1 / 58

Similarity searches in sequence databases

Introduction to Bioinformatics. Similarity searches in sequence databases. Bioinformatics. Sequence search algorithms. Matching a sequence against a database . Example of utilization

eavan
Download Presentation

Similarity searches in sequence databases

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Introduction to Bioinformatics Similarity searches in sequence databases

  2. Bioinformatics Sequence search algorithms

  3. Matching a sequence against a database • Example of utilization • We obtained the sequence of a protein of unknown function, and we would like to search UniProt for all similar proteins, in order to emit hypotheses about the possible function (function prediction by similarity). • Approach: we will align our query sequence to each entry in UniProt. • Problem of size : in Dec 2005, there were 2.500.000 entries in UniProt (Swiss-Prot + TREMBL) • It is possible to apply dynamical programming, but it takes a lot of time or requires high computation power.

  4. Fast algorithms for database matching • FastA • BLAST (Basic Local Alignment Search Tool) • In short • These algorithms are ~50 times faster than Smith-Waterman • They cannot guarantee the optimal solution • However, a comparison with results obtained by dynamical programming has shown that FastA answer is close to the optimum

  5. FastA strategy MVD VDF DFY YYL FYY ... 1 2 3 pos 12345678901234567890123456789012 seq MVDFYYLPGSSMVDVFDFYAKAVGVELNKKLL position index k-tuple positions MVD 1, 12 VDF 2 DFY 3,17 ... ... 3-tuples • FastA builds an index with the positions of all the small words (k-tuples) found in the query sequence. • The program then detects diagonals of k-tuples between the query and the database sequences. • When a significant diagonal is detected, the two sequences are aligned with Smith-Waterman algorithm. • The size of words (k) influences the behaviour of the program: when k increases, the search is faster but one might miss some matches.

  6. Principle of the FastA strategy • Left • Comparison of k-mer positions between query and database sequences. • Center • Highest-density regions ("init regions") are identified. • The best one is highlighted with a star. • Regions with a score below a given threshold appear in dotted lines, for illustation purpose. • Right • Low-scoring regions are filtered out, and the remaining regions are joined. Source: Mount (2000)

  7. BLAST strategies (Altschul et al., 1990; 1997) • Version 1 (1990): gapless BLAST • Prior indexing of all k-mers (words) in the database (formatdb). • When search submitted, builds a dictionary of k-mers found in the query sequence. • Uses a substitution matrix (e.g. BLOSUM) to calculate a score between these words and each possible word of the same length. • Only retains word pairs with sufficient score (threshold on word pair scores). • Each time a word pair from the dictionary passes the threshold (hit), extends it in both directions (without gaps), to obtain a High-scoring Segment Pair (HSP). • The program returns sequences with significant high-scoring segment pairs. • Valeurs par défaut des tailles de mots • Pour les séquences d’acides nucléiques k=11 (cf illustration) • Pour les séquences peptidiques, k=3 • Exercice • Calculer la probabilité de trouver un HSP de taille 11 à une position donnée, dans une séquences nucléique composée de nucléotides équiprobables. GGTAGCAAATGTCCTGTCTGTACTGTACATGGTCAAACTGGTGAAT ||||||||||| ||||||||||| TGTATCAAATGTCCTGTGTGAATGGTAGATGGTCAAACTGGTCAAT

  8. BLAST strategy (gapless version, Altschul et al., 1990) • Version 1 (1990) • Builds a dictionary of k-tuples (small words) found in the query sequence. • Uses a substitution matrix (e.g. BLOSUM) to calculate a score between these words and each possible word of the same length. • Only retains the words with a high score. • Each time a pair of words from the dictionary are found (hits) in the database sequence, extends the hit in both direction (without gaps), to obtain a High-scoring Segment Pair (HSP). • The program returns sequences with significant high-scoring segment pairs. • High scoring pairs (HSP) • For nucleic acid sequences, default k=11 (illustration) • For peptidic sequences, default k=3 • Exercise: compute the probability to find a 11bp HSP starting at a given position, in a nucleic acid sequence with equiprobable nucleotides. GGTAGCAAATGTCCTGTCTGTACTGTACATGGTCAAACTGGTGAAT ||||||||||| ||||||||||| TGTATCAAATGTCCTGTGTGAATGGTAGATGGTCAAACTGGTCAAT

  9. BLAST - Elongation de l’alignement Score d’identité = 1 Score de substitution = -1 The quick brown fox jumps over the lazy dog The quiet brown cat purrs when she sees him

  10. BLAST - Elongation de l’alignement Score d’identité = 1 Score de substitution = -1 The quick brown fox jump The quiet brown cat purr 123 45654 56789 876 5654 <= SCORE

  11. BLAST - Elongation de l’alignement Score d’identité = 1 Score de substitution = -1 The quick brown fox jump The quiet brown cat purr 123 45654 56789 876 5654 <= SCORE 000 00012 10000 123 4345 <=(SCORE(max)-SCORE) L’élongation s’arrête si (SCORE(max)-SCORE) > limite prédéfinie

  12. BLAST - Elongation de l’alignement HSP (High Scoring Pair): The quick brown ||||||| |||||| The quiet brown The quick brown fox jump The quiet brown cat purr 123 45654 56789 876 5654 <= SCORE 000 00012 10000 123 4345 <=(SCORE(max)-SCORE) Ecourter l’alignement jusqu’au dernier Score(max)

  13. BLAST - Elongation de l’alignement • Elongation de l’alignement de deux côtés à partir des mots du dictionnaire • L’élongation s’arrête si le score diminue en-deçà d’une limite prédéfinie par rapport au dernier maximum. • L’alignement est écourté jusqu’au dernier score maximal • (S) => HSP (High Scoring Pair)

  14. BLAST - Exercice • Faites un alignement local entre ces deux séquences en suivant l’algorithme de BLAST version1 • Scores • Identité: 1 • Substitution: -1 • Différence maximale entre le score actuel et le score maximal: 5 TAAATGGTCATGTGATGGTCCTGACTGATGCTGCCTGA GAAATGGTCATGTGATGGTCGTAACGATGCAATTGGGC

  15. BLAST - Exercice • Faites un alignement local entre ces deux séquences en suivant l’algorithme de BLAST version1 • Scores • Identité: 1 • Substitution: -1 • Différence maximale entre le score actuel et le score maximal: 5 TAAATGGTCATGTGATGGTCCTGACTGATGCTGCCTGA GAAATGGTCATGTGATGGTCGTAACGATGCAATTGGGC Noyau

  16. BLAST - Exercice • Faites un alignement local entre ces deux séquences en suivant l’algorithme de BLAST version1 • Scores • Identité: 1 • Substitution: -1 • Différence maximale entre le score actuel et le score maximal: 5 TAAATGGTCATGTGATGGTCCTGACTGATGCTGCCTGA Seq1 GAAATGGTCATGTGATGGTCGTAACGATGCAATTGGGC Seq2 12345678911111111111111211111 Score 01234567898989098765

  17. BLAST - Exercice • Faites un alignement local entre ces deux séquences en suivant l’algorithme de BLAST version1 • Scores • Identité: 1 • Substitution: -1 • Différence maximale entre le score actuel et le score maximal: 5 TAAATGGTCATGTGATGGTCCTGACTGATGCTGCCTGA Seq1 GAAATGGTCATGTGATGGTCGTAACGATGCAATTGGGC Seq2 12345678911111111111111211111 Score 01234567898989098765 00000000000000000001010012345 Score(max)-Score

  18. BLAST - Exercice • Faites un alignement local entre ces deux séquences en suivant l’algorithme de BLAST version1 • Scores • Identité: 1 • Substitution: -1 • Différence maximale entre le score actuel et le score maximal: 5 TAAATGGTCATGTGATGGTCCTGACTGATGCTGCCTGA Seq1 ||||||||||||||||||| | || GAAATGGTCATGTGATGGTCGTAACGATGCAATTGGGC Seq2 12345678911111111111111211111 Score 01234567898989098765 00000000000000000001010012345 Score(max)-Score

  19. BLAST strategies (Altschul et al., 1990; 1997) • Version 1 (1990): gapless BLAST • Prior indexing of all k-mers (words) in the database (formatdb). • When search submitted, builds a dictionary of k-mers found in the query sequence. • Uses a substitution matrix (e.g. BLOSUM) to calculate a score between these words and each possible word of the same length. • Only retains word pairs with sufficient score (threshold on word pair scores). • Each time a word pair from the dictionary passes the threshold (hit), extends it in both directions (without gaps), to obtain a High-scoring Segment Pair (HSP). • The program returns sequences with significant high-scoring segment pairs. • Version 2 (1997): gapped BLAST • Use smaller words, but only extend when there are two hits on the same diagonal. • Extension includes gaps (dynamical programming). • The extension costs more time, but the number of times it is done is reduced, because the extension requires a pair of hits. • Exercise • Compute the probability to find a hit pair starting at a given position, with a spacing comprised between 0 and 30, in a nucleic acid sequence with equiprobable nucleotides.

