An introduction to multiple alignments

Transcription

1 An introduction to multiple alignments original version by Cédric Notredame, updated by Laurent Falquet Overview! Multiple alignments! How-to, Goal, problems, use! Patterns! PROSITE database, syntax, use! PSI-BLAST! BLAST, matrices, use! [ Profiles/HMMs ]

2 Overview! What are multiple alignments?! How can I use my alignments?! How does the computer align the sequences?! The progressive alignment algorithm! What are the difficulties?! Pre-requisite?! How can we compare sequences?! How can we align sequences? Sometimes two sequences are not enough The man with TWO watches NEVER knows the exact time

3 What is a multiple sequence alignment?! What can it do for me?! How can I produce one of these?! How can I use it? chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : What is a multiple sequence alignment?! Structural/biochemical criteria! Residues playing a similar role end up in the same column.! Evolution criteria! Residues having the same ancestor end up in the same column. chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. :

4 How can I use a multiple alignment? chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP unknown -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM unknown AKDDRIRYDNEMKSWEEQMAE * :.*. : Extrapolation Less Than 30 % id BUT Conserved where it MATTERS Unkown Sequence Homology? SwissProt

5 How can I use a multiple alignment? chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : Extrapolation Prosite Patterns P-K-R-[PA]-x(1)-[ST] How can I use a multiple alignment? chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-IQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : L? K>R Extrapolation Prosite Patterns Prosite Profiles A F D E F G H Q I V L W -More Sensitive -More Specific

6 PROSITE profile (see also HMMs) A Substitution Cost For Every Amino Acid, At Every Position How can I use a multiple alignment? chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : Phylogeny chite wheat trybr mouse -Evolution -Paralogy/Orthology

7 How can I use a multiple alignment? chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : Phylogeny Struc. Prediction Column Constraint " Evolution Constraint " Structure Constraint How can I use a multiple alignment? chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : Phylogeny Struc. Prediction PsiPred or PhD For secondary Structure Prediction: 75% Accurate. Threading: is improving but is not yet as good.

8 How can I use a multiple alignment? chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : Phylogeny Struc. Prediction Caution! Automatic Multiple Sequence Alignment methods are not always perfect

9 The problem! why is it difficult to compute a multiple sequence alignment? Biology What is a good alignment? chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * Computation What is the good alignment? The problem! why is it difficult to compute a multiple sequence alignment? CIRCULAR PROBLEM... Good Sequences Good Alignment

10 The problem! Same as pairwise alignment problem! We do NOT know how sequences evolve.! We do NOT understand the relation between structures and sequences.! We would NOT recognize the correct alignment if we had it IN FRONT of our eyes The Charlie Chaplin paradox

11 What do I need to know to make a good multiple alignment?! How do sequences evolve?! How does the computer align the sequences?! How can I choose my sequences?! What is the best program?! How can I use my alignment? An alignment is a story ADKPKRPLSAYMLWLN Insertion Deletion ADKPRRP---LS-YMLWLN ADKPKRPKPRLSAYMLWLN Mutation ADKPKRPLSAYMLWLN Mutations + Selection ADKPKRPLSAYMLWLN ADKPRRPLS-YMLWLN ADKPKRPKPRLSAYMLWLN

12 Homology! Same sequences -> same origin? -> same function? - > same 3D fold? %Sequence Identity Same 3D Fold 30% Twilight Zone Length 100 Residues and mutations! All residues are equal, but some more than others Aliphatic Aromatic L V I F M C P A G G T C S Y H K D N E W Q R Small Hydrophobic Polar Accurate matrices are data driven rather than knowledge driven

13 Substitution matrices Different Flavors: Pam: 250, 350 Blosum: 45, 62 What is the best substitution matrix?! Mutation rates depend on families Family S N Histone Insulin Interleukin I !"Globin Apolipoprot. AI Interferon G Rates in Substitutions/site/Billion Years as measured on Mouse Vs Human (0.08 Billion years)! Choosing the right matrix may be tricky! Gonnet250 > BLOSUM62 > PAM250! Depends on the family, the program used and its tuning

14 Insertions and deletions? Indel Cost Affine Gap Penalty Cost=GOP+GEP*L L Cost Cost L L How to align many sequences?! Exact algorithms are computing time consuming! Needlemann & Wunsch! Smith & Waterman 2 Globins =>1 sec

15 How to align many sequences?! Exact algorithms are computing time consuming! Needlemann & Wunsch! Smith & Waterman 3 Globins =>2 mn How to align many sequences?! Exact algorithms are computing time consuming! Needlemann & Wunsch! Smith & Waterman! -> heuristic wished 4 Globins =>5 hours

