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Introduction to bioinformatics lecture 9 Multiple sequence alignment (II) Scoring a profile position Profile 1 A C D . . Y Profile 2 A C D . . Y At each position (column) we have different residue frequencies for each amino acid (rows) SO: Instead of saying S=M(aa1, aa2) (one residue pair) For frequency f>0 (amino acid is actually there) we take: 20 20 S faai faaj M(aai , aaj ) i j Progressive alignment 1. 2. 3. Perform pair-wise alignments of all of the sequences; Use the alignment scores to produces a dendrogram using neighbour-joining methods (guide-tree); Align the sequences sequentially, guided by the relationships indicated by the tree. Biopat (first method ever) MULTAL (Taylor 1987) DIALIGN PRRP (1&2, Morgenstern 1996) (Gotoh 1996) ClustalW (Thompson et al 1994) PRALINE (Heringa 1999) T Coffee (Notredame 2000) POA (Lee 2002) MUSCLE (Edgar 2004) Progressive multiple alignment 1 2 1 3 Score 1-2 4 5 Score 4-5 Score 1-3 Scores 5×5 Scores to distances Guide tree Similarity matrix Iteration possibilities Multiple alignment General progressive multiple alignment technique (follow generated tree) d 1 3 1 3 2 5 1 3 2 5 root 1 3 2 5 PRALINE progressive strategy d 1 3 1 3 2 1 3 2 5 4 1 3 2 5 4 There are problems … Accuracy is very important !!!! Alignment errors during the construction of the MSA cannot be repaired anymore: propagated into the progressive steps. The comparisons of sequences at early steps during progressive alignments cannot make use of information from other sequences. It is only later during the alignment progression that more information from other sequences (e.g. through profile representation) becomes employed in the alignment steps. “Once a gap, always a gap” Feng & Doolittle, 1987 Additional strategies for multiple sequence alignment • Profile pre-processing • Secondary structure-induced alignment • Globalised local alignment • Matrix extension Objective: try to avoid (early) errors Profile pre-processing 1 2 1 3 4 5 Score 1-2 Score 1-3 Score 4-5 1 1 1 2 3 4 5 A C D . . Y Pi Px Key Sequence Pre-alignment Master-slave (N-to-1) alignment Pre-profile Pre-profile generation 1 2 1 3 Score 1-2 4 5 Score 4-5 Score 1-3 Cut-off 1 1 2 3 4 5 2 2 134 5 5 5 1 2 3 4 Pre-alignments A C D . . Y A C D . . Y A C D . . Y Pre-profiles Pre-profile alignment Pre-profiles 1 2 3 4 5 A C D . . Y A C D . . Y A C D . . Y A C D . . Y Final alignment A C D . . Y 1 2 3 4 5 Pre-profile alignment 1 2 3 4 5 12 3 4 5 21 3 4 5 31 2 4 5 41 2 3 5 5 1 2 3 4 Final alignment 1 2 3 4 5 Pre-profile alignment Alignment consistency 1 2 3 4 5 12 3 4 5 21 3 4 5 1 2 31 2 4 5 41 2 3 5 5 1 2 3 4 5 Ala131 A131 A131 L133 C126 A131 PRALINE pre-profile generation • Idea: use the information from