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Pairwise sequence Alignment

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  1. Pairwise sequence Alignment

  2. Sequence Alignment • Sequence analysis is the process of making biological inferences from the known sequence of monomers in protein, DNA and RNA polymers.

  3. Complete DNA Sequences More than 400 complete genomes have been sequenced

  4. Evolution

  5. Sequence alignment • Comparing DNA/protein sequences for • Similarity • Homology • Prediction of function • Construction of phylogeny • Shotgun assembly • End-space-free alignment / overlap alignment • Finding motifs

  6. Sequence Alignment Procedure of comparing two (pairwise) or more (multiple) sequences by searching for a series of individual characters that are in the same order in the sequencesGCTAGTCAGATCTGACGCTA | |||| ||||| ||| TGGTCACATCTGCCGC

  7. Sequence Alignment AGGCTATCACCTGACCTCCAGGCCGATGCCC TAGCTATCACGACCGCGGTCGATTTGCCCGAC -AGGCTATCACCTGACCTCCAGGCCGA--TGCCC--- TAG-CTATCAC--GACCGC--GGTCGATTTGCCCGAC Definition Given two strings x = x1x2...xM, y = y1y2…yN, an alignment is an assignment of gaps to positions 0,…, M in x, and 0,…, N in y, so as to line up each letter in one sequence with either a letter, or a gap in the other sequence

  8. Sources of variation • Nucleotide substitution • Replication error • Chemical reaction • Insertions or deletions (indels) • Unequal crossing over • Replication slippage • Duplication • a single gene (complete gene duplication) • part of a gene (internal or partial gene duplication) • Domain duplication • Exon shuffling • part of a chromosome (partial polysomy) • an entire chromosome (aneuploidy or polysomy) • the whole genome (polyploidy)

  9. Common mutations in DNA Substitution: A C G T T G A C A C GAT G A C Deletion: A C GT T GA C A C G A C Insertion: A C G T T G A C A C GC A AT T G A C

  10. ? Similar 3D structure ? Similar sequencesproducesimilar proteins Seq.Align. Protein Function Protein1 Protein2 More than 25% sequence identity ? Similar function

  11. Differing rates of DNA evolution • Functional/selective constraints (particular features of coding regions, particular features in 5' untranslated regions) • Variation among different gene regions with different functions (different parts of a protein may evolve at different rates). • Within proteins, variations are observed between • surface and interior amino acids in proteins (order of magnitude difference in rates in haemoglobins) • charged and non-charged amino acids • protein domains with different functions • regions which are strongly constrained to preserve particular functions and regions which are not • different types of proteins -- those with constrained interaction surfaces and those without

  12. Common assumptions • All nucleotide sites change independently • The substitution rate is constant over time and in different lineages • The base composition is at equilibrium • The conditional probabilities of nucleotide substitutions are the same for all sites, and do not change over time • Most of these are not true in many cases…

  13. Pairwise alignments in the 1950s b-corticotropin (sheep) Corticotropin A (pig) ala gly glu asp asp glu asp gly ala glu asp glu CYIQNCPLG CYFQNCPRG Oxytocin Vasopressin

  14. globins: a- b- myoglobin Early example of sequence alignment: globins (1961) H.C. Watson and J.C. Kendrew, “Comparison Between the Amino-Acid Sequences of Sperm Whale Myoglobin and of Human Hæmoglobin.” Nature 190:670-672, 1961.

  15. Pairwise sequence alignment is the most fundamental operation of bioinformatics • • It is used to decide if two proteins (or genes) • are related structurally or functionally • • It is used to identify domains or motifs that • are shared between proteins • It is the basis of BLAST searching (next week) • • It is used in the analysis of genomes

  16. Pairwise alignment: protein sequences can be more informative than DNA • • protein is more informative (20 vs 4 characters); • many amino acids share related biophysical properties • • codons are degenerate: changes in the third position • often do not alter the amino acid that is specified • • protein sequences offer a longer “look-back” time • DNA sequences can be translated into protein, • and then used in pairwise alignments

  17. Page 54

  18. Pairwise alignment: protein sequences can be more informative than DNA • DNA can be translated into six potential proteins 5’ CAT CAA 5’ ATC AAC 5’ TCA ACT 5’ CATCAACTACAACTCCAAAGACACCCTTACACATCAACAAACCTACCCAC 3’ 3’ GTAGTTGATGTTGAGGTTTCTGTGGGAATGTGTAGTTGTTTGGATGGGTG 5’ 5’ GTG GGT 5’ TGG GTA 5’ GGG TAG

