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 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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Multiple Alignments

• Making an optimal alignment between two sequences
  is computationally straightforward, but aligning
  a large number of sequences using the same method
  is almost impossible.
• The problem increases exponentially with the
  number of sequences involved, so it becomes
  computationally expensive (and inefficient) for
  large numbers of sequences.

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Longer Sequences

• What happens to the number of cells in the matrix
  when we add another base to one sequence?
• How about to both?
• cells L1 x L2 or L2 if we use 2
  sequences of the same length.
• So the amount of computing grows with the square
  of seq. length bad but not terrible, because
  the compute time for each cell remains constant

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Align Three Sequences by Dynamic programming
Georg Fullen, VSNS Biocomputing, Univ. Munster
So how many cells (that contain values that must
be computed) do we add for each additional
sequence its a power function! For N
sequences of length L of cells 2n x Ln
This is very bad for computing alignments of a
lot of sequences!
If the calculation takes 1 nanosecond per cell,
then for 6 sequences of length 100, we'll have a
running time of is 26 x 1006 x 10-9 seconds
(64000 seconds). Just add 2 more sequences, and
the running time is 28 x 1008 x 10-9 2.6 x 109
seconds (28 days)
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Global vs. Local Multiple 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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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, averages
  them into a consensus (actually a profile), then
  adds new sequences one at a time to the aligned
  set.
• This is an approximate method!

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CLUSTALW

• CLUSTAL is the most popular multiple alignment
  program
• 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.
• Unix command line program
• Website http//www.ebi.ac.uk/Tools/clustalw2/ind
  ex.html
• There are also Mac and PC versions with a nice
  graphical interface (CLUSTALX).

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http//www.ebi.ac.uk/Tools/clustalw2/index.html
CLUSTALW2 at the EBI website
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Other Multiple Alignment Tools

• MUSCLE
• http//www.ebi.ac.uk/Tools/muscle/index.html
• TCOFFE http//www.ebi.ac.uk/Tools/t-coffee/
• MSA

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Editing Multiple Alignments

• There are a variety of tools that can be used to
  modify and display 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 an
  alignment program.

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Alignment 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
• Sequencher, MacVector, DS Gene, Vector NTI, all
  include a built-in version of CLUSTAL

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SeqVu
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JalView

• Install on your machine
• or run as a Java WebStart application

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• 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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Analysis of Alignments

• Once you have a multiple alignment, what can you
  do with it?
• 1) Identify regions of similarity and difference
• conserved regions may be functionally important,
  and/or sites for inclusive (cross species) primer
  design
• Variable regions may be functionally important,
  and/or sites for gene/allele-specific primer
  design
• 2) Create a sequence logo
• 3) Build a Phylogenetic Tree (next week)

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Format a Multiple Alignment

• The concept of a consensus sequence is implied
  by any multiple alignment. There can be various
  rules for building the consensus simple majority
  rules, plurality by a specific , etc.
• The alignment may look nicer by showing how each
  letter matches the consensus highlight the
  differences.

• PLOTSIMILARITY (a graph of overall similarity
  across the alignment) EMBOSS plotcon
• Show match to consensus showalign
• Shade by similarity prettyplot/Boxshade

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Plurality 2.00 Threshold 4 AveWeight 0.55
AveMatch 2.91 AvMisMatch -2.00 PRETTY of
_at_pretty.list October 7, 1998 1035 ..
1
50 fa10.ugly .......... ..........
.......... ..TTttGESA D.PvtTtVE. fa12.ugly
.......... .......... .......... ..TTatGESA
D.PvtTtVE. fo1k.ugly .......... ..........
.......... ..TTsaGESA D.PvtTtVE. e.ugly
Gvenae.kgv tEnTna.Tad fvaqpvyLPe .nqT......
kv.Affynrs p1m.ugly GlgqmlEsmI .dnTvreTvg
AatsrdaLPn teasGPthSk eiPALTAVET p1s.ugly
GlgqmlEsmI .dnTvreTvg AatsrdaLPn teasGPahSk
eiPALTAVET p2s.ugly GigdmiEgav .Egitknalv
pptstnsLPg hkpsGPahSk eiPALTAVET p3s.ugly
Giedliseva .qgal..Tls lpkqqdsLPd tkasGPahSk
evPALTAVET cb3.ugly ...gpvEdaI .......T..
Aaigr..vad tvgTGPtnSe aiPALTAaET r14.ugly
GlgdelEevI vEkT.kqTv. Asi....... ..ssGPkhtq
kvPiLTAnET r2.ugly ...npvEnyI dEvlnevlv.
.......vPn inssnPttSn saPALdAaET Consensus
G-----E--I -E-T---T-- A------LP- --TTGPGESA
D-PALTAVET //////////////////////////////////////
/////////////////////////// 301

349 fa10.ugly aElyCPRPll AIkvtsqdRy KqKI.iAPa.
..KQll.... ......... fa12.ugly aElyCPRPll
AIevssqdRh KqKI.iAPg. ..KQll....
......... fo1k.ugly aEtyCPRPll AIhpt.eaRh
KqKI.vAPv. ..KQTl.... ......... e.ugly
krvfCPRPtv ffPwpTsG.D Kidmtpragv lmlespnald
isrty.... p1m.ugly irvWCPRPPR AlaYygpGvD
ykdgtltPls tkdlTTy... ......... p1s.ugly
irvWCPRPPR AvaYygpGvD ykdgtltPls tkdlTTy...
......... p2s.ugly VrvWCPRPPR AvPYfgpGvD
ykdg.ltPlp ekglTTy... ......... p3s.ugly
VrvWCPRPPR AvPYygpGvD yrn.nldPls ekglTTy...
......... cb3.ugly VkaWiPRPPR lcqYekakn.
vnfrssgvtt trqsiTtmtn tgaiwtti. r14.ugly
VEaWiPRaPR AlPY.Tsigr tny..pknte pvikkrk.gd
i.ksy.... r2.ugly VkaWCPRPPR AleY.Trahr
tnfkiedrsi qtaivTrpii ttagpsdmy Consensus
VE-WCPRPPR AIPY-T-GRD K-KI--AP-- --KQTT----
---------
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Boxshade
Shade each letter of the alignment based on its
match to the consensus highlights conserved
regions much more informative for protein
alignments (shades of grey for similar amino
acids)
http//mobyle.pasteur.fr/cgi-bin/MobylePortal/port
al.py?formboxshade
http//www.ch.embnet.org/software/BOX_form.html
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Sequence Logos
http//weblogo.berkeley.edu/logo.cgi
http//weblogo.threeplusone.com/create.cgi
http//genome.tugraz.at/Logo/
T. D. Schneider and R. M. Stephens. Sequence
logos a new way to display consensus sequences.
Nucleic Acids Research, Vol. 18, No 20, p.
6097-6100.
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Buidling on Alignments

• Multiple Alignments are the starting point for
  calculating phylogenetic trees
• Motifs and Profiles are calculated from multiple
  alignments