all query sequences to make a pre-profile for each query sequence that contains information from other sequences • You can use all sequences in each pre-profile, or use only those sequences that will probably align ‘correctly’. Incorrectly aligned sequences in the preprofiles will increase the noise level. • Select using alignment score: only allow sequences in pre-profiles if their alignment with the score higher than a given threshold value. In PRALINE, this threshold is given as prepro=1500 (alignment score threshold value is 1500 – see next two slides) Flavodoxin-cheY consistency scores (PRALINE prepro=0) 1fx1 FLAV_DESVH FLAV_DESDE FLAV_DESGI FLAV_DESSA 4fxn FLAV_MEGEL 2fcr FLAV_ANASP FLAV_ECOLI FLAV_AZOVI FLAV_ENTAG FLAV_CLOAB 3chy --7899999999999TEYTAETIARQL8776-6657777777777777553799VL999ST97775599989-435566677798998878AQGRKVACF -46788999999999TEYTAETIAREL7777-7757777777777777553799VL999ST97775599989-435566677798998878AQGRKVACF -47899999999999999999999988776695658888777777778763YDAVL999SAW9877789877753556666669777776789GRKVAAF -46788999999999TEGVAEAIAKTL9997-76678888777777887539DVVL999ST987776--9889546667776697776557777888888 93677799999999999999999999988759765777888888888876399999999STW77765--9999536666677797998779999999999 -878779999999999999999999776666967567788888888888777999999988777776--9889577788888897773237888888888 9776779999999999999999997777766-665666677788899976799999999987777669--887362334466695555455778888888 --87899999999999TEVADFIGK996541900300000112233355679DLLF99999855312888111224555555407777777888888888 -47899LFYGTQTGKTESVAEIIR9777653922356677777777897779999999999988843--9998555778777899998879999999999 997789999GSDTGNTENIAKMIQ8774222922456678889999995569999999999755553----99262225555495777767778999999 --79IGLFFGSNTGKTRKVAKSIK99887759657577888888999777899999999999877761112222222244555-5555555778999999 94789999999999999999999998755229223234555555555555688899999998875521111111133477777-7777777999999999 -86999ILYSSKTGKTERVAK9997555555057678887888887777765778899998522223--9888342234455597777777777777777 0122222223333335666665555555222922222222222221112163335555755553222888877674533344493332222222222222 Avrg Consist Conservation 8667778888888889999999998776554844455566666666665557888888888766544887666334445566586666556778888888 0125538675848969746963946463343045244355446543473516658868567554455000000314365446505575435547747759 1fx1 FLAV_DESVH FLAV_DESDE FLAV_DESGI FLAV_DESSA 4fxn FLAV_MEGEL 2fcr FLAV_ANASP FLAV_ECOLI FLAV_AZOVI FLAV_ENTAG FLAV_CLOAB 3chy