  20. BLAST strategies (Altschul et al., 1990; 1997) • Version 1 (1990): gapless BLAST • Prior indexing of all k-mers (words) in the database (formatdb). • When search submitted, builds a dictionary of k-mers found in the query sequence. • Uses a substitution matrix (e.g. BLOSUM) to calculate a score between these words and each possible word of the same length. • Only retains word pairs with sufficient score (threshold on word pair scores). • Each time a word pair from the dictionary passes the threshold (hit), extends it in both directions (without gaps), to obtain a High-scoring Segment Pair (HSP). • The program returns sequences with significant high-scoring segment pairs. • Version 2 (1997): gapped BLAST • Use smaller words, but only extend when there are two hits on the same diagonal. • Extension includes gaps (dynamical programming). • The extension costs more time, but the number of times it is done is reduced, because the extension requires a pair of hits. • PSI-BLAST (in the 1997 article as well) • A second step after the proper BLAST process. • Once the gapped BLAST has returned a set of sequences, these sequences are aligned and used to build a profile motif. • The database is then scanned with this profile motif to collect additional similarities. • The process can be iterated several times • collect sequences > build a profile -> collect sequences -> build a profile ...

  21. Some traps for BLAST searches • Spurious domains • Some domains are found in many proteins. This does not mean that these proteins have the same function. The width of the alignment should thus be analyzed to assess whether the alignment covers most of the sequence length, or only a small segment. • Low complexity regions (repetitive sequences). • return multiple matches with no apparent functional relationship. • Cloning vectors • Some entries in the database contain a fragment of the cloning vector. This can return many apparent matches, where the matching region is restricted to the cloning vector.

  22. Bioinformatics Statistics of sequence similarities

  23. Matching statistics - raw score • The raw score is computed by summing the scores (obtained from the substitution matrix) for each pair of residues (r1,i and r2,i) over the length of the alignment (L). R L A S V E T D M P L T L R Q H T L T S L Q T T L K A H L G T H

  24. Matching statistics - raw score • The raw score is computed by summing the scores (obtained from the substitution matrix) for each pair of residues (r1,i and r2,i) over the length of the alignment (L). R L A S V E T D M P L T L R Q H . | . | : : | . : . . . | . . | T L T S L Q T T L K A H L G T H -1 +4 +0 +4 +1 +2 +5 -1 +2 -1 -1 -2 +4 -2 -1 +8 = 21

  25. MSP-wise P-value and bit score • The P-value of a matching segment pair (MSP) with score Sis the probability to observe a score of at least S by chance. • Karlin and Altschul (1990) defined a way to calculate the P-value of an MSP. • The P-value follows an exponential distribution, with two parameters : lambda and K. These two parameters depend on the substitution matrix chosen. They have thus to be estimated for each substitution matrix separately. • The analytic way to determine the parameters lambda and L is only valid for gapless alignments. • For alignment with gaps, Altschul et al (1997) propose to estimate these parameters on the basis of empirical observations • Bit score of an MSP • Karlin and Altschul (1990) also propose to convert the raw score S into a bit score S’. • This facilitates the interpretation of the score, because the P-value can be directly calculated from the bit score, irrespective of the substitution matrix used for the alignment. .

  26. Matching statistics- the E-value • Let us imagine that we align a random word with another random word. The score is likely to be generally low. • However, if this is repeated billions of times, some high scores will occasionally occur by chance. • In a database scan, each word of the query sequence is compared to each word of the database. • For a query sequence of size m and a database of size n, the search space (number of word pair comparisons) is thus N=nm. • FastA and BLAST estimate, for each score, the number of matches that would be expected by chance, given the size of the database. This is called the E-value. • The E-value is the product of the nominal P-value (i.e. the risk of false positives for a single comparison) by the size of the search space. • For a given score S, the expected number of random matches thus increases with the size of the database.

  27. Threshold on E-value • The lower is the E-value, the more significant is the match. • High E-value ( > 1) indicate that the match should not be trusted too much. • One essential parameter of FastA and BLAST is the threshold on E-value. • Very low values (e.g.: 1e-21) • Indicate that the match is very unlikely to result from chance. • It is thus likely that it results from a common ancestry between the aligned sequences. • In such case, we can thus admit the hypothesis of homology. • Beware • On the BLAST Web server at NCBI, the default threshold value is 10 • This means that each query would return ~10 matches by chance alone. • If this default value is used, we already know that the answer is likely to contain ~10false positive.

  28. Matching statistics - database-wise P-value (=Family-Wise Error Rate) • From the E-value (E), one can estimate the probability to observe at least X matches by chance in random sequences. • This is a simple application of the Poisson distribution : calculate the probability to observe X occurrences of an event whilst expecting E. • A particular case is the probability to observe at least one match by chance • P(X>=1) • This probability is generally called Family-Wise Error Rate (FWER). • In the case of similarity searches, one can call it database-wise P-value. • This P-value represents the probability to find at least one spurious match in the whole database search, with a score greater or equal to S.

  29. Distribution of probability for matching scores Source: W.P.Pearson (2000). Protein sequence comparison and Protein evolution. ISMB Tutorial. • When one performs a database similarity search, the distribution of scores follows an extreme value distribution. This distribution is asymmetric, and should thus not be modelled with a normal (Gaussian) distribution.

  30. Interpreting similarity search results

  31. Score distribution • The histogram shows the number of database matches for each score. • For scores higher than 92, the number of matches is very small. • A higher resolution histogram is shown besides the main histogram. • Asterisks (*) represent the random expectation (E-value) for each score zoom FastA output from Pearson (2000)