16 How to align many sequences?! Exact algorithms are computing time consuming! Needlemann & Wunsch! Smith & Waterman! -> heuristic really wished! 5 Globins =>3 weeks How to align many sequences?! Exact algorithms are computing time consuming! Needlemann & Wunsch! Smith & Waterman! -> heuristic required! 6 Globins =>9 years

17 How to align many sequences?! Exact algorithms are computing time consuming! Needlemann & Wunsch! Smith & Waterman! -> heuristic definitely required! 7 Globins =>1000 years Existing methods 1-Carillo and Lipman: -MSA, DCA. -Few Small Closely Related Sequence. -Do Well When They Can Run. 4-Progressive: -ClustalW, Pileup, Multalign - Fast and Sensitive 2-Segment Based: 3-Iterative: -DIALIGN, MACAW. -May Align Too Few Residues -HMMs, HMMER, SAM. -Slow, Sometimes Inacurate -Good Profile Generators 5-Mixtures: -T-Coffee, MAFFT, MUSCLE, ProbCons, Psi-Praline, - Very sensitive

18 Progressive alignment Feng and Dolittle, 1980; Taylor 1981 Dynamic Programming Using A Substitution Matrix Progressive alignment Feng and Dolittle, 1980; Taylor Depends on the CHOICE of the sequences. -Depends on the ORDER of the sequences (Tree). -Depends on the PARAMETERS: Substitution Matrix. Penalties (Gop, Gep). Sequence Weight. Tree making Algorithm.

19 Progressive alignment! Works well when phylogeny is dense! No outlayer sequence! Example: river crossing Selecting sequences from a BLAST output

20 A common mistake!!! Sequences too closely related PRVA_MACFU PRVA_HUMAN PRVA_GERSP PRVA_MOUSE PRVA_RAT PRVA_RABIT SMTDLLNAEDIKKAVGAFSAIDSFDHKKFFQMVGLKKKSADDVKKVFHILDKDKSGFIEE SMTDLLNAEDIKKAVGAFSATDSFDHKKFFQMVGLKKKSADDVKKVFHMLDKDKSGFIEE SMTDLLSAEDIKKAIGAFAAADSFDHKKFFQMVGLKKKTPDDVKKVFHILDKDKSGFIEE SMTDVLSAEDIKKAIGAFAAADSFDHKKFFQMVGLKKKNPDEVKKVFHILDKDKSGFIEE SMTDLLSAEDIKKAIGAFTAADSFDHKKFFQMVGLKKKSADDVKKVFHILDKDKSGFIEE AMTELLNAEDIKKAIGAFAAAESFDHKKFFQMVGLKKKSTEDVKKVFHILDKDKSGFIEE :**::*.*******:***:* :****************..::******:*********** PRVA_MACFU PRVA_HUMAN PRVA_GERSP PRVA_MOUSE PRVA_RAT PRVA_RABIT DELGFILKGFSPDARDLSAKETKTLMAAGDKDGDGKIGVDEFSTLVAES DELGFILKGFSPDARDLSAKETKMLMAAGDKDGDGKIGVDEFSTLVAES DELGFILKGFSSDARDLSAKETKTLLAAGDKDGDGKIGVEEFSTLVSES DELGSILKGFSSDARDLSAKETKTLLAAGDKDGDGKIGVEEFSTLVAES DELGSILKGFSSDARDLSAKETKTLMAAGDKDGDGKIGVEEFSTLVAES EELGFILKGFSPDARDLSVKETKTLMAAGDKDGDGKIGADEFSTLVSES :*** ******.******.**** *:************.:******:** Identical sequences brings no information Multiple sequence alignments thrive on diversity