G888799955555559888888888899777----7777797787787978---555555566776555677777778888799-----G888799955555559888888888899777----7777797787787978---555555566776555677777778888799-----A88878685555555999988888889998879--8777788-98777777--8555555554433245667777777777599-----87775977755555677777777777777778---88888887667778777775555555555542424667888887777-------977768777555556777777777777777767887777777778888-978985555555556536556888888888877-------867777555555552666666666555555577887767999877777977777665555555555444466666666555798-----8577775666666525556777778888888689977888988776558677885544333222222212233223355557-------877773573333333777766667777765533333333333333322833333333332244444567777777888777633-----977773775333344777888888777777733334444444444433833333344444444444455577777788777734-----977743786444444777788888888888833334444444444444244444555554555775667788888888877734110000 97776355333333466666667777777773333444444444444482333355555555555545558888888877772311---977773886555555866666666677666633333333333333322123333344444444455555665566666555582-----766627222222212444444444455555587882222222222222111111122222222222344443333333233399-----222227222222224111355431113324578-87778997666556877776322222222222322222323344444422------ Avrg Consist Conservation 866656564444444666666666666666656665555565555555655565444443444443344455666666666666889999 73663057433334163464534444*746710000011010011000000010434744645443225474454448434301000000 Consistency are scored 0 toSId= 10;3838 the value 10 is represented by the corresponding amino acid (red) Iteration 0values SP= 135136.00 AvSP=from 10.473 AvSId= 0.297 Flavodoxin-cheY consistency scores (PRALINE prepro=1500) 1fx1 FLAV_DESVH FLAV_DESSA FLAV_DESGI FLAV_DESDE 4fxn FLAV_MEGEL 2fcr FLAV_ANASP FLAV_AZOVI FLAV_ENTAG FLAV_ECOLI FLAV_CLOAB 3chy -42444IVYGSTTGNTEYTAETIARQL886666666577777775667888DLVLLGCSTW77766----995476666769-77888788AQGRKVACF -34444IVYGSTTGNTEYTAETIAREL776666666577777775667888DLVLLGCSTW77766----995476666769-77888788AQGRKVACF -33444IVYGSTTGNTET99999888777655777668888899666686YDIVLFGCSTW77777----996466666779-88SL98ADLKGKKVSVF -34444IVYGSTTGNTEGVA9999999999765555677777886666678DVVLLGCSTW77777----995466666779-88887688888KKVGVF -44777IVFGSSTGNTE988777666655566777778899999777777YDAVLFGCSAW88877----997587777779-8887766777GRKVAAF -32222IVYWSGTGNTE8888888876666778888888888NI8888586DILILGCSA888888------8-8888886--66665378ISGKKVALF -12222IVYWSGTGNTEAMA8888888888888888555555555555485DVILLGCPAMGSE77------572222288--8888755588GKKVGLF -41456IFFSTSTGNTTEVA999998865432222765554443244779YDLLFLGAPT944411999-111112454441-8DKLPEVDMKDLPVAIF -00456LFYGTQTGKTESVAEII987755323322427776666623589YQYLIIGCPTW55532--999843678W988899998888888GKLVAYF -42445LFFGSNTGKTRKVAKSIK87777434333536666665467777YQFLILGTPTLGEG862222222222355558-45666666888KTVALF -266IGIFFGSDTGQTRKVAKLIHQKL6664664424DVRRATR88888SYPVLLLGTPT88888644444444446WQEF8-8NTLSEADLTGKTVALF -51114IFFGSDTGNTENIAKMI987743311111555555588355599YDILLLGIPT954431----88355225544--44666666779KLVALF -63666ILYSSKTGKTERVAKLIE63333333333333333333366LQESEGIIFGTPTY63--6--------66SWE33333333333333GKLGAAF ADKELKFLVVDDFSTMRRIVRNLLKELGFNNVEEAEDGVDALNKLQ-AGGYGFVI---SDWNMPNM----------DGLEL--LKTIRADGAMSALPVLM Avrg Consist Conservation 9334459999999999999999988776655555555666667756667889999999999767658888775555566668967777677889999999 0236428675848969746963946463344354312564565414344366588685675544550000003144654460055575345547747759 1fx1 FLAV_DESVH FLAV_DESSA FLAV_DESGI FLAV_DESDE 4fxn FLAV_MEGEL 2fcr FLAV_ANASP FLAV_AZOVI FLAV_ENTAG FLAV_ECOLI FLAV_CLOAB 3chy G98879-89-999877977--7788899999999955--88888-9988887798999777778766553344588776666222266899899 G98879-89-999877977--7788899999999955--88888-9988887798999777778766553344588776666222266899899 G98878-688688888-88--88999999999999979988888887788889-89-9787777666756645577776666654466899899 G98879-898688888987--788888999GATLV7698899-9998789888-8899787878776663122477788888333276899899 AS8888-68-888888899--9999999999988888-99988888988778897888776668854222212255555555333277999999 GS2228-228222222222--2388888888888888888888888888888888888887778866765535577555533221288888888 G4888--28-8888882MD--AWKQRTEDTGATVI77---------------------77222--224444222222244222112-------GLGDA5-8Y5DNFC88-88--8877777777777765444555555555544385555777774465333357799999987555333899899 GTGDQ5-GY5899999-99--99EEKISQRGG99975555544444444433284444466665555555556666676666433333899899 GLGDQ5-885777555-55--55555788888888555555555555555554855555555555666555555888855555544442--288 GLGDQL-NYSKNFVSA-MR--ILYDLVIARGACVVG8888EGYKFSFSAA6664NEFVGLPLDQEN88888EERIDSWLE88842242688688 GC99549784688888987997777777778888855444444444444444114444777774455775567788888887433322100100 STANS6366663333333333336666666666666666663333363366336663333336EDENARIFGERIANKVKQI333333666666 VTAEA---KKENIIAA-----------AQAGAS-------------------------GYVVK-----PFTAATLEEKLNKIFEKLGM------ Avrg Consist Conservation 9988779787777777777997788888888888866777777777767766677777676667766655455577776666433355788788 746640037154545706300354534444*745753000001010010000000010683760144442335574454448434301000000 Iteration 0 SP= 136702.00 AvSP= 10.654 SId= 3955 AvSId= 0.308 Consistency values are scored from 0 to 10; the value 10 is represented by the corresponding amino acid (red) Strategies for multiple sequence alignment • Profile pre-processing • Secondary structure-induced alignment • Globalised local alignment • Matrix extension Objective: integrate secondary structure information to anchor alignments and avoid errors Protein structure hierarchical levels PRIMARY STRUCTURE (amino acid sequence) SECONDARY STRUCTURE (helices, strands) VHLTPEEKSAVTALWGKVNVDE VGGEALGRLLVVYPWTQRFFE SFGDLSTPDAVMGNPKVKAHG KKVLGAFSDGLAHLDNLKGTFA TLSELHCDKLHVDPENFRLLGN