Database Similarity Searching

Transcription

1 An Introduction to Bioinformatics BSC4933/ISC5224 Florida State University Feb. 23, 2009 Database Similarity Searching Steven M. Thompson Florida State University of Department Scientific Computing How can you search the databases for similar sequences, if pair-wise alignments take N 2 time?! Significance and heuristics... 1

2 But, why even do database searches? We can imagine screening databases for sequences similar to ours using the concepts of dynamic programming and log-odds scoring matrices and some yet to be described tricks. But what do database searches tell us; what can we gain from them? Why even bother? Inference through homology is a fundamental principle of biology. When a sequence is found to fall into a preexisting family we may be able to infer function, mechanism, evolution, perhaps even structure, based on homology with its neighbors. That is, homology is transitive. If no significant similarity can be found, the fact that your sequence is new and different could be very important. Granted, its characterization may prove difficult, but it could be well worth it. Homology and similarity Don t confuse homology with similarity: there is a huge difference! Similarity is a statistic that describes how much two (sub)sequences are alike according to some set scoring criteria. It can be normalized to ascertain statistical significance, but it s still just a number. Homology, in contrast and by definition, implies an evolutionary relationship more than just the fact that we ve all evolved from the same old primordial ooze. To demonstrate homology reconstruct the phylogeny of the organisms or genes of interest. Better yet, show experimental evidence structural, morphological, genetic, or fossil that corroborates your assertion. There is no such thing as percent homology; something is either homologous or it is not. Walter Fitch is credited with homology is like pregnancy you can t be 45% pregnant, just like something can t be 45% homologous. You either are or you are not. Highly significant similarity can argue for homology, but never the other way around. 2

3 Independent of all that, what is a good alignment? So, first Significance: when is any alignment worth anything biologically? An old statistics trick Monte Carlo simulations: Z score = [ ( actual score ) - ( mean of randomized scores ) ] ( standard deviation of randomized score distribution ) The Normal (Abby Normal?) distribution Z scores often measure the distance from the mean using this simplistic Monte Carlo model assuming a Gaussian distribution, aka the Normal distribution which is a distribution of the sum of independent variables ( in spite of the fact that sequence-space actually follows what is know as the Extreme Value distribution. However, the Monte Carlo method does approximate significance estimates fairly well. 3

4 Sequence-space (Huh, what s that?) actually follows the Extreme Value distribution which is a distribution of the maximum of independent variables (align scores) ( < :== : := :* :* :* :===* :=========* :==================*== :===============================*===== :===========================================*==== :=====================================================*=== :==========================================================* :===========================================================* :======================================================== * :=================================================== * :=============================================* :====================================== * :============================== * :========================= * :=====================* :=================* :=============*= :==========* :========* :======* :=====* :====* :===* :==* :=* :=* :=* :* :* :* :* :* :* :* :* :* :* :* :* :* :* :* :* :* > :*= Based on this known statistical distribution, and robust statistical methodology, a realistic Expectation function, the E Value, can be calculated from database searches. The particulars of how this is done will wait, but the take-home message is the same... The Expectation Value! The higher the E value is, the more probable that the observed match is due to chance in a search of the same size database, and the lower its Z score will be, i.e. is NOT significant. Therefore, the smaller the E value, i.e. the closer it is to zero, the more significant it is and the higher its Z score will be! The E value is the number that really matters. But, where s it come from? 4

5 The Extreme Value distribution also see For local, pairwise alignment: E P (S x) = 1 - exp[kmne -λs ] Kmne -λs Where E is the Expectation value, P is Probability, S is a particular alignment score, x is a random alignment score, K is an empirically derived correction constant based on the search space size, m and n are the lengths of the two sequences involved in the comparison, e is the usual natural log base, and λ is another empirically derived constant based on the scoring system used. For database searches: E N/n (Kmne -λs ) P number of comparisons Where N is the total length of the entire database being searched. However, all this theory really only applies to ungapped alignments, but it all works well enough when gaps are allowed as long as K and λ are adjusted to compensate. Probability relates to Expectation P = 1 - e -E with E = Expectation Value E= 10 -> P= E= > P=0.01 E= 1 -> P=0.63 E= > P=0.001 E= 0.1 -> P=0.095 E= > P= So, with E Values < 0.001, P Values E Values Check out these sites, among hundreds, for the derivation of these statistics: nt.pdf, week3_statistics.pdf, and 5