21 Respect information! PRVA_MACFU PRVA_HUMAN PRVA_GERSP PRVA_MOUSE PRVA_RAT PRVA_RABIT TPCC_MOUSE PRVA_MACFU PRVA_HUMAN PRVA_GERSP PRVA_MOUSE PRVA_RAT PRVA_RABIT TPCC_MOUSE PRVA_MACFU PRVA_HUMAN PRVA_GERSP PRVA_MOUSE PRVA_RAT PRVA_RABIT TPCC_MOUSE SMTDLLN----AEDIKKA SMTDLLN----AEDIKKA SMTDLLS----AEDIKKA SMTDVLS----AEDIKKA SMTDLLS----AEDIKKA AMTELLN----AEDIKKA MDDIYKAAVEQLTEEQKNEFKAAFDIFVLGAEDGCISTKELGKVMRMLGQNPTPEELQEM : :*..*:::: VGAFSAIDS--FDHKKFFQMVG------LKKKSADDVKKVFHILDKDKSGFIEEDELGFI VGAFSATDS--FDHKKFFQMVG------LKKKSADDVKKVFHMLDKDKSGFIEEDELGFI IGAFAAADS--FDHKKFFQMVG------LKKKTPDDVKKVFHILDKDKSGFIEEDELGFI IGAFAAADS--FDHKKFFQMVG------LKKKNPDEVKKVFHILDKDKSGFIEEDELGSI IGAFTAADS--FDHKKFFQMVG------LKKKSADDVKKVFHILDKDKSGFIEEDELGSI IGAFAAAES--FDHKKFFQMVG------LKKKSTEDVKKVFHILDKDKSGFIEEEELGFI IDEVDEDGSGTVDFDEFLVMMVRCMKDDSKGKSEEELSDLFRMFDKNADGYIDLDELKMM :.. *.*..:*: *: * *. :::..:*:::**:.*:*: :** : -This alignment is not informative about the relation between TPCC MOUSE and the rest of the sequences. -A better spread of the sequences is needed Selecting diverse sequences PRVB_CYPCA PRVB_BOACO PRV1_SALSA PRVB_LATCH PRVB_RANES PRVA_MACFU PRVA_ESOLU PRVB_CYPCA PRVB_BOACO PRV1_SALSA PRVB_LATCH PRVB_RANES PRVA_MACFU PRVA_ESOLU -AFAGVLNDADIAAALEACKAADSFNHKAFFAKVGLTSKSADDVKKAFAIIDQDKSGFIE -AFAGILSDADIAAGLQSCQAADSFSCKTFFAKSGLHSKSKDQLTKVFGVIDRDKSGYIE MACAHLCKEADIKTALEACKAADTFSFKTFFHTIGFASKSADDVKKAFKVIDQDASGFIE -AVAKLLAAADVTAALEGCKADDSFNHKVFFQKTGLAKKSNEELEAIFKILDQDKSGFIE -SITDIVSEKDIDAALESVKAAGSFNYKIFFQKVGLAGKSAADAKKVFEILDRDKSGFIE -SMTDLLNAEDIKKAVGAFSAIDSFDHKKFFQMVGLKKKSADDVKKVFHILDKDKSGFIE --AKDLLKADDIKKALDAVKAEGSFNHKKFFALVGLKAMSANDVKKVFKAIDADASGFIE : *:.:..*.:*. * ** *: * : * :* * **:** LKGFSPDARDLSAKETKTLMAAGDKDGDGKIGVDEFSTLVAES- LKGFSPDARDLSAKETKMLMAAGDKDGDGKIGVDEFSTLVAES- LKGFSSDARDLSAKETKTLLAAGDKDGDGKIGVEEFSTLVSES- LKGFSSDARDLSAKETKTLLAAGDKDGDGKIGVEEFSTLVAES- LKGFSSDARDLSAKETKTLMAAGDKDGDGKIGVEEFSTLVAES- LKGFSPDARDLSVKETKTLMAAGDKDGDGKIGADEFSTLVSES- LQ---ATGETITEDDIEELMKDGDKNNDGRIDYDEFLEFMKGVE *:... ::.: : *: ***:.**:*. :** :: EDELKLFLQNFKADARALTDGETKTFLKAGDSDGDGKIGVDEFTALVKA- EDELKKFLQNFDGKARDLTDKETAEFLKEGDTDGDGKIGVEEFVVLVTKG VEELKLFLQNFCPKARELTDAETKAFLKAGDADGDGMIGIDEFAVLVKQ- DEELELFLQNFSAGARTLTKTETETFLKAGDSDGDGKIGVDEFQKLVKA- QDELGLFLQNFRASARVLSDAETSAFLKAGDSDGDGKIGVEEFQALVKA- EDELGFILKGFSPDARDLSAKETKTLMAAGDKDGDGKIGVDEFSTLVAES EEELKFVLKSFAADGRDLTDAETKAFLKAADKDGDGKIGIDEFETLVHEA :**.*:.*.* *: ** ::.* **** **::** ** -A REASONABLE model now exists. -Going further:remote homologues.