VLVCVLAHHFGKEFTPPVQAAY QKVVAGVANALAHKYH QUATERNARY STRUCTURE (oligomers) TERTIARY STRUCTURE (fold) Why use (predicted) structural information • “Structure more conserved than sequence” – Many structural protein families (e.g. globins) have family members with very low sequence similarities. For example, globin sequences identities can be as low as 10% while still having an identical fold. • This means that you can still observe equivalent secondary structures in homologous proteins even if sequence similarities are extremely low. • But you are dependent on the quality of prediction methods. For example, secondary structure prediction is currently at 76% correctness. So, 1 out of 4 predicted amino acids is still incorrect. Two superposed protein structures with two wellsuperposed helices Red: well superposed Blue: low match quality C5 anaphylatoxin -- human (PDB code 1kjs) and pig (1c5a)) proteins are superposed How to combine ss and aa info Amino acid substitution matrices Dynamic programming search matrix M D A A S T I L C G S MDAGSTVILCFV HHHCCCEEEEEE H H H H H C C C E E E C C H E E C Default In terms of scoring… • So how would you score a profile using this extra information? – Same formula as in lecture 6, but you can use sec. struct. specific substitution scores in various combinations. • Where does it fit in? – Very important: structure is always more conserved than sequence so it can help with the insertion(or not) of gaps. Sequences to be aligned Predict secondary structure Secondary structure HHHHCCEEECCCEEECCHH CCCCCCEECCCEEEECCHH HHHCCCCEECCCEEHHH HHHHHCCEEEECCCEECCC HHHHHHHHHHHHHCCCEEEE Align sequences using secondary structure Multiple alignment Using predicted secondary structure 1fx1 FLAV_DESVH FLAV_DESGI FLAV_DESSA FLAV_DESDE 2fcr FLAV_ANASP FLAV_ECOLI FLAV_AZOVI FLAV_ENTAG 4fxn FLAV_MEGEL FLAV_CLOAB 3chy 1fx1 FLAV_DESVH FLAV_DESGI FLAV_DESSA FLAV_DESDE 2fcr FLAV_ANASP FLAV_ECOLI FLAV_AZOVI FLAV_ENTAG 4fxn FLAV_MEGEL FLAV_CLOAB 3chy -PK-ALIVYGSTTGNTEYTAETIARQLANAG-YEVDSRDAASVEAGGLFEGFDLVLLGCSTWGDDSI------ELQDDFIPLFDS-LEETGAQGRKVACF e eeee b ssshhhhhhhhhhhhhhttt eeeee stt tttttt seeee b ee sss ee ttthhhhtt ttss tt eeeee MPK-ALIVYGSTTGNTEYTaETIARELADAG-YEVDSRDAASVEAGGLFEGFDLVLLgCSTWGDDSI------ELQDDFIPLFDS-LEETGAQGRKVACf e eeeeee hhhhhhhhhhhhhhh eeeeee eeeeee hhhhhh eeeee MPK-ALIVYGSTTGNTEGVaEAIAKTLNSEG-METTVVNVADVTAPGLAEGYDVVLLgCSTWGDDEI------ELQEDFVPLYED-LDRAGLKDKKVGVf e eeeeee hhhhhhhhhhhhhh eeeeee hhhhhh eeeeeee hhhhhh eeeeee MSK-SLIVYGSTTGNTETAaEYVAEAFENKE-IDVELKNVTDVSVADLGNGYDIVLFgCSTWGEEEI------ELQDDFIPLYDS-LENADLKGKKVSVf eeeeee hhhhhhhhhhhhhh eeeee eeeee hhhhhhh h eeeee MSK-VLIVFGSSTGNTESIaQKLEELIAAGG-HEVTLLNAADASAENLADGYDAVLFgCSAWGMEDL------EMQDDFLSLFEE-FNRFGLAGRKVAAf eeee hhhhhhhhhhhhhh