Multiple Alignment

1
Multiple Alignment

• Stuart M. Brown
• NYU School of Medicine

2
(No Transcript)
3
Pairwise Alignment

• The alignment of two sequences (DNA or protein)
  is a relatively straightforward computational
  problem.
• Can be done in a word processor
• But how to evaluate the results?
• A natural job for a computer
• The best solution seems to be an approach called
  Dynamic Programming.

TGCCATAGAGCGTAGTCGTTCCCT lt gt
CTAGAGAGCGTAGTCAGAGTGTCTTTGAGTTCC
4
Protein or DNA

• Most of the examples that I will show illustrate
  protein alignments
• All of the tools can handle either protein or DNA
• More distantly related protein sequences can be
  aligned because different amino acids can be
  similar

5
Dynamic Programming

• Dynamic Programming is a very general programming
  technique.
• It is applicable when a large search space can be
  structured into a succession of stages, such
  that
• the initial stage contains trivial solutions to
  sub-problems
• each partial solution in a later stage can be
  calculated by recurring a fixed number of partial
  solutions in an earlier stage
• the final stage contains the overall solution

6
(No Transcript)
7
Global vs. Local Alignments

• Global alignment algorithms start at the
  beginning of two sequences and add gaps to each
  until the end of one is reached.
• Local alignment algorithms finds the region (or
  regions) of highest similarity between two
  sequences and build the alignment outward from
  there.

8
(No Transcript)
9
Global Alignment

• Global algorithms are often not effective for
  highly diverged sequences and do not reflect the
  biological reality that two sequences may only
  share limited regions of conserved sequence.
• Sometimes two sequences may be derived from
  ancient recombination events where only a single
  functional domain is shared.
• Global alignment is useful when you want to force
  two sequences to align over their entire length

10
Global Alignment Programs

• The Fasta program Align implements the Needleman
  and Wunsch Global alignment algorithm.
• EMBOSS has the same program called Needle, but it
  also has an improved version called Stretcher
• (Myers and Miller, CABIOS, 1989)

11
Local Alignment

• The Fasta program LALIGN implements the
  Smith-Waterman local alignment algorithm.
• FASTA and BLAST are local alignment algorithms
• NCBI has a BLAST 2 Sequences feature on its
  website
• http//www.ncbi.nlm.nih.gov/gorf/bl2.html

12
Pairwise Alignment on the Web

• The ALIGN global alignment program is available
  at several servers
• http//molbiol.soton.ac.uk/compute/align.html
• http//www2.igh.cnrs.fr/bin/align-guess.cgi
• LALIGN local alignment program is available at
  several servers
• http//www2.igh.cnrs.fr/bin/lalign-guess.cgi
• http//www.ch.embnet.org/software/LALIGN_form.html
  
• LFASTA uses FASTA for local alignment of 2
  sequences
• http//pbil.univ-lyon1.fr/lfasta.html
• BLAST 2 Sequences (NCBI)
• http//www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html

13
(No Transcript)
14
Multiple Alignments

• In theory, making an optimal alignment between
  two sequences is computationally straightforward
  (Smith-Waterman algorithm), but aligning a large
  number of sequences using the same method is
  almost impossible.
• The problem increases exponentially with the
  number of sequences involved
• (the product of the sequence lengths)

15
Optimal Alignment

• For a given group of sequences, there is no
  single "correct" alignment, only an alignment
  that is "optimal" according to some set of
  calculations.
• Determining what alignment is best for a given
  set of sequences is really up to the judgment of
  the investigator.

16
Progressive PairwiseMethods

• Most of the available multiple alignment programs
  use some sort of incremental or progressive
  method that makes pairwise alignments, then adds
  new sequences one at a time to these
  aligned groups.
• This is an approximate method!

17
Common Programs

• CLUSTAL is the most popular alignment program
  that uses a progressive pairwise algorithm.
• PILEUP is the multiple alignment program in the
  GCG package

18
The CLUSTAL Algorithm

• First, CLUSTAL calculates approximate pairwise
  similarity scores between all sequences to be
  aligned, and they are clustered into a dendrogram
  (tree structure).
• Then the most similar pairs of sequences are
  aligned.
• Averages (similar to consensus sequences) are
  calculated for the aligned pairs.
• New sequences and clusters of sequences are added
  one by one, according to the branching order in
  the dendrogram.

19
CLUSTAL

• CLUSTAL is a stand-alone multiple alignment
  program (UNIX) that has been improved on an ad
  hoc basis to deal with the real biology of
  sequence alignment
• Gap penalties can be adjusted based on specific
  amino acid residues, regions of hydrophobicity,
  proximity to other gaps, or secondary structure.
• it can re-align just selected sequences or
  selected regions in an existing alignment
• It can compute phylogenetic trees from a set of
  aligned sequences.
• There are also Mac and PC versions with a nice
  graphical interface (CLUSTALX).