  19. Pairwise alignment: protein sequences can be more informative than DNA • Many times, DNA alignments are appropriate • --to confirm the identity of a cDNA • --to study noncoding regions of DNA • --to study DNA polymorphisms • --example: Neanderthal vs modern human DNA Query: 181 catcaactacaactccaaagacacccttacacccactaggatatcaacaaacctacccac 240 |||||||| |||| |||||| ||||| | ||||||||||||||||||||||||||||||| Sbjct: 189 catcaactgcaaccccaaagccacccct-cacccactaggatatcaacaaacctacccac 247

  20. b-lactoglobulin (P02754) retinol-binding protein (NP_006735) Page 42

  21. Definitions Pairwise alignment The process of lining up two or more sequences to achieve maximal levels of identity (and conservation, in the case of amino acid sequences) for the purpose of assessing the degree of similarity and the possibility of homology.

  22. Definitions Homology Similarity attributed to descent from a common ancestor. Page 42

  23. Definitions Homology Similarity attributed to descent from a common ancestor. Identity The extent to which two (nucleotide or amino acid) sequences are invariant. RBP: 26 RVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWD- 84 + K ++ + + + GTW++ MA + L + AVT + +L+ W+ glycodelin: 23 QTKQDLELPKLAGTWHSMAMA-TNNISLMATLKAPLRVHITSLLPTPEDNLEI V LHRWEN 81 Page 44

  24. Definitions: two types of homology Orthologs Homologous sequences in different species that arose from a common ancestral gene during speciation; may or may not be responsible for a similar function. Paralogs Homologous sequences within a single species that arose by gene duplication. Page 43

  25. common carp Orthologs: members of a gene (protein) family in various organisms. This tree shows RBP orthologs. zebrafish rainbow trout teleost African clawed frog chicken human mouse rat horse pig cow rabbit 10 changes Page 43

  26. apolipoprotein D Paralogs: members of a gene (protein) family within a species retinol-binding protein 4 Complement component 8 Alpha-1 Microglobulin /bikunin prostaglandin D2 synthase progestagen- associated endometrial protein neutrophil gelatinase- associated lipocalin Odorant-binding protein 2A 10 changes Lipocalin 1 Page 44

  27. Pairwise alignment of retinol-binding protein and b-lactoglobulin 1 MKWVWALLLLAAWAAAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEG 50 RBP . ||| | . |. . . | : .||||.:| : 1 ...MKCLLLALALTCGAQALIVT..QTMKGLDIQKVAGTWYSLAMAASD. 44 lactoglobulin 51 LFLQDNIVAEFSVDETGQMSATAKGRVR.LLNNWD..VCADMVGTFTDTE 97 RBP : | | | | :: | .| . || |: || |. 45 ISLLDAQSAPLRV.YVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTK 93 lactoglobulin 98 DPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAV...........QYSC 136 RBP || ||. | :.|||| | . .| 94 IPAVFKIDALNENKVL........VLDTDYKKYLLFCMENSAEPEQSLAC 135 lactoglobulin 137 RLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQ.EELCLARQYRLIV 185 RBP . | | | : || . | || | 136 QCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI....... 178 lactoglobulin Page 46

  28. Definitions Similarity The extent to which nucleotide or protein sequences are related. It is based upon identity plus conservation. Identity The extent to which two sequences are invariant. Conservation Changes at a specific position of an amino acid or (less commonly, DNA) sequence that preserve the physico-chemical properties of the original residue.

  29. Pairwise alignment of retinol-binding protein and b-lactoglobulin 1 MKWVWALLLLAAWAAAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEG 50 RBP . ||| | . |. . . | : .||||.:| : 1 ...MKCLLLALALTCGAQALIVT..QTMKGLDIQKVAGTWYSLAMAASD. 44 lactoglobulin 51 LFLQDNIVAEFSVDETGQMSATAKGRVR.LLNNWD..VCADMVGTFTDTE 97 RBP : | | | | :: | .| . || |: || |. 45 ISLLDAQSAPLRV.YVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTK 93 lactoglobulin 98 DPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAV...........QYSC 136 RBP || ||. | :.|||| | . .| 94 IPAVFKIDALNENKVL........VLDTDYKKYLLFCMENSAEPEQSLAC 135 lactoglobulin 137 RLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQ.EELCLARQYRLIV 185 RBP . | | | : || . | || | 136 QCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI....... 178 lactoglobulin Identity (bar) Page 46