  32. BLAST result • The text shows the result of a BLAST search, • Query: the E.coli protein MetL, a bifunctional enzyme combining aspartokinase and homoserine dehydrogenase activities. • Database: all proteins from Escherichia coli K12. • The BLAST result file starts with a summary of • the parameters used for the search • The matching sequences and the score of each match. BLASTP 2.2.6 [Apr-09-2003] Reference: Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402. Query= metL gi|16131778|ref|NP_418375.1| aspartokinase II and homoserine dehydrogenase II; bifunctional: aspartokinase II (N-terminal); homoserine dehydrogenase II (C-terminal) [Escherichia coli K12] (810 letters) Database: /Users/jvanheld/rsa- tools/data/genomes/Escherichia_coli_K12/genome/NC_000913.faa 4242 sequences; 1,351,322 total letters Searching.........done Score E Sequences producing significant alignments: (bits) Value gi|16131778|ref|NP_418375.1| aspartokinase II and homoserine deh... 1596 0.0 gi|16127996|ref|NP_414543.1| bifunctional: aspartokinase I (N-te... 344 2e-95 gi|16131850|ref|NP_418448.1| aspartokinase III, lysine sensitive... 122 7e-29 gi|16128228|ref|NP_414777.1| gamma-glutamate kinase [Escherichia... 31 0.28 >gi|16131778|ref|NP_418375.1| aspartokinase II and homoserine dehydrogenase II; bifunctional: aspartokinase II (N-terminal); homoserine dehydrogenase II (C-terminal) [Escherichia coli K12] Length = 810 Score = 1596 bits (4132), Expect = 0.0 Identities = 810/810 (100%), Positives = 810/810 (100%) Query: 1 MSVIAQAGAKGRQLHKFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINW 60 MSVIAQAGAKGRQLHKFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINW Sbjct: 1 MSVIAQAGAKGRQLHKFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINW 60 Query: 61 LKLSQTDRLSAHQVQQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDA 120 LKLSQTDRLSAHQVQQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDA Sbjct: 61 LKLSQTDRLSAHQVQQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDA 120 Query: 121 VYAEVVGHGEVWSARLMSAVLNQQGLPAAWLDAREFLRAERAAQPQVDEGLSYPLLQQLL 180 VYAEVVGHGEVWSARLMSAVLNQQGLPAAWLDAREFLRAERAAQPQVDEGLSYPLLQQLL Sbjct: 121 VYAEVVGHGEVWSARLMSAVLNQQGLPAAWLDAREFLRAERAAQPQVDEGLSYPLLQQLL 180 Query: 181 VQHPGKRLVVTGFISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADP 240 VQHPGKRLVVTGFISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADP Sbjct: 181 VQHPGKRLVVTGFISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADP 240 Query: 241 RKVKDACLLPLLRLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRIER 300 RKVKDACLLPLLRLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRIER Sbjct: 241 RKVKDACLLPLLRLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRIER 300 Query: 301 VLASGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLL 360 VLASGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLL Sbjct: 301 VLASGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLL 360 Query: 361 QFCYTSEVADSALKILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPV 420 QFCYTSEVADSALKILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPV Sbjct: 361 QFCYTSEVADSALKILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPV 420 Query: 421 EFTWQSDDGISLVAVLRTGPTESLIQGLHQSVFRAEKRIGLVLFGKGNIGSRWLELFARE 480 EFTWQSDDGISLVAVLRTGPTESLIQGLHQSVFRAEKRIGLVLFGKGNIGSRWLELFARE Sbjct: 421 EFTWQSDDGISLVAVLRTGPTESLIQGLHQSVFRAEKRIGLVLFGKGNIGSRWLELFARE 480 Query: 481 QSTLSARTGFEFVLAGVVDSRRSLLSYDGLDASRALAFFNDEAVEQDEESLFLWMRAHPY 540 QSTLSARTGFEFVLAGVVDSRRSLLSYDGLDASRALAFFNDEAVEQDEESLFLWMRAHPY Sbjct: 481 QSTLSARTGFEFVLAGVVDSRRSLLSYDGLDASRALAFFNDEAVEQDEESLFLWMRAHPY 540 Query: 541 DDLVVLDVTASQQLADQYLDFASHGFHVISANKLAGASDSNKYRQIHDAFEKTGRHWLYN 600 DDLVVLDVTASQQLADQYLDFASHGFHVISANKLAGASDSNKYRQIHDAFEKTGRHWLYN Sbjct: 541 DDLVVLDVTASQQLADQYLDFASHGFHVISANKLAGASDSNKYRQIHDAFEKTGRHWLYN 600 Query: 601 ATVGAGLPINHTVRDLIDSGDTILSISGIFSGTLSWLFLQFDGSVPFTELVDQAWQQGLT 660 ATVGAGLPINHTVRDLIDSGDTILSISGIFSGTLSWLFLQFDGSVPFTELVDQAWQQGLT Sbjct: 601 ATVGAGLPINHTVRDLIDSGDTILSISGIFSGTLSWLFLQFDGSVPFTELVDQAWQQGLT 660 Query: 661 EPDPRDDLSGKDVMRKLVILAREAGYNIEPDQVRVESLVPAHCEGGSIDHFFENGDELNE 720 EPDPRDDLSGKDVMRKLVILAREAGYNIEPDQVRVESLVPAHCEGGSIDHFFENGDELNE Sbjct: 661 EPDPRDDLSGKDVMRKLVILAREAGYNIEPDQVRVESLVPAHCEGGSIDHFFENGDELNE 720 Query: 721 QMVQRLEAAREMGLVLRYVARFDANGKARVGVEAVREDHPLASLLPCDNVFAIESRWYRD 780 QMVQRLEAAREMGLVLRYVARFDANGKARVGVEAVREDHPLASLLPCDNVFAIESRWYRD Sbjct: 721 QMVQRLEAAREMGLVLRYVARFDANGKARVGVEAVREDHPLASLLPCDNVFAIESRWYRD 780 Query: 781 NPLVIRGPGAGRDVTAGAIQSDINRLAQLL 810 NPLVIRGPGAGRDVTAGAIQSDINRLAQLL Sbjct: 781 NPLVIRGPGAGRDVTAGAIQSDINRLAQLL 810 >gi|16127996|ref|NP_414543.1| bifunctional: aspartokinase I (N-terminal); homoserine dehydrogenase I (C-terminal) [Escherichia coli K12] Length = 820 Score = 344 bits (882), Expect = 2e-95 Identities = 247/821 (30%), Positives = 410/821 (49%), Gaps = 44/821 (5%) Query: 16 KFGGSSLADVKCYLRVAGIMAEYSQPDDMM-VVSAAGSTTNQLINWLKLSQTDRLSAHQV 74 KFGG+S+A+ + +LRVA I+ ++ + V+SA TN L+ ++ + + + + + Sbjct: 5 KFGGTSVANAERFLRVADILESNARQGQVATVLSAPAKITNHLVAMIEKTISGQDALPNI 64 Query: 75 QQTLRRYQCDLISGLLPAEEADSL--ISAFVSDLERLAALLDSGIN------DAVYAEVV 126 R + +L++GL A+ L + FV + GI+ D++ A ++ Sbjct: 65 SDAERIF-AELLTGLAAAQPGFPLAQLKTFVDQEFAQIKHVLHGISLLGQCPDSINAALI 123 Query: 127 GHGEVWSARLMSAVLNQQGLPAAWLDAREFLRAER---AAQPQVDEGLSYPLLQQLLVQH 183 GE S +M+ VL +G +D E L A + + E ++ H Sbjct: 124 CRGEKMSIAIMAGVLEARGHNVTVIDPVEKLLAVGHYLESTVDIAESTRRIAASRIPADH 