Multiple Alignment

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Multiple Alignment

• Stuart M. Brown
• NYU School of Medicine

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Pairwise Alignment

• The alignment of two sequences (DNA or protein)
  is a relatively straightforward computational
  problem.
• The best solution seems to be an approach called
  Dynamic Programming.

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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

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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.

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GAP

• The GCG program GAP implements the Needleman and
  Wunsch Global alignment algorithm.
• 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.
• GAP is useful when you want to force two
  sequences to align over their entire length

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BESTFIT

• The GCG program BESTFIT 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

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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

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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)

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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 judgement of
  the investigator.

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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!

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PILEUP

• PILEUP is the multiple alignment program in the
  GCG package
• CLUSTAL is another popular program (also
  available on the RCR server) that uses a similar
  algorithm.

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The PILEUP Algorithm

• First, PILEUP 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.

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PILEUP Considerations

• Since the alignment is calculated on a
  progressive basis, the order of the initial
  sequences can affect the final alignment.
• PILEUP paramaters 2 gap penalties (gap insert
  and gap extend) and an amino acid comparison
  matrix.
• PILEUP will refuse to align sequences that
  require too many gaps or mismatches.
• PILEUP will take quite a while to align more than
  about 10 sequences

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Instructions for running PILEUP

• PILEUP uses a list of sequence files as input
• You can use output from a FASTA or LOOKUP search
  as a list or make your own list in a text editor
• A list file can include files from your own
  directory and/or GCG database files.

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LIST file format

• List files always begin with two dots ..
• ..
• gpS31321
• gpYno3_Yeast
• S51900.pep
• Yan2_Schpo
• Ypd1_Caeel
• A36205
• Mpp1_Rat begin100 end345
• B46665.pep
• Ymxg_Bacsu begin150 end464
• A48043.pep
• List files can also include Begin and End
  positions within a sequence

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PILEUP _at_myseqs.list

• Now at the gt prompt, type PILEUP and the name of
  the file that is your list of sequence names.
• However, GCG requires that you must precede the
  name of your list file with the _at_ character.
• So the command looks like this
  
• gt PILEUP _at_myseqs.list

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PILEUP Output
gt more myseqs.msf
1501
1550 Hsirf2 SERPSKKGKK PKTEKEDKVK
HIKQEPVESS LGLSNGVSDL SPEYAVLTST Muirf2
SERPSKKGKK PKTEKEERVK HIKQEPVESS LGLSNGVSGF
SPEYAVLTSA Chirf2 SERPSKKGKK TKSEKDDKFK
QIKQEPVESS FGI.NGLNDV TSDY.FLSSS Muirf1
LTRNQRKERK SKSSRDTKSK TKRKLCGDVS PDTFS..DGL
SSSTLPDDHS Ratirf1 LTKNQRKERK SKSSRDTKSK
TKRKLCGDSS PDTLS..DGL SSSTLPDDHS Hsirf1
LTKNQRKERK SKSSRDAKSK AKRKSCGDSS PDTFS..DGL
SSSTLPDDHS Chkirf1a LTKDQKKERK SKSSREARNK
SKRKLYEDMR MEESA..ERL TSTPLPDDHS Hsirf3a

Mmuirf3
Hsirf5
GPAPTDSQPP EDYSFGAGEE EEEEEELQRM LPSLSLTDAV
QSGPHMTPYS Mmuirf6 IPQPQGS.VI NPGSTGSAPW
DEKDNDVDED EEEDELEQSQ HHVPIQDTFP Hump48
...PPGIVSG QPGTQKVPSK RQHSSVSSER KEEEDAMQNC
TLSPSVLQDS Mup48 ...PAGTLPN QPRNQKSPCK
RSISCVSPER EEN...MENG RTNGVVNHSD Hsirf4
...PEGAKKG AKQLTLEDPQ MSMSHPYTMT TPYPSLPA.Q
VHNYMMPPLD Mupip ...PEGAKKG AKQLTLDDTQ
MAMGHPYPMT APYGSLPAQQ VHNYMMPPHD Huicsbp
...PEEDQK. .......... .......... CKLGVATAGC
VNEVTEMECG Muicsbp ...PEEEQK. ..........
.......... CKLGVAPAGC MSEVPEMECG Chkicsbp
...PEEEQK. .......... .......... CKIGVGNGSS
LTDVGDMDCS 1551
1600 Hsirf2 IKNEVDSTVN
IIVVGQSHLD SNIENQEIVT NPPDICQVVE VTTESDEQPV
Muirf2 IKNEVDSTVN IIVVGQSHLD SNIEDQEIVT
NPPDICQVVE VTTESDDQPV Chirf2 IKNEVDSTVN
IVVVGQPHLD GSSEEQVIVA NPPDVCQVVE VTTESDEQPL
Muirf1 SYTTQGYLGQ DLDMER.DIT PALSPCVVSS
SLSEWHMQMD I.IPDSTTDL Ratirf1 SYTAQGYLGQ
DLDMDR.DIT PALSPCVVSS SLSEWHMQMD I.MPDSTTDL
Hsirf1 SYTVPGYM.Q DLEVEQ.ALT PALSPCAVSS
TLPDWHIPVE V.VPDSTSDL Chkirf1a SYTAHDYTGQ
EVEVENTSIT LDLSSCEVSG SLTDWRMPME IAMADSTNDI
Hsirf3a
Mmuirf3

Hsirf5 LLKEDVKWPP TLQPPTLQPP VVLGPPAPDP
SPLAPPPGNP AGFRELLSEV Mmuirf6 FL........
NINGSPMAPA SVGNCSVGNC SPESVWP... ......KTEP
Hump48 LNNEEEGASG GAVHSDIGSS SSSSSPEPQE
VTDTTEAPFQ ........GD Mup48 SGSNIGGGGN
GSNRSD...S NSNCNSELEE GAGTTEATIR ........ED
Hsirf4 RSWRDYVPDQ PHPEIPYQCP MTFGPRGHHW
QGPACENGCQ VTGTFYACAP Mupip RSWRDYAPDQ
SHPEIPYQCP VTFGPRGHHW QGPSCENGCQ VTGTFYACAP
Huicsbp RSEIDELIKE .PSVDDYMGM IKRSPSP...
P.DACRS..Q LLPDWWAHEP Muicsbp RSEIEELIKE
.PSVDEYMGM TKRSPSP... P.EACRS..Q ILPDWWVQQP
Chkicsbp PSAIDDLMKE PPCVDEYLGI IKRSPSP...
PQETCRN..P PIPDWWMQQP
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PILEUP options

• For a first try, take the default options, but
  give the output file a meaningful name.
• If you dont get a good alignment, try a less
  stringent matrix and/or gap penalties.
• gt PILEUP -matroldpep.cmp
• It is a good idea to run PILEUP in batch mode if
  you have more than 10 sequences to align
• gt PILEUP -bat

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CLUSTAL

• CLUSTAL is a stand-alone (i.e. not integrated
  into GCG) multiple alignment program that is
  superior in some respects to PILEUP
• 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).

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Using CLUSTAL

• On mcrcr0 type clustal
• CLUSTAL can only work with sequences in
  multi-sequence FASTA format.
• The GCG program TOFASTA can convert lists of file
  names into FASTA multi-sequence format.

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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

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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/

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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.

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GCG alignment editors

• Alignments produced with PILEUP (or CLUSTAL) can
  be adjusted with LINEUP.
• Nicely shaded printouts can be produced with
  PRETTYBOX
• GCG's SeqLab X-Windows interface has a superb
  multiple sequence editor - the best editor
  of any kind.

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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

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SeqVu
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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/
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