eeeee hhhhhhhhhhheeeee hhhhhhh hh eeeee --K-IGIFFSTSTGNTTEVADFIGKTLGAK---ADAPIDVDDVTDPQALKDYDLLFLGAPTWNTGAD----TERSGTSWDEFLYDKLPEVDMKDLPVAIF eeeee ssshhhhhhhhhhhhhggg b eeggg s gggggg seeeeeee stt s s s sthhhhhhhtggg tt eeeee SKK-IGLFYGTQTGKTESVaEIIRDEFGND--VVTL-HDVSQAE-VTDLNDYQYLIIgCPTWNIGEL--------QSDWEGLYSE-LDDVDFNGKLVAYf eeeee hhhhhhhhhhhh eee hhh hhhhhhheeeeee hhhhhhhhh eeeeee -AI-TGIFFGSDTGNTENIaKMIQKQLGKD--VADV-HDIAKSS-KEDLEAYDILLLgIPTWYYGEA--------QCDWDDFFPT-LEEIDFNGKLVALf eee hhhhhhhhhhhh eee hhh hhhhhhheeeee hhhhh eeeeee -AK-IGLFFGSNTGKTRKVaKSIKKRFDDET-MSDA-LNVNRVS-AEDFAQYQFLILgTPTLGEGELPGLSSDCENESWEEFLPK-IEGLDFSGKTVALf eee hhhhhhhhhhhhh hhh hhhhhhheeeee hhhhhhhhh eeeeee MAT-IGIFFGSDTGQTRKVaKLIHQKLDG---IADAPLDVRRAT-REQFLSYPVLLLgTPTLGDGELPGVEAGSQYDSWQEFTNT-LSEADLTGKTVALf eeee hhhhhhhhhhhh hhh hhhhhhheeeee hhhhh eeeee ----MKIVYWSGTGNTEKMAELIAKGIIESG-KDVNTINVSDVNIDELLNE-DILILGCSAMGDEVL------E-ESEFEPFIEE-IST-KISGKKVALF eeeee ssshhhhhhhhhhhhhhhtt eeeettt sttttt seeeeee btttb ttthhhhhhh hst t tt eeeee M---VEIVYWSGTGNTEAMaNEIEAAVKAAG-ADVESVRFEDTNVDDVASK-DVILLgCPAMGSEEL------E-DSVVEPFFTD-LAP-KLKGKKVGLf hhhhhhhhhhhhhh eeeee hhhhhhhh eeeee eeeee M-K-ISILYSSKTGKTERVaKLIEEGVKRSGNIEVKTMNL-DAVDKKFLQESEGIIFgTPTY-YANI--------SWEMKKWIDE-SSEFNLEGKLGAAf eee hhhhhhhhhhhhhh eeeeee hhhhhhhhhh eeee hhhhhhhhh eeeee ADKELKFLVVDDFSTMRRIVRNLLKELGFNN-VEEAEDGV-DALNKLQAGGYGFVISD---WNMPNM----------DGLELLKTIRADGAMSALPVLMV tt eeee s hhhhhhhhhhhhhht eeeesshh hhhhhhhh eeeee s sss hhhhhhhhhh ttttt eeee GCGDS-SY-EYFCGAVDAIEEKLKNLGAEIVQD---------------------GLRIDGD--PRAARDDIVGWAHDVRGAI-------eee s ss sstthhhhhhhhhhhttt ee s eeees gggghhhhhhhhhhhhhh GCGDS-SY-EYFCGAVDAIEEKLKNLgAEIVQD---------------------GLRIDGD--PRAARDDIVGwAHDVRGAI-------eee hhhhhhhhhhhh eeeee eeeee hhhhhhhhhhhhhh GCGDS-SY-TYFCGAVDVIEKKAEELgATLVAS---------------------SLKIDGE--P--DSAEVLDwAREVLARV-------eee hhhhhhhhhhhh eeeee hhhhhhhhhhh GCGDS-DY-TYFCGAVDAIEEKLEKMgAVVIGD---------------------SLKIDGD--P--ERDEIVSwGSGIADKI-------hhhhhhhhhhhh eeeee e eee ASGDQ-EY-EHFCGAVPAIEERAKELgATIIAE---------------------GLKMEGD--ASNDPEAVASfAEDVLKQL-------e hhhhhhhhhhhhhh eeeee ee hhhhhhhhhhh GLGDAEGYPDNFCDAIEEIHDCFAKQGAKPVGFSNPDDYDYEESKSVRD-GKFLGLPLDMVNDQIPMEKRVAGWVEAVVSETGV-----eee ttt ttsttthhhhhhhhhhhtt eee b gggs s tteet teesseeeettt ss hhhhhhhhhhhhhhhht GTGDQIGYADNFQDAIGILEEKISQRgGKTVGYWSTDGYDFNDSKALR-NGKFVGLALDEDNQSDLTDDRIKSwVAQLKSEFGL-----hhhhhhhhhhhhhh eeee hhhhhhhhhhhhhhhh GCGDQEDYAEYFCDALGTIRDIIEPRgATIVGHWPTAGYHFEASKGLADDDHFVGLAIDEDRQPELTAERVEKwVKQISEELHLDEILNA hhhhhhhhhhhhhh eeee hhhhhhhhhhhhhhhhhh GLGDQVGYPENYLDALGELYSFFKDRgAKIVGSWSTDGYEFESSEAVVD-GKFVGLALDLDNQSGKTDERVAAwLAQIAPEFGLS--L-e hhhhhhhhhhhhhh eeeee hhhhhhhhhhh GLGDQLNYSKNFVSAMRILYDLVIARgACVVGNWPREGYKFSFSAALLENNEFVGLPLDQENQYDLTEERIDSwLEKLKPAV-L-----hhhhhhhhhhhhhhh