183 Query: 184 PGKRLVVTGFISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKV 243 +++ GF + N GE V+LGRNGSDYSA + A IW+DV GVY+ DPR+V Sbjct: 184 ---MVLMAGFTAGNEKGELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTCDPRQV 240 Query: 244 KDACLLPLLRLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQ-----GSTRI 298 DA LL + EA EL+ A VLH RT+ P++ +I ++ + P G++R Sbjct: 241 PDARLLKSMSYQEAMELSYFGAKVLHPRTITPIAQFQIPCLIKNTGNPQAPGTLIGASRD 300 Query: 299 ERVLASGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQ 358 E L + +++ +++ + P + + + RA++ + + + Sbjct: 301 EDELP----VKGISNLNNMAMFSVSGPGMKGMVGMAARVFAAMSRARISVVLITQSSSEY 356 Query: 359 LLQFCYTSEVADSALKILDEA-------GLPGELRLRQGLALVAMVGAGVTRNPLHCHRF 411 + FC A + + E GL L + + LA++++VG G+ +F Sbjct: 357 SISFCVPQSDCVRAERAMQEEFYLELKEGLLEPLAVTERLAIISVVGDGMRTLRGISAKF 416 Query: 412 WQQLKGQPVEFTW--QSDDGISLVAVLRTGPTESLIQGLHQSVFRAEKRIGLVLFGKGNI 469 + L + Q S+ V+ + ++ HQ +F ++ I + + G G + Sbjct: 417 FAALARANINIVAIAQGSSERSISVVVNNDDATTGVRVTHQMLFNTDQVIEVFVIGVGGV 476 Query: 470 GSRWLELFAREQSTLSARTGFEFVLAGVVDSRRSLLSYDGLDASRALAFFNDEAVEQDEE 529 G LE R+QS L + + + GV +S+ L + GL+ L + +E + E Sbjct: 477 GGALLEQLKRQQSWLKNKH-IDLRVCGVANSKALLTNVHGLN----LENWQEELAQAKEP 531 Query: 530 ----SLFLWMRAHPYDDLVVLDVTASQQLADQYLDFASHGFHVISANKLAGASDSNKYRQ 585 L ++ + + V++D T+SQ +ADQY DF GFHV++ NK A S + Y Q Sbjct: 532 FNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQYADFLREGFHVVTPNKKANTSSMDYYHQ 591 Query: 586 IHDAFEKTGRHWLYNATVGAGLPINHTVRDLIDSGDTILSISGIFSGTLSWLFLQFDGSV 645 + A EK+ R +LY+ VGAGLP+ +++L+++GD ++ SGI SG+LS++F + D + Sbjct: 592 LRYAAEKSRRKFLYDTNVGAGLPVIENLQNLLNAGDELMKFSGILSGSLSYIFGKLDEGM 651 Query: 646 PFTELVDQAWQQGLTEPDPRDDLSGKDVMRKLVILAREAGYNIEPDQVRVESLVPAHCEG 705 F+E A + G TEPDPRDDLSG DV RKL+ILARE G +E + +E ++PA Sbjct: 652 SFSEATTLAREMGYTEPDPRDDLSGMDVARKLLILARETGRELELADIEIEPVLPAEFNA 711 Query: 706 -GSIDHFFENGDELNEQMVQRLEAAREMGLVLRYVARFDANGKARVGVEAVREDHPLASL 764 G + F N +L++ R+ AR+ G VLRYV D +G RV + V + PL + Sbjct: 712 EGDVAAFMANLSQLDDLFAARVAKARDEGKVLRYVGNIDEDGVCRVKIAEVDGNDPLFKV 771 Query: 765 LPCDNVFAIESRWYRDNPLVIRGPGAGRDVTAGAIQSDINR 805 +N A S +Y+ PLV+RG GAG DVTA + +D+ R Sbjct: 772 KNGENALAFYSHYYQPLPLVLRGYGAGNDVTAAGVFADLLR 812 >gi|16131850|ref|NP_418448.1| aspartokinase III, lysine sensitive; aspartokinase III, lysine-sensitive [Escherichia coli K12] Length = 449 Score = 122 bits (307), Expect = 7e-29 Identities = 121/452 (26%), Positives = 194/452 (42%), Gaps = 25/452 (5%) Query: 16 KFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINWLK-LSQTDRLSAHQV 74 KFGG+S+AD R A I+ + ++V+SA+ TN L+ + L +R + Sbjct: 8 KFGGTSVADFDAMNRSADIVLSDANVR-LVVLSASAGITNLLVALAEGLEPGERF---EK 63 Query: 75 QQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDAVYAEVVGHGEVWSA 134 +R Q ++ L I + ++ LA + A+ E+V HGE+ S Sbjct: 64 LDAIRNIQFAILERLRYPNVIREEIERLLENITVLAEAAALATSPALTDELVSHGELMST 123 Query: 135 RLMSAVLNQQGLPAAWLDAREFLRA-ERAAQPQVDEGLSYPLLQQLLVQHPGKRLVVT-G 192 L +L ++ + A W D R+ +R +R + + D L L+ + LV+T G Sbjct: 124 LLFVEILRERDVQAQWFDVRKVMRTNDRFGRAEPDIAALAELAALQLLPRLNEGLVITQG 183 Query: 193 FISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKVKDACLLPLL 252 FI N G T LGR GSDY+A + SRV IW+DV G+Y+ DPR V A + + Sbjct: 184 FIGSENKGRTTTLGRGGSDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEI 243 Query: 253 RLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRI---------ERVLA 303 EA+E+A A VLH TL P S+I + + S P G T + R LA Sbjct: 244 AFAEAAEMATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFRALA 303 Query: 304 SGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLLQFC 363 ++T H L A LA I L +A+ L Sbjct: 304 LRRNQTLLTLHSLNMLHSRGFLAEVFGILARHNISVDLITTSEVSVAL-------TLDTT 356 Query: 364 YTSEVADSAL--KILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPVE 421 ++ D+ L +L E + + +GLALVA++G +++ + L+ + Sbjct: 357 GSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKACGVGKEVFGVLEPFNIR 416 Query: 422 FTWQSDDGISLVAVLRTGPTESLIQGLHQSVF 453 +L ++ E ++Q LH ++F Sbjct: 417 MICYGASSHNLCFLVPGEDAEQVVQKLHSNLF 448 >gi|16128228|ref|NP_414777.1| gamma-glutamate kinase [Escherichia coli K12] Length = 367 Score = 31.2 bits (69), Expect = 0.28 Identities = 17/56 (30%), Positives = 29/56 (51%) Query: 194 ISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKVKDACLL 249 I+ N+A T + +D + LAG ++ + +D G+Y+ADPR A L+ Sbjct: 133 INENDAVATAEIKVGDNDNLSALAAILAGADKLLLLTDQKGLYTADPRSNPQAELI 188 Database: /Users/jvanheld/rsa- tools/data/genomes/Escherichia_coli_K12/genome/NC_000913.faa Posted date: Sep 8, 2004 12:13 PM Number of letters in database: 1,351,322 Number of sequences in database: 4242 Lambda K H 0.320 0.136 0.397 Gapped Lambda K H 0.267 0.0410 0.140 Matrix: BLOSUM62 Gap Penalties: Existence: 11, Extension: 1 Number of Hits to DB: 2,199,628 Number of Sequences: 4242 Number of extensions: 96525 Number of successful extensions: 290 Number of sequences better than 1.0: 4 Number of HSP's better than 1.0 without gapping: 4 Number of HSP's successfully gapped in prelim test: 0 Number of HSP's that attempted gapping in prelim test: 279 Number of HSP's gapped (non-prelim): 5 length of query: 810 length of database: 1,351,322 effective HSP length: 92 effective length of query: 718 effective length of database: 961,058 effective search space: 690039644 effective search space used: 690039644 T: 11 A: 40 X1: 16 ( 7.4 bits) X2: 38 (14.6 bits) X3: 64 (24.7 bits) S1: 41 (21.8 bits) S2: 65 (29.6 bits)