22 Aligning remote homologues PRVA_MACFU PRVA_ESOLU PRVB_CYPCA PRVB_BOACO PRV1_SALSA PRVB_LATCH PRVB_RANES TPCS_RABIT TPCS_PIG TPCC_MOUSE PRVA_MACFU PRVA_ESOLU PRVB_CYPCA PRVB_BOACO PRV1_SALSA PRVB_LATCH PRVB_RANES TPCS_RABIT TPCS_PIG TPCC_MOUSE SMTDLLNA----EDIKKA AKDLLKA----DDIKKA AFAGVLND----ADIAAA AFAGILSD----ADIAAG MACAHLCKE----ADIKTA AVAKLLAA----ADVTAA SITDIVSE----KDIDAA -TDQQAEARSYLSEEMIAEFKAAFDMFDADGG-GDISVKELGTVMRMLGQTPTKEELDAI -TDQQAEARSYLSEEMIAEFKAAFDMFDADGG-GDISVKELGTVMRMLGQTPTKEELDAI MDDIYKAAVEQLTEEQKNEFKAAFDIFVLGAEDGCISTKELGKVMRMLGQNPTPEELQEM : :: VGAFSAIDS--FDHKKFFQMVG------LKKKSADDVKKVFHILDKDKSGFIEEDELGFI LDAVKAEGS--FNHKKFFALVG------LKAMSANDVKKVFKAIDADASGFIEEEELKFV LEACKAADS--FNHKAFFAKVG------LTSKSADDVKKAFAIIDQDKSGFIEEDELKLF LQSCQAADS--FSCKTFFAKSG------LHSKSKDQLTKVFGVIDRDKSGYIEEDELKKF LEACKAADT--FSFKTFFHTIG------FASKSADDVKKAFKVIDQDASGFIEVEELKLF LEGCKADDS--FNHKVFFQKTG------LAKKSNEELEAIFKILDQDKSGFIEDEELELF LESVKAAGS--FNYKIFFQKVG------LAGKSAADAKKVFEILDRDKSGFIEQDELGLF IEEVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELAECFRIFDRNADGYIDAEELAEI IEEVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELAECFRIFDRNMDGYIDAEELAEI IDEVDEDGSGTVDFDEFLVMMVRCMKDDSKGKSEEELSDLFRMFDKNADGYIDLDELKMM :..:... *: * : * :* :.*:*: :**. Going further PRVA_MACFU PRVB_BOACO PRV1_SALSA TPCS_RABIT TPCS_PIG TPCC_MOUSE TPC_PATYE PRVA_MACFU PRVB_BOACO PRV1_SALSA TPCS_RABIT TPCS_PIG TPCC_MOUSE TPC_PATYE VGAFSAIDS--FDHKKFFQMVG------LKKKSADDVKKVFHILDKDKSGFIEEDELGFI LQSCQAADS--FSCKTFFAKSG------LHSKSKDQLTKVFGVIDRDKSGYIEEDELKKF LEACKAADT--FSFKTFFHTIG------FASKSADDVKKAFKVIDQDASGFIEVEELKLF IEEVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELAECFRIFDRNADGYIDAEELAEI IEEVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELAECFRIFDRNMDGYIDAEELAEI IDEVDEDGSGTVDFDEFLVMMVRCMKDDSKGKSEEELSDLFRMFDKNADGYIDLDELKMM SDEMDEEATGRLNCDAWIQLFER---KLKEDLDERELKEAFRVLDKEKKGVIKVDVLRWI. :... ::. : * :* :.* *. : *. PRVA_MACFU LKGFSPDARDLSAKETKTLMAAGDKDGDGKIGVDEFSTLVAES- PRVA_ESOLU LKSFAADGRDLTDAETKAFLKAADKDGDGKIGIDEFETLVHEA- PRVB_CYPCA LQNFKADARALTDGETKTFLKAGDSDGDGKIGVDEFTALVKA-- PRVB_BOACO LQNFDGKARDLTDKETAEFLKEGDTDGDGKIGVEEFVVLVTKG- PRV1_SALSA LQNFCPKARELTDAETKAFLKAGDADGDGMIGIDEFAVLVKQ-- PRVB_LATCH LQNFSAGARTLTKTETETFLKAGDSDGDGKIGVDEFQKLVKA-- PRVB_RANES LQNFRASARVLSDAETSAFLKAGDSDGDGKIGVEEFQALVKA-- TPCS_RABIT FR---ASGEHVTDEEIESLMKDGDKNNDGRIDFDEFLKMMEGVQ TPCS_PIG Swiss Institute FR---ASGEHVTDEEIESIMKDGDKNNDGRIDFDEFLKMMEGVQ of Bioinf ormatics TPCC_MOUSE Institut Suisse LQ---ATGETITEDDIEELMKDGDKNNDGRIDYDEFLEFMKGVE de Bioinf ormatique ::.. :: : ::.* :.** *. :** :: LKGFSPDARDLSAKETKTLMAAGDKDGDGKIGVDEFSTLVAES-- LQNFDGKARDLTDKETAEFLKEGDTDGDGKIGVEEFVVLVTKG-- LQNFCPKARELTDAETKAFLKAGDADGDGMIGIDEFAVLVKQ--- FR---ASGEHVTDEEIESLMKDGDKNNDGRIDFDEFLKMMEGVQ- FR---ASGEHVTDEEIESIMKDGDKNNDGRIDFDEFLKMMEGVQ- LQ---ATGETITEDDIEELMKDGDKNNDGRIDYDEFLEFMKGVE- LS---SLGDELTEEEIENMIAETDTDGSGTVDYEEFKCLMMSSDA :. :: : :: * :..* :. :** ::