eeee hhhhhhh hhhhhhhhhhhh G-----SYGWGDGKWMRDFEERMNGYGCVVVET---------------------PLIVQNE--PDEAEQDCIEFGKKIANI--------e eesss shhhhhhhhhhhhtt ee s eeees ggghhhhhhhhhhhht G-----SYGWGSGEWMDAWKQRTEDTgATVIGT----------------------AIVNEM--PDNAPE-CKElGEAAAKA--------hhhhhhhhhhh eeeee eeee h hhhhhhhh STANSIA-GGSDIALLTILNHLMVK-gMLVYSG----GVAFGKPKTHLG-----YVHINEI--QENEDENARIfGERiANkV--KQIF-hhhhhhhhhhhhhh eeeee hhhh hhh hhhhhhhhhhhh h -----------TAEAKKENIIAAAQAGASGY-------------------------VVK----P-FTAATLEEKLNKIFEKLGM-----ess hhhhhhhhhtt see ees s hhhhhhhhhhhhhhht G Strategies for multiple sequence alignment • Profile pre-processing • Secondary structure-induced alignment • Globalised local alignment • Matrix extension Objectives: Instead of single amino acid positions, focus on local alignments Consider best local alignment through each cell in DP matrix Try to avoid (early) errors Globalised local alignment 1. Local (SW) alignment (M + Po,e) + = 2. Global (NW) alignment (no M or Po,e) Double dynamic programming Strategies for multiple sequence alignment • Profile pre-processing • Secondary structure-induced alignment • Globalised local alignment • Matrix extension Objective: try to avoid (early) errors Integrating alignment methods and alignment information with T-Coffee • Integrating different pair-wise alignment techniques (NW, SW, ..) • Combining different multiple alignment methods (consensus multiple alignment) • Combining sequence alignment methods with structural alignment techniques • Plug in user knowledge Matrix extension T-Coffee Tree-based Consistency Objective Function For alignmEnt Evaluation Cedric Notredame Des Higgins Jaap Heringa J. Mol. Biol., 302, 205-217;2000 Using different sources of alignment information Clustal Clustal Structure alignments Dialign Lalign Manual T-Coffee Search matrix extension – alignment transitivity T-Coffee Other sequences Direct alignment Search matrix extension but..... T-COFFEE (V1.23) multiple sequence alignment Flavodoxin-cheY 1fx1 FLAV_DESVH FLAV_DESGI FLAV_DESSA FLAV_DESDE 4fxn FLAV_MEGEL FLAV_CLOAB 2fcr FLAV_ENTAG FLAV_ANASP FLAV_AZOVI FLAV_ECOLI 3chy 1fx1 FLAV_DESVH FLAV_DESGI FLAV_DESSA FLAV_DESDE 4fxn FLAV_MEGEL FLAV_CLOAB 2fcr FLAV_ENTAG FLAV_ANASP FLAV_AZOVI FLAV_ECOLI 3chy ----PKALIVYGSTTGNTEYTAETIARQLANAG-YEVDSRDAASVE-AGGLFEGFDLVLLGCSTWGDDSIE------LQDDFIPL-FDSLEETGAQGRK-------MPKALIVYGSTTGNTEYTAETIARELADAG-YEVDSRDAASVE-AGGLFEGFDLVLLGCSTWGDDSIE------LQDDFIPL-FDSLEETGAQGRK-------MPKALIVYGSTTGNTEGVAEAIAKTLNSEG-METTVVNVADVT-APGLAEGYDVVLLGCSTWGDDEIE------LQEDFVPL-YEDLDRAGLKDKK-------MSKSLIVYGSTTGNTETAAEYVAEAFENKE-IDVELKNVTDVS-VADLGNGYDIVLFGCSTWGEEEIE------LQDDFIPL-YDSLENADLKGKK-------MSKVLIVFGSSTGNTESIAQKLEELIAAGG-HEVTLLNAADAS-