  33. BLAST result - first match • The first match is the query sequence itself (metL). This is not surprising since we scanned the set of all E.coli proteins with a protein from E.coli. • The E-value (0) means that, with this level of similarity; one would expect 0 false positive by chance. >gi|16131778|ref|NP_418375.1| aspartokinase II and homoserine dehydrogenase II; bifunctional: aspartokinase II (N-terminal); homoserine dehydrogenase II (C-terminal) [Escherichia coli K12] Length = 810 Score = 1596 bits (4132), Expect = 0.0 Identities = 810/810 (100%), Positives = 810/810 (100%) Query: 1 MSVIAQAGAKGRQLHKFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINW 60 MSVIAQAGAKGRQLHKFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINW Sbjct: 1 MSVIAQAGAKGRQLHKFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINW 60 Query: 61 LKLSQTDRLSAHQVQQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDA 120 LKLSQTDRLSAHQVQQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDA Sbjct: 61 LKLSQTDRLSAHQVQQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDA 120 Query: 121 VYAEVVGHGEVWSARLMSAVLNQQGLPAAWLDAREFLRAERAAQPQVDEGLSYPLLQQLL 180 VYAEVVGHGEVWSARLMSAVLNQQGLPAAWLDAREFLRAERAAQPQVDEGLSYPLLQQLL Sbjct: 121 VYAEVVGHGEVWSARLMSAVLNQQGLPAAWLDAREFLRAERAAQPQVDEGLSYPLLQQLL 180 Query: 181 VQHPGKRLVVTGFISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADP 240 VQHPGKRLVVTGFISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADP Sbjct: 181 VQHPGKRLVVTGFISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADP 240 Query: 241 RKVKDACLLPLLRLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRIER 300 RKVKDACLLPLLRLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRIER Sbjct: 241 RKVKDACLLPLLRLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRIER 300 Query: 301 VLASGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLL 360 VLASGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLL Sbjct: 301 VLASGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLL 360 Query: 361 QFCYTSEVADSALKILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPV 420 QFCYTSEVADSALKILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPV Sbjct: 361 QFCYTSEVADSALKILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPV 420 Query: 421 EFTWQSDDGISLVAVLRTGPTESLIQGLHQSVFRAEKRIGLVLFGKGNIGSRWLELFARE 480 EFTWQSDDGISLVAVLRTGPTESLIQGLHQSVFRAEKRIGLVLFGKGNIGSRWLELFARE Sbjct: 421 EFTWQSDDGISLVAVLRTGPTESLIQGLHQSVFRAEKRIGLVLFGKGNIGSRWLELFARE 480 Query: 481 QSTLSARTGFEFVLAGVVDSRRSLLSYDGLDASRALAFFNDEAVEQDEESLFLWMRAHPY 540 QSTLSARTGFEFVLAGVVDSRRSLLSYDGLDASRALAFFNDEAVEQDEESLFLWMRAHPY Sbjct: 481 QSTLSARTGFEFVLAGVVDSRRSLLSYDGLDASRALAFFNDEAVEQDEESLFLWMRAHPY 540 Query: 541 DDLVVLDVTASQQLADQYLDFASHGFHVISANKLAGASDSNKYRQIHDAFEKTGRHWLYN 600 DDLVVLDVTASQQLADQYLDFASHGFHVISANKLAGASDSNKYRQIHDAFEKTGRHWLYN Sbjct: 541 DDLVVLDVTASQQLADQYLDFASHGFHVISANKLAGASDSNKYRQIHDAFEKTGRHWLYN 600 Query: 601 ATVGAGLPINHTVRDLIDSGDTILSISGIFSGTLSWLFLQFDGSVPFTELVDQAWQQGLT 660 ATVGAGLPINHTVRDLIDSGDTILSISGIFSGTLSWLFLQFDGSVPFTELVDQAWQQGLT Sbjct: 601 ATVGAGLPINHTVRDLIDSGDTILSISGIFSGTLSWLFLQFDGSVPFTELVDQAWQQGLT 660 Query: 661 EPDPRDDLSGKDVMRKLVILAREAGYNIEPDQVRVESLVPAHCEGGSIDHFFENGDELNE 720 EPDPRDDLSGKDVMRKLVILAREAGYNIEPDQVRVESLVPAHCEGGSIDHFFENGDELNE Sbjct: 661 EPDPRDDLSGKDVMRKLVILAREAGYNIEPDQVRVESLVPAHCEGGSIDHFFENGDELNE 720 Query: 721 QMVQRLEAAREMGLVLRYVARFDANGKARVGVEAVREDHPLASLLPCDNVFAIESRWYRD 780 QMVQRLEAAREMGLVLRYVARFDANGKARVGVEAVREDHPLASLLPCDNVFAIESRWYRD Sbjct: 721 QMVQRLEAAREMGLVLRYVARFDANGKARVGVEAVREDHPLASLLPCDNVFAIESRWYRD 780 Query: 781 NPLVIRGPGAGRDVTAGAIQSDINRLAQLL 810 NPLVIRGPGAGRDVTAGAIQSDINRLAQLL Sbjct: 781 NPLVIRGPGAGRDVTAGAIQSDINRLAQLL 810 >gi|16127996|ref|NP_414543.1| bifunctional: aspartokinase I (N-terminal); homoserine dehydrogenase I (C-terminal) [Escherichia coli K12] Length = 820 Score = 344 bits (882), Expect = 2e-95 Identities = 247/821 (30%), Positives = 410/821 (49%), Gaps = 44/821 (5%) Query: 16 KFGGSSLADVKCYLRVAGIMAEYSQPDDMM-VVSAAGSTTNQLINWLKLSQTDRLSAHQV 74 KFGG+S+A+ + +LRVA I+ ++ + V+SA TN L+ ++ + + + + + Sbjct: 5 KFGGTSVANAERFLRVADILESNARQGQVATVLSAPAKITNHLVAMIEKTISGQDALPNI 64 Query: 75 QQTLRRYQCDLISGLLPAEEADSL--ISAFVSDLERLAALLDSGIN------DAVYAEVV 126 R + +L++GL A+ L + FV + GI+ D++ A ++ Sbjct: 65 SDAERIF-AELLTGLAAAQPGFPLAQLKTFVDQEFAQIKHVLHGISLLGQCPDSINAALI 123 Query: 127 GHGEVWSARLMSAVLNQQGLPAAWLDAREFLRAER---AAQPQVDEGLSYPLLQQLLVQH 183 GE S +M+ VL +G +D E L A + + E ++ H Sbjct: 124 CRGEKMSIAIMAGVLEARGHNVTVIDPVEKLLAVGHYLESTVDIAESTRRIAASRIPADH 183 Query: 184 PGKRLVVTGFISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKV 243 +++ GF + N GE V+LGRNGSDYSA + A IW+DV GVY+ DPR+V Sbjct: 184 ---MVLMAGFTAGNEKGELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTCDPRQV 240 Query: 244 KDACLLPLLRLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQ-----GSTRI 298 DA LL + EA EL+ A VLH RT+ P++ +I ++ + P G++R Sbjct: 241 PDARLLKSMSYQEAMELSYFGAKVLHPRTITPIAQFQIPCLIKNTGNPQAPGTLIGASRD 300 Query: 299 ERVLASGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQ 358 E L + +++ +++ + P + + + RA++ + + + Sbjct: 301 EDELP----VKGISNLNNMAMFSVSGPGMKGMVGMAARVFAAMSRARISVVLITQSSSEY 356 Query: 359 LLQFCYTSEVADSALKILDEA-------GLPGELRLRQGLALVAMVGAGVTRNPLHCHRF 411 + FC A + + E GL L + + LA++++VG G+ +F Sbjct: 357 SISFCVPQSDCVRAERAMQEEFYLELKEGLLEPLAVTERLAIISVVGDGMRTLRGISAKF 416 Query: 412 WQQLKGQPVEFTW--QSDDGISLVAVLRTGPTESLIQGLHQSVFRAEKRIGLVLFGKGNI 469 + L + Q S+ V+ + ++ HQ +F ++ I + + G G + Sbjct: 417 FAALARANINIVAIAQGSSERSISVVVNNDDATTGVRVTHQMLFNTDQVIEVFVIGVGGV 476 Query: 470 GSRWLELFAREQSTLSARTGFEFVLAGVVDSRRSLLSYDGLDASRALAFFNDEAVEQDEE 529 G LE R+QS L + + + GV +S+ L + GL+ L + +E + E Sbjct: 477 GGALLEQLKRQQSWLKNKH-IDLRVCGVANSKALLTNVHGLN----LENWQEELAQAKEP 531 Query: 530 ----SLFLWMRAHPYDDLVVLDVTASQQLADQYLDFASHGFHVISANKLAGASDSNKYRQ 585 L ++ + + V++D T+SQ +ADQY DF GFHV++ NK A S + Y Q Sbjct: 532 FNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQYADFLREGFHVVTPNKKANTSSMDYYHQ 591 Query: 586 IHDAFEKTGRHWLYNATVGAGLPINHTVRDLIDSGDTILSISGIFSGTLSWLFLQFDGSV 645 + A EK+ R +LY+ VGAGLP+ +++L+++GD ++ SGI SG+LS++F + D + Sbjct: 592 LRYAAEKSRRKFLYDTNVGAGLPVIENLQNLLNAGDELMKFSGILSGSLSYIFGKLDEGM 651 Query: 646 PFTELVDQAWQQGLTEPDPRDDLSGKDVMRKLVILAREAGYNIEPDQVRVESLVPAHCEG 705 F+E A + G TEPDPRDDLSG DV RKL+ILARE G +E + +E ++PA Sbjct: 652 SFSEATTLAREMGYTEPDPRDDLSGMDVARKLLILARETGRELELADIEIEPVLPAEFNA 711 Query: 706 -GSIDHFFENGDELNEQMVQRLEAAREMGLVLRYVARFDANGKARVGVEAVREDHPLASL 764 G + F N +L++ R+ AR+ G VLRYV D +G RV + V + PL + Sbjct: 712 EGDVAAFMANLSQLDDLFAARVAKARDEGKVLRYVGNIDEDGVCRVKIAEVDGNDPLFKV 771 Query: 765 LPCDNVFAIESRWYRDNPLVIRGPGAGRDVTAGAIQSDINR 805 +N A S +Y+ PLV+RG GAG DVTA + +D+ R Sbjct: 772 KNGENALAFYSHYYQPLPLVLRGYGAGNDVTAAGVFADLLR 812 >gi|16131850|ref|NP_418448.1| aspartokinase III, lysine sensitive; aspartokinase III, lysine-sensitive [Escherichia coli K12] Length = 449 Score = 122 bits (307), Expect = 7e-29 Identities = 121/452 (26%), Positives = 194/452 (42%), Gaps = 25/452 (5%) Query: 16 KFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINWLK-LSQTDRLSAHQV 74 KFGG+S+AD R A I+ + ++V+SA+ TN L+ + L +R + Sbjct: 8 KFGGTSVADFDAMNRSADIVLSDANVR-LVVLSASAGITNLLVALAEGLEPGERF---EK 63 Query: 75 QQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDAVYAEVVGHGEVWSA 134 +R Q ++ L I + ++ LA + A+ E+V HGE+ S Sbjct: 64 LDAIRNIQFAILERLRYPNVIREEIERLLENITVLAEAAALATSPALTDELVSHGELMST 123 Query: 135 RLMSAVLNQQGLPAAWLDAREFLRA-ERAAQPQVDEGLSYPLLQQLLVQHPGKRLVVT-G 192 L +L ++ + A W D R+ +R +R + + D L L+ + LV+T G Sbjct: 124 LLFVEILRERDVQAQWFDVRKVMRTNDRFGRAEPDIAALAELAALQLLPRLNEGLVITQG 183 Query: 193 FISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKVKDACLLPLL 252 FI N G T LGR GSDY+A + SRV IW+DV G+Y+ DPR V A + + Sbjct: 184 FIGSENKGRTTTLGRGGSDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEI 243 Query: 253 RLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRI---------ERVLA 303 EA+E+A A VLH TL P S+I + + S P G T + R LA Sbjct: 244 AFAEAAEMATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFRALA 303 Query: 304 SGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLLQFC 363 ++T H L A LA I L +A+ L Sbjct: 304 LRRNQTLLTLHSLNMLHSRGFLAEVFGILARHNISVDLITTSEVSVAL-------TLDTT 356 Query: 364 YTSEVADSAL--KILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPVE 421 ++ D+ L +L E + + +GLALVA++G +++ + L+ + Sbjct: 357 GSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKACGVGKEVFGVLEPFNIR 416 Query: 422 FTWQSDDGISLVAVLRTGPTESLIQGLHQSVF 453 +L ++ E ++Q LH ++F Sbjct: 417 MICYGASSHNLCFLVPGEDAEQVVQKLHSNLF 448 >gi|16128228|ref|NP_414777.1| gamma-glutamate kinase [Escherichia coli K12] Length = 367 Score = 31.2 bits (69), Expect = 0.28 Identities = 17/56 (30%), Positives = 29/56 (51%) Query: 194 ISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKVKDACLL 249 I+ N+A T + +D + LAG ++ + +D G+Y+ADPR A L+ Sbjct: 133 INENDAVATAEIKVGDNDNLSALAAILAGADKLLLLTDQKGLYTADPRSNPQAELI 188 Database: /Users/jvanheld/rsa- tools/data/genomes/Escherichia_coli_K12/genome/NC_000913.faa Posted date: Sep 8, 2004 12:13 PM Number of letters in database: 1,351,322 Number of sequences in database: 4242 Lambda K H 0.320 0.136 0.397 Gapped Lambda K H 0.267 0.0410 0.140 Matrix: BLOSUM62 Gap Penalties: Existence: 11, Extension: 1 Number of Hits to DB: 2,199,628 Number of Sequences: 4242 Number of extensions: 96525 Number of successful extensions: 290 Number of sequences better than 1.0: 4 Number of HSP's better than 1.0 without gapping: 4 Number of HSP's successfully gapped in prelim test: 0 Number of HSP's that attempted gapping in prelim test: 279 Number of HSP's gapped (non-prelim): 5 length of query: 810 length of database: 1,351,322 effective HSP length: 92 effective length of query: 718 effective length of database: 961,058 effective search space: 690039644 effective search space used: 690039644 T: 11 A: 40 X1: 16 ( 7.4 bits) X2: 38 (14.6 bits) X3: 64 (24.7 bits) S1: 41 (21.8 bits) S2: 65 (29.6 bits)

