from protein databases
    – Using pairwise similarity present in multiple pairs
    – Allowing the user to specify regions that are to be
    aligned
    T-Coffee
    • Tree-based Consistency Objective Function
    for Alignment Evaluation
    • Cédric Notredame, 2000
    • Derives constraints from libraries of pairwise
    alignments
    • Slow, but accurate
    • 3D-Coffee: Extends T-Coffee with structure
    information from PDB files
    • Libraries contain pair-
    wise alignments
    • Each AA-pair in them
    is a constraint
    • Weights: Percent identity
    of alignments
    • Fitting a set of weighted
    constrains onto a MSA is
    NP-complete
     heuristic solution:
    Extension
    What to use
    • For small numbers of sequences (<20) with relatively
    high identity (>40%), any tool works
    • Large number of sequences may require fast
    methods: MAFFT (progressive)
    • Low identity (<35%): T-Coffee, Probcons, MAFFT (L-
    ins-i)
    – Low-identity alignments generally don‘t work well
    – Long N- or C-Terminal extensions: T-Coffee and MAFFT (E-
    ins-i)
    – Using structure information may help: 3D-Coffee
    MSA Editors
    • JalView: Java Alignment Editor
    Sources
    • http://de.wikipedia.org/wiki/Substitutionsmatrix
    • http://en.wikipedia.org/wiki/BLAST
    • William R. Pearson, [5] Rapid and sensitive sequence comparison with FASTP and FASTA, Methods in
    Enzymology
    • Stephen F. Altschul, Warren Gish, Webb Miller, Eugene W. Myers, David J. Lipman, Basic local alignment
    search tool, Journal of Molecular Biology
    • http://en.wikipedia.org/wiki/HHpred_/_HHsearch
    • Jimin Pei, Multiple protein sequence alignment, Current Opinion in Structural Biology
    • Chuong B. Do, Kazutaka Katoh, Protein Multiple Sequence Alignment, Methods in Molecular Biology
    • Thompson et al, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through
    sequence weighting, position specific gap penalties and weight matrix choice, Nucleic Acids Research
    • Notredame et al, T-Coffee: A Novel Method for Fast and Accurate Multiple Sequence Alignment, Journal of
    Molecular Biology
    • Robert C. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic
    Acids Research
    • Chuong et al, ProbCons: Probabilistic Consistency-Based Multiple Sequence Alignment, Genome Research
    • Katoh et al, MAFFT Version 5: Improvement in accuracy of multiple sequence alignment, Nucleic Acids
    Research
    • Thompson et al, A Comprehensive Benchmark Study of Multiple Sequence Alignment Methods, PLoS One
    • Papadopoulos et al, COBALT: Constraint-based alignment tool for multiple protein sequences,
    Bioinformatics
    • Katoh et al, MAFFT: A novel method for rapid multiple sequence alignment based on fast Forier transform,
    Nucleic Acids Research

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