AENLADGYDAVLFGCSAWGMEDLE------MQDDFLSL-FEEFNRFGLAGRK----------MKIVYWSGTGNTEKMAELIAKGIIESG-KDVNTINVSDVN-IDELL-NEDILILGCSAMGDEVLE-------ESEFEPF-IEEIS-TKISGKK---------MVEIVYWSGTGNTEAMANEIEAAVKAAG-ADVESVRFEDTN-VDDVA-SKDVILLGCPAMGSEELE-------DSVVEPF-FTDLA-PKLKGKK--------MKISILYSSKTGKTERVAKLIEEGVKRSGNIEVKTMNLDAVD-KKFLQ-ESEGIIFGTPTYYAN---------ISWEMKKW-IDESSEFNLEGKL---------KIGIFFSTSTGNTTEVADFIGKTLGAKA---DAPIDVDDVTDPQAL-KDYDLLFLGAPTWNTGA----DTERSGTSWDEFLYDKLPEVDMKDLP-------MATIGIFFGSDTGQTRKVAKLIHQKLDGIA---DAPLDVRRAT-REQF-LSYPVLLLGTPTLGDGELPGVEAGSQYDSWQEF-TNTLSEADLTGKT-------SKKIGLFYGTQTGKTESVAEIIRDEFGNDV---VTLHDVSQAE-VTDL-NDYQYLIIGCPTWNIGEL--------QSDWEGL-YSELDDVDFNGKL--------AKIGLFFGSNTGKTRKVAKSIKKRFDDET-M-SDALNVNRVS-AEDF-AQYQFLILGTPTLGEGELPGLSSDCENESWEEF-LPKIEGLDFSGKT--------AITGIFFGSDTGNTENIAKMIQKQLGKDV---ADVHDIAKSS-KEDL-EAYDILLLGIPTWYYGEA--------QCDWDDF-FPTLEEIDFNGKL----ADKELKFLVVD--DFSTMRRIVRNLLKELGFN-NVE-EAEDGVDALNKLQ-AGGYGFVISDWNMPNMDGLE--------------LLKTIRADGAMSALPVLMV :. . . : . :: ---------VACFGCGDSS--YEYFCGA-VDAIEEKLKNLGAEIVQDG---------------------LRIDGDPRAA--RDDIVGWAHDVRGAI----------------VACFGCGDSS--YEYFCGA-VDAIEEKLKNLGAEIVQDG---------------------LRIDGDPRAA--RDDIVGWAHDVRGAI----------------VGVFGCGDSS--YTYFCGA-VDVIEKKAEELGATLVASS---------------------LKIDGEPDSA----EVLDWAREVLARV----------------VSVFGCGDSD--YTYFCGA-VDAIEEKLEKMGAVVIGDS---------------------LKIDGDPE----RDEIVSWGSGIADKI----------------VAAFASGDQE--YEHFCGA-VPAIEERAKELGATIIAEG---------------------LKMEGDASND--PEAVASFAEDVLKQL----------------VALFGS------YGWGDGKWMRDFEERMNGYGCVVVETP---------------------LIVQNEPD--EAEQDCIEFGKKIANI-----------------VGLFGS------YGWGSGEWMDAWKQRTEDTGATVIGTA---------------------IV--NEMP--DNAPECKELGEAAAKA-----------------GAAFSTANSI--AGGSDIA-LLTILNHLMVKGMLVY----SGGVAFGKPKTHLGYVHINEIQENEDENARIFGERIANKVKQIF-------------------VAIFGLGDAEGYPDNFCDA-IEEIHDCFAKQGAKPVGFSNPDDYDYEESKSVRDG-KFLGLPLDMVNDQIPMEKRVAGWVEAVVSETGV--------------VALFGLGDQLNYSKNFVSA-MRILYDLVIARGACVVGNWPREGYKFSFSAALLENNEFVGLPLDQENQYDLTEERIDSWLEKLKPAVL---------------VAYFGTGDQIGYADNFQDA-IGILEEKISQRGGKTVGYWSTDGYDFNDSKALRNG-KFVGLALDEDNQSDLTDDRIKSWVAQLKSEFGL--------------VALFGLGDQVGYPENYLDA-LGELYSFFKDRGAKIVGSWSTDGYEFESSEAVVDG-KFVGLALDLDNQSGKTDERVAAWLAQIAPEFGLSL------------VALFGCGDQEDYAEYFCDA-LGTIRDIIEPRGATIVGHWPTAGYHFEASKGLADDDHFVGLAIDEDRQPELTAERVEKWVKQISEELHLDEILNA TAEAKKENIIAAAQAGASGYVVKPFT---AATLEEKLNKIFEKLGM---------------------------------------------------------. Multiple alignment methods Multi-dimensional dynamic programming > extension of pairwise sequence alignment. Progressive alignment > incorporates phylogenetic information to guide the alignment process Iterative alignment > correct for problems with progressive alignment by repeatedly realigning subgroups of sequence