  34. BLAST result - second match • The second match is another bifunctional protein, product of the gene thrA. • This protein contains the same two domains as metA (aspartokinase and homoserine dehydrogenase). • The alignment covers almost the complete sequences (820 aa), with 30% identities and 49% similarity. • The E-value is very low (2e-95), indicating that thrA and metL are likely to be true homologs. >gi|16127996|ref|NP_414543.1| bifunctional: aspartokinase I (N-terminal); homoserine dehydrogenase I (C-terminal) [Escherichia coli K12] Length = 820 Score = 344 bits (882), Expect = 2e-95 Identities = 247/821 (30%), Positives = 410/821 (49%), Gaps = 44/821 (5%) Query: 16 KFGGSSLADVKCYLRVAGIMAEYSQPDDMM-VVSAAGSTTNQLINWLKLSQTDRLSAHQV 74 KFGG+S+A+ + +LRVA I+ ++ + V+SA TN L+ ++ + + + + + Sbjct: 5 KFGGTSVANAERFLRVADILESNARQGQVATVLSAPAKITNHLVAMIEKTISGQDALPNI 64 Query: 75 QQTLRRYQCDLISGLLPAEEADSL--ISAFVSDLERLAALLDSGIN------DAVYAEVV 126 R + +L++GL A+ L + FV + GI+ D++ A ++ Sbjct: 65 SDAERIF-AELLTGLAAAQPGFPLAQLKTFVDQEFAQIKHVLHGISLLGQCPDSINAALI 123 Query: 127 GHGEVWSARLMSAVLNQQGLPAAWLDAREFLRAER---AAQPQVDEGLSYPLLQQLLVQH 183 GE S +M+ VL +G +D E L A + + E ++ H Sbjct: 124 CRGEKMSIAIMAGVLEARGHNVTVIDPVEKLLAVGHYLESTVDIAESTRRIAASRIPADH 183 Query: 184 PGKRLVVTGFISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKV 243 +++ GF + N GE V+LGRNGSDYSA + A IW+DV GVY+ DPR+V Sbjct: 184 ---MVLMAGFTAGNEKGELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTCDPRQV 240 Query: 244 KDACLLPLLRLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQ-----GSTRI 298 DA LL + EA EL+ A VLH RT+ P++ +I ++ + P G++R Sbjct: 241 PDARLLKSMSYQEAMELSYFGAKVLHPRTITPIAQFQIPCLIKNTGNPQAPGTLIGASRD 300 Query: 299 ERVLASGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQ 358 E L + +++ +++ + P + + + RA++ + + + Sbjct: 301 EDELP----VKGISNLNNMAMFSVSGPGMKGMVGMAARVFAAMSRARISVVLITQSSSEY 356 Query: 359 LLQFCYTSEVADSALKILDEA-------GLPGELRLRQGLALVAMVGAGVTRNPLHCHRF 411 + FC A + + E GL L + + LA++++VG G+ +F Sbjct: 357 SISFCVPQSDCVRAERAMQEEFYLELKEGLLEPLAVTERLAIISVVGDGMRTLRGISAKF 416 Query: 412 WQQLKGQPVEFTW--QSDDGISLVAVLRTGPTESLIQGLHQSVFRAEKRIGLVLFGKGNI 469 + L + Q S+ V+ + ++ HQ +F ++ I + + G G + Sbjct: 417 FAALARANINIVAIAQGSSERSISVVVNNDDATTGVRVTHQMLFNTDQVIEVFVIGVGGV 476 Query: 470 GSRWLELFAREQSTLSARTGFEFVLAGVVDSRRSLLSYDGLDASRALAFFNDEAVEQDEE 529 G LE R+QS L + + + GV +S+ L + GL+ L + +E + E Sbjct: 477 GGALLEQLKRQQSWLKNKH-IDLRVCGVANSKALLTNVHGLN----LENWQEELAQAKEP 531 Query: 530 ----SLFLWMRAHPYDDLVVLDVTASQQLADQYLDFASHGFHVISANKLAGASDSNKYRQ 585 L ++ + + V++D T+SQ +ADQY DF GFHV++ NK A S + Y Q Sbjct: 532 FNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQYADFLREGFHVVTPNKKANTSSMDYYHQ 591 Query: 586 IHDAFEKTGRHWLYNATVGAGLPINHTVRDLIDSGDTILSISGIFSGTLSWLFLQFDGSV 645 + A EK+ R +LY+ VGAGLP+ +++L+++GD ++ SGI SG+LS++F + D + Sbjct: 592 LRYAAEKSRRKFLYDTNVGAGLPVIENLQNLLNAGDELMKFSGILSGSLSYIFGKLDEGM 651 Query: 646 PFTELVDQAWQQGLTEPDPRDDLSGKDVMRKLVILAREAGYNIEPDQVRVESLVPAHCEG 705 F+E A + G TEPDPRDDLSG DV RKL+ILARE G +E + +E ++PA Sbjct: 652 SFSEATTLAREMGYTEPDPRDDLSGMDVARKLLILARETGRELELADIEIEPVLPAEFNA 711 Query: 706 -GSIDHFFENGDELNEQMVQRLEAAREMGLVLRYVARFDANGKARVGVEAVREDHPLASL 764 G + F N +L++ R+ AR+ G VLRYV D +G RV + V + PL + Sbjct: 712 EGDVAAFMANLSQLDDLFAARVAKARDEGKVLRYVGNIDEDGVCRVKIAEVDGNDPLFKV 771 Query: 765 LPCDNVFAIESRWYRDNPLVIRGPGAGRDVTAGAIQSDINR 805 +N A S +Y+ PLV+RG GAG DVTA + +D+ R Sbjct: 772 KNGENALAFYSHYYQPLPLVLRGYGAGNDVTAAGVFADLLR 812 >gi|16131850|ref|NP_418448.1| aspartokinase III, lysine sensitive; aspartokinase III, lysine-sensitive [Escherichia coli K12] Length = 449 Score = 122 bits (307), Expect = 7e-29 Identities = 121/452 (26%), Positives = 194/452 (42%), Gaps = 25/452 (5%) Query: 16 KFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINWLK-LSQTDRLSAHQV 74 KFGG+S+AD R A I+ + ++V+SA+ TN L+ + L +R + Sbjct: 8 KFGGTSVADFDAMNRSADIVLSDANVR-LVVLSASAGITNLLVALAEGLEPGERF---EK 63 Query: 75 QQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDAVYAEVVGHGEVWSA 134 +R Q ++ L I + ++ LA + A+ E+V HGE+ S Sbjct: 64 LDAIRNIQFAILERLRYPNVIREEIERLLENITVLAEAAALATSPALTDELVSHGELMST 123 Query: 135 RLMSAVLNQQGLPAAWLDAREFLRA-ERAAQPQVDEGLSYPLLQQLLVQHPGKRLVVT-G 192 L +L ++ + A W D R+ +R +R + + D L L+ + LV+T G Sbjct: 124 LLFVEILRERDVQAQWFDVRKVMRTNDRFGRAEPDIAALAELAALQLLPRLNEGLVITQG 183 Query: 193 FISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKVKDACLLPLL 252 FI N G T LGR GSDY+A + SRV IW+DV G+Y+ DPR V A + + Sbjct: 184 FIGSENKGRTTTLGRGGSDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEI 243 Query: 253 RLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRI---------ERVLA 303 EA+E+A A VLH TL P S+I + + S P G T + R LA Sbjct: 244 AFAEAAEMATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFRALA 303 Query: 304 SGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLLQFC 363 ++T H L A LA I L +A+ L Sbjct: 304 LRRNQTLLTLHSLNMLHSRGFLAEVFGILARHNISVDLITTSEVSVAL-------TLDTT 356 Query: 364 YTSEVADSAL--KILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPVE 421 ++ D+ L +L E + + +GLALVA++G +++ + L+ + Sbjct: 357 GSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKACGVGKEVFGVLEPFNIR 416 Query: 422 FTWQSDDGISLVAVLRTGPTESLIQGLHQSVF 453 +L ++ E ++Q LH ++F Sbjct: 417 MICYGASSHNLCFLVPGEDAEQVVQKLHSNLF 448 >gi|16128228|ref|NP_414777.1| gamma-glutamate kinase [Escherichia coli K12] Length = 367 Score = 31.2 bits (69), Expect = 0.28 Identities = 17/56 (30%), Positives = 29/56 (51%) Query: 194 ISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKVKDACLL 249 I+ N+A T + +D + LAG ++ + +D G+Y+ADPR A L+ Sbjct: 133 INENDAVATAEIKVGDNDNLSALAAILAGADKLLLLTDQKGLYTADPRSNPQAELI 188 Database: /Users/jvanheld/rsa- tools/data/genomes/Escherichia_coli_K12/genome/NC_000913.faa Posted date: Sep 8, 2004 12:13 PM Number of letters in database: 1,351,322 Number of sequences in database: 4242 Lambda K H 0.320 0.136 0.397 Gapped Lambda K H 0.267 0.0410 0.140 Matrix: BLOSUM62 Gap Penalties: Existence: 11, Extension: 1 Number of Hits to DB: 2,199,628 Number of Sequences: 4242 Number of extensions: 96525 Number of successful extensions: 290 Number of sequences better than 1.0: 4 Number of HSP's better than 1.0 without gapping: 4 Number of HSP's successfully gapped in prelim test: 0 Number of HSP's that attempted gapping in prelim test: 279 Number of HSP's gapped (non-prelim): 5 length of query: 810 length of database: 1,351,322 effective HSP length: 92 effective length of query: 718 effective length of database: 961,058 effective search space: 690039644 effective search space used: 690039644 T: 11 A: 40 X1: 16 ( 7.4 bits) X2: 38 (14.6 bits) X3: 64 (24.7 bits) S1: 41 (21.8 bits) S2: 65 (29.6 bits)