20
Using Clustal

• Custal requires that sequences be input as a
  single multi-sequence Fasta file
• This is where an integrated software platform
  like GCG is nice
• Output can be in many formats including GCG/MSF,
  Clustal, and multi-Fasta.

21

• gtPYCDA07TF input_file_1
• ATGCCCATACTACTCTTCTGGTAGTTGGAATGAAGCCCAAAATATGATAA
  AACCTTTTCT
• TACTAAAGTTTGTCAGGAAGTAGAAAGAATTGCTCATTGTGGAAAATGGG
  AAGAATGGAG
• TGAATGTTCTACTACTTGT
• gtPYCDA08TR input_file_2
• TATAGAAATAAAACTCCATTAAAAAATATTTTCCTTTTTCCTAATTATTT
  CTCTAAAATA
• TAACAATCTAATTCATATAATATCATTACAATCACATATATATCTCTTTA
  AATTTTGTTC
• CCTTTTTCCCTACGAGTTGTATCAGCAATAATCTCCTACAAGGTTAGACG
  TTGCTTCAAG
• TTATTTTCAACAAATTTGGTCATTTTCAGCAAATTTTGCCATTTTCAGCA
  AATTTTGCCA
• TTTTCAACAAATTTTGCCATTTTCAACAGATTTTGCCATTTTCAACAGAT
  TTTGCCATTT
• TCAGCAAATTTTGCCATATTCAACAAATTTTGCCATTTTCAGCAAATTTT
  ACCATTTTTA
• GCAAATTAGTATACCGTGTTAT
• gtPYCDA09TRB input_file_3
• GCGGGAATATAGAAATAAAACTCCATTAAAAAATATAAACCTTTTTTTTA
  ATTATCACCC
• TAAAACATAACAATCTAATTCATATAATATCATTACAATCACATATATAT
  CTCTTTAAAT
• AATGATCCCTTTTTCCCTACGAGTTGTATCAGCAATAATCTCCTACAACG
  GATAGACGTT
• GCTTCAAGTTCTTTTCAACAAATTGGGTCATTTTCAGCGAATTTTGCCAT
  TTTCAGCAAA
• TTTGGCCATACTCAACAAATTTTGCCATTGGCAACAGATTTTGCCATTTT
  CAACAGATTT
• TGCCGTTGTCAGCAAATTTTGCCATATTCAACAAATTTTGCCAATCTCAG
  CAAATTTTAC

22
Multiple Alignment tools on the Web

• There are a variety of multiple alignment tools
  available for free on the web.
• CLUSTAL is available from a number of sites (with
  a variety of restrictions)
• Other algorithms are available too
• Watch out for experimental algorithms there
  may be a good reason why you have never heard of
  some oddball program

23
Some URLs

• EMBL-EBI
• http//www.ebi.ac.uk/clustalw/
• BCM Search Launcher Multiple Alignment
• http//dot.imgen.bcm.tmc.edu9331/multi-align/mult
  i-align.html
• Multiple Sequence Alignment for Proteins (Wash.
  U. St. Louis)
• http//www.ibc.wustl.edu/service/msa/

24
Editing Multiple Alignments

• There are a variety of tools that can be used to
  modify a multiple alignment.
• These programs can be very useful in formatting
  and annotating an alignment for publication.
• An editor can also be used to make modifications
  by hand to improve biologically significant
  regions in a multiple alignment created by one of
  the automated alignment programs.

25
Challenge of Formats

• There are many different file formats for
  multiple alignments.
• Hopefully you can get your alignment program and
  your formatting program to agree on one.
• The GCG MSF format is very common
• You can also represent aligned sequences in
  multi-sequence Fasta format with gaps.

26
BOXSHADE

• Shades by similarity
• UNIX, Mac, and Windows versions, built into GCG
• On the web at
• http//www.ch.embnet.org/software/BOX_form.html
• http//bioweb.pasteur.fr/seqanal/interfaces/boxsha
  de.html
• http//huge.eng.uiowa.edu/tscheetz/sequence-analy
  sis/examples/BoxShade/BOX_form.html

27
(No Transcript)
28
(No Transcript)
29
Other editors

• The MACAW and SeqVu program for Macintosh and
  GeneDoc and DCSE for PCs are free and provide
  excellent editor functionality.
• Many comprehensive molecular biology programs
  include multiple alignment functions
• MacVector, OMIGA, Vector NTI, and
  GeneTool/PepTool all include a built-in version
  of CLUSTAL

30
SeqVu
31
Editors on the Web

• Check out CINEMA (Colour INteractive Editor for
  Multiple Alignments)
• It is an editor created completely in JAVA (old
  browsers beware)
• It includes a fully functional version of
  CLUSTAL, BLAST, and a DotPlot module

http//www.bioinf.man.ac.uk/dbbrowser/CINEMA2.1/
32
(No Transcript)