6 Rules of thumb for a protein search The Z score represents the number of standard deviations some particular alignment is from a distribution of random alignments (often, but not always, based on the normal distribution). They very roughly correspond to the listed E Values (based on the Extreme Value distribution) for a typical protein sequence similarity search through a database with ~125,000 protein entries. On to the searches But N 2 is way too slow! How can it be done? Database searching programs use the two concepts of dynamic programming and log-odds scoring matrices; however, dynamic programming takes far too long when used against most sequence databases with a normal computer. Remember how big the databases are! Therefore, the programs use tricks to make things happen faster. These tricks fall into two main categories, that of hashing, and that of approximation. 6

7 Corn beef hash? Huh... Hashing is the process of breaking your sequence into small words or k-tuples (think all chopped up, just like corn beef hash) of a set size and creating a look-up table with those words keyed to position numbers. Computers can deal with numbers way faster than they can deal with strings of letters, and this preprocessing step happens very quickly. Then when any of the word positions match part of an entry in the database, that match, the offset, is saved. In general, hashing reduces the complexity of the search problem from N 2 for dynamic programming to N, the length of all the sequences in the database. A simple hash table The sequence FAMLGFIKYLPGCM and a word size of one, would produce this query lookup position hash table: word A C F G I K L M P Y Pos comparing it to the database sequence TGFIKYLPGACT, would yield the following offset table: T G F I K Y L P G A C T (example from Krane and Raymer, 2003, Fundamental Concepts of Bioinformatics p. 50) 7

8 Hmmm & some interpretation The offset numbers come from the difference between the positions of the words in the query sequence and the position of the occurrence of that word in the target sequence. Then.... Look at all of the offsets equal to three in the previous table. Therefore, offset the alignment by three: FAMLGFIKYLPGCM TGFIKYLPGACT Quick and easy. Computers can compare these sorts of tables very fast. The trick is to know how far to attempt to extend the alignment out. OK. Heuristics... What s that? Approximation techniques are collectively known as heuristics. Webster s defines heuristic as serving to guide, discover, or reveal;... but unproved or incapable of proof. In database similarity searching techniques the heuristic usually restricts the necessary search space by calculating some sort of a statistic that allows the program to decide whether further scrutiny of a particular match should be pursued. This statistic may miss things depending on the parameters set that s what makes it heuristic. Worthwhile results at the end are compiled and the longest alignment within the program s restrictions is created. The exact implementation varies between the different programs, but the basic idea follows in most all of them. 8

9 Two predominant versions exist: BLAST and Fast Both return local alignments, and are not a single program, but rather a family of programs with implementations designed to compare a sequence to a database in about every which way imaginable. These include: 1) a DNA sequence against a DNA database (not recommended unless forced to do so because you are dealing with a non-translated region of the genome DNA is just too darn noisy, only identity & four bases!), 2) a translated (where the translation is done on-the-fly in all six frames) version of a DNA sequence against a translated ( on-the-fly six-frame) version of the DNA database (not available in the Fast package), 3) a translated ( on-the-fly six-frame) version of a DNA sequence against a protein database, 4) a protein sequence against a translated ( on-the-fly six-frame) version of a DNA database, 5) or a protein sequence against a protein database. Many implementations allow for the possibility of frame shifts in translated comparisons and don t penalize the score for doing so. The BLAST and Fast programs some generalities BLAST Basic Local Alignment Search Tool, developed at NCBI. 1) Normally NOT a good idea to use for DNA against DNA searches w/o translation (not optimized); 2) Pre-filters repeat and low complexity sequence regions; 4) Can find more than one region of gapped similarity; 5) Very fast heuristic and parallel implementation; 6) Restricted to precompiled, specially formatted databases; FastA and its family of relatives, developed by Bill Pearson at the University of Virginia. 1) Works well for DNA against DNA searches (within limits of possible sensitivity); 2) Can find only one gapped region of similarity; 3) Relatively slow, should often be run in the background; 4) Does not require specially prepared, preformatted databases. 9