  35. BLAST result - third match • The third match is the product of the gene lysC: aspartokinase III. • This protein contains the aspartokinase domain, but not the homoserine dehydrogenase. • Consequently, the alignment only extends over the first half of the query protein (453aa). • On this segment, there is a good level of identity (26%) and similarity (42%). • The E-value is very low (7e–29), indicating that the two domains are likely to be true homologs. >gi|16131850|ref|NP_418448.1| aspartokinase III, lysine sensitive; aspartokinase III, lysine-sensitive [Escherichia coli K12] Length = 449 Score = 122 bits (307), Expect = 7e-29 Identities = 121/452 (26%), Positives = 194/452 (42%), Gaps = 25/452 (5%) Query: 16 KFGGSSLADVKCYLRVAGIMAEYSQPDDMMVVSAAGSTTNQLINWLK-LSQTDRLSAHQV 74 KFGG+S+AD R A I+ + ++V+SA+ TN L+ + L +R + Sbjct: 8 KFGGTSVADFDAMNRSADIVLSDANVR-LVVLSASAGITNLLVALAEGLEPGERF---EK 63 Query: 75 QQTLRRYQCDLISGLLPAEEADSLISAFVSDLERLAALLDSGINDAVYAEVVGHGEVWSA 134 +R Q ++ L I + ++ LA + A+ E+V HGE+ S Sbjct: 64 LDAIRNIQFAILERLRYPNVIREEIERLLENITVLAEAAALATSPALTDELVSHGELMST 123 Query: 135 RLMSAVLNQQGLPAAWLDAREFLRA-ERAAQPQVDEGLSYPLLQQLLVQHPGKRLVVT-G 192 L +L ++ + A W D R+ +R +R + + D L L+ + LV+T G Sbjct: 124 LLFVEILRERDVQAQWFDVRKVMRTNDRFGRAEPDIAALAELAALQLLPRLNEGLVITQG 183 Query: 193 FISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKVKDACLLPLL 252 FI N G T LGR GSDY+A + SRV IW+DV G+Y+ DPR V A + + Sbjct: 184 FIGSENKGRTTTLGRGGSDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEI 243 Query: 253 RLDEASELARLAAPVLHARTLQPVSGSEIDLQLRCSYTPDQGSTRI---------ERVLA 303 EA+E+A A VLH TL P S+I + + S P G T + R LA Sbjct: 244 AFAEAAEMATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFRALA 303 Query: 304 SGTGARIVTSHDDVCLIEFQVPASQDFKLAHKEIDQILKRAQVRPLAVGVHNDRQLLQFC 363 ++T H L A LA I L +A+ L Sbjct: 304 LRRNQTLLTLHSLNMLHSRGFLAEVFGILARHNISVDLITTSEVSVAL-------TLDTT 356 Query: 364 YTSEVADSAL--KILDEAGLPGELRLRQGLALVAMVGAGVTRNPLHCHRFWQQLKGQPVE 421 ++ D+ L +L E + + +GLALVA++G +++ + L+ + Sbjct: 357 GSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKACGVGKEVFGVLEPFNIR 416 Query: 422 FTWQSDDGISLVAVLRTGPTESLIQGLHQSVF 453 +L ++ E ++Q LH ++F Sbjct: 417 MICYGASSHNLCFLVPGEDAEQVVQKLHSNLF 448 >gi|16128228|ref|NP_414777.1| gamma-glutamate kinase [Escherichia coli K12] Length = 367 Score = 31.2 bits (69), Expect = 0.28 Identities = 17/56 (30%), Positives = 29/56 (51%) Query: 194 ISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKVKDACLL 249 I+ N+A T + +D + LAG ++ + +D G+Y+ADPR A L+ Sbjct: 133 INENDAVATAEIKVGDNDNLSALAAILAGADKLLLLTDQKGLYTADPRSNPQAELI 188 Database: /Users/jvanheld/rsa- tools/data/genomes/Escherichia_coli_K12/genome/NC_000913.faa Posted date: Sep 8, 2004 12:13 PM Number of letters in database: 1,351,322 Number of sequences in database: 4242 Lambda K H 0.320 0.136 0.397 Gapped Lambda K H 0.267 0.0410 0.140 Matrix: BLOSUM62 Gap Penalties: Existence: 11, Extension: 1 Number of Hits to DB: 2,199,628 Number of Sequences: 4242 Number of extensions: 96525 Number of successful extensions: 290 Number of sequences better than 1.0: 4 Number of HSP's better than 1.0 without gapping: 4 Number of HSP's successfully gapped in prelim test: 0 Number of HSP's that attempted gapping in prelim test: 279 Number of HSP's gapped (non-prelim): 5 length of query: 810 length of database: 1,351,322 effective HSP length: 92 effective length of query: 718 effective length of database: 961,058 effective search space: 690039644 effective search space used: 690039644 T: 11 A: 40 X1: 16 ( 7.4 bits) X2: 38 (14.6 bits) X3: 64 (24.7 bits) S1: 41 (21.8 bits) S2: 65 (29.6 bits)