  30. Pairwise alignment of retinol-binding protein and b-lactoglobulin 1 MKWVWALLLLAAWAAAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEG 50 RBP . ||| | . |. . . | : .||||.:| : 1 ...MKCLLLALALTCGAQALIVT..QTMKGLDIQKVAGTWYSLAMAASD. 44 lactoglobulin 51 LFLQDNIVAEFSVDETGQMSATAKGRVR.LLNNWD..VCADMVGTFTDTE 97 RBP : | | | | :: | .| . || |: || |. 45 ISLLDAQSAPLRV.YVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTK 93 lactoglobulin 98 DPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAV...........QYSC 136 RBP || ||. | :.|||| | . .| 94 IPAVFKIDALNENKVL........VLDTDYKKYLLFCMENSAEPEQSLAC 135 lactoglobulin 137 RLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQ.EELCLARQYRLIV 185 RBP . | | | : || . | || | 136 QCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI....... 178 lactoglobulin Very similar (two dots) Somewhat similar (one dot) Page 46

  31. Definitions Pairwise alignment The process of lining up two or more sequences to achieve maximal levels of identity (and conservation, in the case of amino acid sequences) for the purpose of assessing the degree of similarity and the possibility of homology. Page 47

  32. Pairwise alignment of retinol-binding protein and b-lactoglobulin 1 MKWVWALLLLAAWAAAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEG 50 RBP . ||| | . |. . . | : .||||.:| : 1 ...MKCLLLALALTCGAQALIVT..QTMKGLDIQKVAGTWYSLAMAASD. 44 lactoglobulin 51 LFLQDNIVAEFSVDETGQMSATAKGRVR.LLNNWD..VCADMVGTFTDTE 97 RBP : | | | | :: | .| . || |: || |. 45 ISLLDAQSAPLRV.YVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTK 93 lactoglobulin 98 DPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAV...........QYSC 136 RBP || ||. | :.|||| | . .| 94 IPAVFKIDALNENKVL........VLDTDYKKYLLFCMENSAEPEQSLAC 135 lactoglobulin 137 RLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQ.EELCLARQYRLIV 185 RBP . | | | : || . | || | 136 QCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI....... 178 lactoglobulin Internal gap Terminal gap Page 46

  33. Gaps • Positions at which a letter is paired with a null are called gaps. • Gap scores are typically negative. • Since a single mutational event may cause the insertion or deletion of more than one residue, the presence of a gap is ascribed more significance than the length of the gap. Thus there are separate penalties for gap creation and gap extension. • In BLAST, it is rarely necessary to change gap values from the default.

  34. Pairwise alignment of retinol-binding protein and b-lactoglobulin 1 MKWVWALLLLAAWAAAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEG 50 RBP . ||| | . |. . . | : .||||.:| : 1 ...MKCLLLALALTCGAQALIVT..QTMKGLDIQKVAGTWYSLAMAASD. 44 lactoglobulin 51 LFLQDNIVAEFSVDETGQMSATAKGRVR.LLNNWD..VCADMVGTFTDTE 97 RBP : | | | | :: | .| . || |: || |. 45 ISLLDAQSAPLRV.YVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTK 93 lactoglobulin 98 DPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAV...........QYSC 136 RBP || ||. | :.|||| | . .| 94 IPAVFKIDALNENKVL........VLDTDYKKYLLFCMENSAEPEQSLAC 135 lactoglobulin 137 RLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQ.EELCLARQYRLIV 185 RBP . | | | : || . | || | 136 QCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI....... 178 lactoglobulin