23 What makes a good alignment! The more divergent the sequences, the better! The fewer indels, the better! Nice ungapped blocks separated with indels! Different classes of residues within a block:! Completely conserved (*)! Size and hydropathy conserved (:)! Size or hydropathy conserved (.)! The ultimate evaluation is a matter of personal judgment and knowledge Avoiding pitfalls

24 Naming your sequences the right way! Never use white spaces in your sequence names! Never use special symbols. Stick to plain letters, numbers and the underscore sign (_) to replace spaces. Avoid ALL other signs, especially the most tempting ones #,, *, >, <! Never use names longer than 15 characters! Never give the same name to 2 different sequences in your set. Some programs accept it, others like ClustalW don t. Do not use too many sequences!

25 Beware of Repeats! There is a problem when two sequences do not contain the same number of repeats! It is then better to manually extract the repeats and to align them separately. Individual repeats can be recognized using Dotlet or Dotter. Keep a biological perspective chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : chite AD--K----PKR-PLYMLWLNS-ARESIKRENPDFK-VT-EVAKKGGELWRGLwheat -DPNK----PKRAP-FFVFMGE-FREEFKQKNPKNKSVA-AVGKAAGERWKSLS trybr -K--KDSNAPKR-AMT-MFFSSDFR-S-KH-S-DLS-IV-EMSKAAGAAWKELG mouse ----K----PKR-PRYNIYVSESFQEA-K--D-D-S-AQGKL-KLVNEAWKNLS * ***.:: ::... : *... : *. *: * DIFFERENT PARAMETERS chite KSEWEAKAATAKQNY-I--RALQE-YERNG-Gwheat KAPYVAKANKLKGEY-N--KAIAA-YNK-GESA trybr RKVYEEMAEKDKERY----K--RE-M mouse KQAYIQLAKDDRIRYDNEMKSWEEQMAE----- : : * :.* :

26 Do not overtune!!! chite ---ADKPKRPLSAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAPSAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPRSAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. :::.:... :.. *. *: * chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : chite ---ADKPKRPL-SAYMLWLNSARESIKRENPDFK-VTEVAKKGGELWRGLKD wheat --DPNKPKRAP-SAFFVFMGEFREEFKQKNPKNKSVAAVGKAAGERWKSLSE trybr KKDSNAPKRAMTSFMFFSSDFRS-----KHSDLS-IVEMSKAAGAAWKELGP mouse -----KPKRPR-SAYNIYVSESFQ----EAKDDS-AQGKLKLVNEAWKNLSP ***. *.:... :.. *. *: * DO NOT PLAY WITH PARAMETERS! IF YOU KNOW THE ALIGNMENT YOU WANT: MAKE IT YOURSELF! chite AATAKQNYIRALQEYERNGGwheat ANKLKGEYNKAIAAYNKGESA trybr AEKDKERYKREM mouse AKDDRIRYDNEMKSWEEQMAE * :.*. : BaliBase classification and benchmark PROBLEM Description Even Phylogenic Spread. One Outlayer Sequence Two Distantly related Groups Long Internal Indel Long Terminal Indel

27 Choosing the right method Source: BaliBase Thompson et al, NAR, (1999) Do et al, Genome Res. (2005) PROBLEM Program Strategy ClustalW, T-coffee, MUSCLE, ProbCons T-Coffee, MUSCLE, ProbCons ProbCons, MUSCLE, MAFFT Dialign II, ProbCons, T-Coffee Dialign II, ProbCons, MAFFT Some interesting links

28 More links! MUSCLE! MAFFT! PROBCONS! PSI-PRALINE! 3D-COFFEE! Conclusion! The best alignment method:! Your brain! The right data! The best evaluation method:! Your eyes! Experimental information (SwissProt)! Choosing the sequences well is important! Beware of repeated elements! What can I conclude?! Homology -> information extrapolation! How can I go further?! Patterns! Profiles! HMMs!