  36. BLAST result - fourth match • The fourth match is a gamma-glutamate kinase, product of proB. • The match has the same level of identity (30%) and similarity (51%) as the second match (thrA). • However, this match only extends over 56aa, whereas the alignment between thrA and metL extends over 821aa. • The significance of the match is thus much lower: the E-value is quite high (0.28) suggesting that the similarity could be an artefact. • This clearly illustrates the fact that the important parameter to evaluate the significance of an alignment is the E-value, not the percentage of similarity ! >gi|16128228|ref|NP_414777.1| gamma-glutamate kinase [Escherichia coli K12] Length = 367 Score = 31.2 bits (69), Expect = 0.28 Identities = 17/56 (30%), Positives = 29/56 (51%) Query: 194 ISRNNAGETVLLGRNGSDYSATQIGALAGVSRVTIWSDVAGVYSADPRKVKDACLL 249 I+ N+A T + +D + LAG ++ + +D G+Y+ADPR A L+ Sbjct: 133 INENDAVATAEIKVGDNDNLSALAAILAGADKLLLLTDQKGLYTADPRSNPQAELI 188

  37. BLAST result - summary • The last part of the BLAST result gives some statistics about the search: • Number of hits • Number of sequences in the DB • … Database: /Users/jvanheld/rsa- tools/data/genomes/Escherichia_coli_K12/genome/NC_000913.faa Posted date: Sep 8, 2004 12:13 PM Number of letters in database: 1,351,322 Number of sequences in database: 4242 Lambda K H 0.320 0.136 0.397 Gapped Lambda K H 0.267 0.0410 0.140 Matrix: BLOSUM62 Gap Penalties: Existence: 11, Extension: 1 Number of Hits to DB: 2,199,628 Number of Sequences: 4242 Number of extensions: 96525 Number of successful extensions: 290 Number of sequences better than 1.0: 4 Number of HSP's better than 1.0 without gapping: 4 Number of HSP's successfully gapped in prelim test: 0 Number of HSP's that attempted gapping in prelim test: 279 Number of HSP's gapped (non-prelim): 5 length of query: 810 length of database: 1,351,322 effective HSP length: 92 effective length of query: 718 effective length of database: 961,058 effective search space: 690039644 effective search space used: 690039644 T: 11 A: 40 X1: 16 ( 7.4 bits) X2: 38 (14.6 bits) X3: 64 (24.7 bits) S1: 41 (21.8 bits) S2: 65 (29.6 bits)

  38. BLAST – examples of results

  39. BLAST (NCBI)

  40. BLAST (NCBI)

  41. BLASTn paramètres

  42. BLASTp paramètres

  43. Checking expected values with random sequences (“negative control”)

  44. Searching a sequence database with a random sequence as query • Empirical test of the expected value: • We generated a random sequence of 1588 aa using the tool random-sequence (http://rsat.ulb.ac.be/rsat/ ). • This random sequence mimics the amino acid and dipeptide composition of yeast proteins (generated with First order Markov model). • A blast search against the non-redundant database returns several hits. • These hits however have low scores. Not surprisingly, the corresponding expected values are higher than 1.

  45. blastp with random sequences • pblast of 10 random sequences against the non-redundant database. • Between 1 and 15 matches per trial. • Was this to be expected ? • This corresponds pretty well to the expectation • On the NCBI BLAST web server, the default threshold on expect has been set to 10. • We thus expect, for each request, an average of 10 matches by chance. • We indeed observe this order of magnitude when submitting random sequences.

  46. The modalities of BLAST

  47. DNA versus protein searches • When the query is a coding DNA sequence, it is recommended to apply searches with the translated rather than raw DNA sequences • This allows to introduce a substitution matrix (PAM, BLOSUM, ...), which better reflects the evolutionary changes. • It has been shown that some distant relationships can be detected with translated searches, but escape detection with the DNA search. • It is easier to filter out low complexity regions from proteins than from DNA sequences.

  48. Les modalités de BLAST Séquence requête Base de données Peptidique Peptidique BLASTp tBLASTn BLASTx BLASTn Nucléique Nucléique tBLASTx

  49. BLAST - a family of purpose-specific programs ATTGTGAGTCCTGATGATGGT TAACTCTCAGGACTACTACCA • Different program names exist, depending on the type (protein or nucleic acid) of query and database sequences. • For comparison between nucleic acids and proteins, the nucleic acid is translated in the 6 frames (3 frames per strand) 6-frames translation

More Related