  35. Pairwise alignment of retinol-binding protein from human (top) and rainbow trout (O. mykiss) 1 .MKWVWALLLLA.AWAAAERDCRVSSFRVKENFDKARFSGTWYAMAKKDP 48 :: || || || .||.||. .| :|||:.|:.| |||.||||| 1 MLRICVALCALATCWA...QDCQVSNIQVMQNFDRSRYTGRWYAVAKKDP 47 . . . . . 49 EGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTED 98 |||| ||:||:|||||.|.|.||| ||| :||||:.||.| ||| || | 48 VGLFLLDNVVAQFSVDESGKMTATAHGRVIILNNWEMCANMFGTFEDTPD 97 . . . . . 99 PAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADS 148 ||||||:||| ||:|| ||||||::||||| ||: |||| ..||||| | 98 PAKFKMRYWGAASYLQTGNDDHWVIDTDYDNYAIHYSCREVDLDGTCLDG 147 . . . . . 149 YSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRSERNLL 199 |||:||| | || || |||| :..|:| .|| : | |:|: 148 YSFIFSRHPTGLRPEDQKIVTDKKKEICFLGKYRRVGHTGFCESS...... 192

  36. Pairwise sequence alignment allows us to look back billions of years ago (BYA) Origin of life Earliest fossils Origin of eukaryotes Eukaryote/ archaea Fungi/animal Plant/animal insects 4 3 2 1 0 Page 48

  37. Multiple sequence alignment of glyceraldehyde 3-phosphate dehydrogenases fly GAKKVIISAP SAD.APM..F VCGVNLDAYK PDMKVVSNAS CTTNCLAPLA human GAKRVIISAP SAD.APM..F VMGVNHEKYD NSLKIISNAS CTTNCLAPLA plant GAKKVIISAP SAD.APM..F VVGVNEHTYQ PNMDIVSNAS CTTNCLAPLA bacterium GAKKVVMTGP SKDNTPM..F VKGANFDKY. AGQDIVSNAS CTTNCLAPLA yeast GAKKVVITAP SS.TAPM..F VMGVNEEKYT SDLKIVSNAS CTTNCLAPLA archaeon GADKVLISAP PKGDEPVKQL VYGVNHDEYD GE.DVVSNAS CTTNSITPVA fly KVINDNFEIV EGLMTTVHAT TATQKTVDGP SGKLWRDGRG AAQNIIPAST human KVIHDNFGIV EGLMTTVHAI TATQKTVDGP SGKLWRDGRG ALQNIIPAST plant KVVHEEFGIL EGLMTTVHAT TATQKTVDGP SMKDWRGGRG ASQNIIPSST bacterium KVINDNFGII EGLMTTVHAT TATQKTVDGP SHKDWRGGRG ASQNIIPSST yeast KVINDAFGIE EGLMTTVHSL TATQKTVDGP SHKDWRGGRT ASGNIIPSST archaeon KVLDEEFGIN AGQLTTVHAY TGSQNLMDGP NGKP.RRRRA AAENIIPTST fly GAAKAVGKVI PALNGKLTGM AFRVPTPNVS VVDLTVRLGK GASYDEIKAK human GAAKAVGKVI PELNGKLTGM AFRVPTANVS VVDLTCRLEK PAKYDDIKKV plant GAAKAVGKVL PELNGKLTGM AFRVPTSNVS VVDLTCRLEK GASYEDVKAA bacterium GAAKAVGKVL PELNGKLTGM AFRVPTPNVS VVDLTVRLEK AATYEQIKAA yeast GAAKAVGKVL PELQGKLTGM AFRVPTVDVS VVDLTVKLNK ETTYDEIKKV archaeon GAAQAATEVL PELEGKLDGM AIRVPVPNGS ITEFVVDLDD DVTESDVNAA Page 49

  38. Multiple sequence alignment of human lipocalin paralogs ~~~~~EIQDVSGTWYAMTVDREFPEMNLESVTPMTLTTL.GGNLEAKVTM lipocalin 1 LSFTLEEEDITGTWYAMVVDKDFPEDRRRKVSPVKVTALGGGNLEATFTF odorant-binding protein 2a TKQDLELPKLAGTWHSMAMATNNISLMATLKAPLRVHITSEDNLEIVLHR progestagen-assoc. endo. VQENFDVNKYLGRWYEIEKIPTTFENGRCIQANYSLMENGNQELRADGTV apolipoprotein D VKENFDKARFSGTWYAMAKDPEGLFLQDNIVAEFSVDETGNWDVCADGTF retinol-binding protein LQQNFQDNQFQGKWYVVGLAGNAI.LREDKDPQKMYATIDKSYNVTSVLF neutrophil gelatinase-ass. VQPNFQQDKFLGRWFSAGLASNSSWLREKKAALSMCKSVDGGLNLTSTFL prostaglandin D2 synthase VQENFNISRIYGKWYNLAIGSTCPWMDRMTVSTLVLGEGEAEISMTSTRW alpha-1-microglobulin PKANFDAQQFAGTWLLVAVGSACRFLQRAEATTLHVAPQGSTFRKLD... complement component 8 Page 49

  39. General approach to pairwise alignment • Choose two sequences • Select an algorithm that generates a score • Allow gaps (insertions, deletions) • Score reflects degree of similarity • Alignments can be global or local • Estimate probability that the alignment • occurred by chance

  40. Calculation of an alignment score

  41. Where we’re heading in the next 10 minutes: creating a set of “scoring matrices” that let us assign scores for each aligned amino acid in a pairwise alignment. What should the score be when a serine matches a serine, or a threonine, or a valine? Can we devise “lenient” scoring systems to help us align distantly related proteins, and more conservative scoring systems to align closely related proteins?

  42. lys found at 58% of arg sites Emile Zuckerkandl and Linus Pauling (1965) considered substitution frequencies in 18 globins (myoglobins and hemoglobins from human to lamprey). Black: identity Gray: very conservative substitutions (>40% occurrence) White: fairly conservative substitutions (>21% occurrence) Red: no substitutions observed Page 80

  43. Page 80

  44. Dayhoff’s 34 protein superfamilies Accepted point mutations ProteinPAMs per 100 million years Ig kappa chain 37 Kappa casein 33 Lactalbumin 27 Hemoglobin a 12 Myoglobin 8.9 Insulin 4.4 Histone H4 0.10 Ubiquitin 0.00 400 fold From 1978 Page 50

  45. Pairwise alignment of human (NP_005203) versus mouse (NP_031812) ubiquitin

  46. Multiple sequence alignment of glyceraldehyde 3-phosphate dehydrogenases fly GAKKVIISAP SAD.APM..F VCGVNLDAYK PDMKVVSNAS CTTNCLAPLA human GAKRVIISAP SAD.APM..F VMGVNHEKYD NSLKIISNAS CTTNCLAPLA plant GAKKVIISAP SAD.APM..F VVGVNEHTYQ PNMDIVSNAS CTTNCLAPLA bacterium GAKKVVMTGP SKDNTPM..F VKGANFDKY. AGQDIVSNAS CTTNCLAPLA yeast GAKKVVITAP SS.TAPM..F VMGVNEEKYT SDLKIVSNAS CTTNCLAPLA archaeon GADKVLISAP PKGDEPVKQL VYGVNHDEYD GE.DVVSNAS CTTNSITPVA fly KVINDNFEIV EGLMTTVHAT TATQKTVDGP SGKLWRDGRG AAQNIIPAST human KVIHDNFGIV EGLMTTVHAI TATQKTVDGP SGKLWRDGRG ALQNIIPAST plant KVVHEEFGIL EGLMTTVHAT TATQKTVDGP SMKDWRGGRG ASQNIIPSST bacterium KVINDNFGII EGLMTTVHAT TATQKTVDGP SHKDWRGGRG ASQNIIPSST yeast KVINDAFGIE EGLMTTVHSL TATQKTVDGP SHKDWRGGRT ASGNIIPSST archaeon KVLDEEFGIN AGQLTTVHAY TGSQNLMDGP NGKP.RRRRA AAENIIPTST fly GAAKAVGKVI PALNGKLTGM AFRVPTPNVS VVDLTVRLGK GASYDEIKAK human GAAKAVGKVI PELNGKLTGM AFRVPTANVS VVDLTCRLEK PAKYDDIKKV plant GAAKAVGKVL PELNGKLTGM AFRVPTSNVS VVDLTCRLEK GASYEDVKAA bacterium GAAKAVGKVL PELNGKLTGM AFRVPTPNVS VVDLTVRLEK AATYEQIKAA yeast GAAKAVGKVL PELQGKLTGM AFRVPTVDVS VVDLTVKLNK ETTYDEIKKV archaeon GAAQAATEVL PELEGKLDGM AIRVPVPNGS ITEFVVDLDD DVTESDVNAA

  47. Dayhoff’s numbers of “accepted point mutations”: what amino acid substitutions occur in proteins? From closely related protein sequences (at least 85% identity) Numbers of APM, multiplied by 10, in 1572 cases of amino acid substitutions from closely related sequences