10 The algorithms, in brief BLAST: Two word hits on the same diagonal above some similarity threshold triggers ungapped extension until the score isn t improved enough above another threshold: the HSP. Initiate gapped extensions using dynamic programming for those HSP s above a third threshold up to the point where the score starts to drop below a fourth threshold: yields alignment. Fast: Combine nonoverlapping init regions on different diagonals: initn. Find all ungapped exact word hits; maximize the ten best continuous regions scores: init1. Use dynamic programming in a band for all regions with initn scores better than some threshold: opt score. BLAST the algorithm in more detail 1) After BLAST has sorted its lookup table, it tries to find all double word hits along the same diagonal within some specified distance using what NCBI calls a Discrete Finite Automaton (DFA). These word hits of size W do not have to be identical; rather, they have to be better than some threshold value T. To identify these double word hits, the DFA scans through all strings of words (typically W=3 for peptides) that score at least T (usually 11 for peptides). 2) Each double word hit that passes this step then triggers a process called un-gapped extension in both directions, such that each diagonal is extended as far as it can, until the running score starts to drop below a pre-defined value X within a certain range A. The result of this pass is called a High-Scoring segment Pair or HSP. 3) Those HSPs that pass this step with a score better than S then begin a gapped extension step utilizing dynamic programming. Those gapped alignments with Expectation values better than the user specified cutoff are reported. The extreme value distribution of BLAST Expectation value parameters is pre-computed against each precompiled database this is one area that speeds up the algorithm considerably. 10

11 The BLAST algorithm, continued The math can be generalized thus: for any two sequences of length m and n, local, best alignments are identified as HSPs. HSPs are stretches of sequence pairs that cannot be further improved by extension or trimming, as described above. For ungapped alignments, the number of expected HSPs with a score of at least S is given by the formula from before: E = Kmne λs This is called an E-value for the score S. In a database search n is the size of the database in residues, so N=mn is the search space size. K and λ are be supplied by statistical theory, and, as mentioned above, can be calculated by comparison to pre-computed, simulated distributions. These two parameters define the statistical significance of an E-value. The E-value defines the significance of the search. As mentioned above, the smaller an E-value is, the more likely it is significant. A value of 0.01 to is a good starting point for significance in most typical searches. In other words, in order to assess whether a given alignment constitutes evidence for homology, it helps to know how strong an alignment can be expected from chance alone. The Fast algorithm in more detail Fast is an older algorithm than BLAST. The original Fast paper came out in 1988, based on David Lipman s work in a 1983 paper; the original BLAST paper was published in Both algorithms have been upgraded substantially since originally released. Fast was the first widely used, powerful sequence database searching algorithm. Bill Pearson continually refines the programs such that they remain a viable alternative to BLAST, especially if one is restricted to searching DNA against DNA without translation. They are also very helpful in situations where BLAST finds no significant alignments arguably, Fast may be more sensitive than BLAST in these situations. Fast is also a hashing style algorithm and builds words of a set k- tuple size, by default two for peptides. It then identifies all exact word matches between the sequence and the database members. Note that the word matches must be exact for Fast and only similar, above some threshold, for BLAST. 11

12 The Fast algorithm, continued From these exact word matches: 1) Scores are assigned to each continuous, ungapped, diagonal by adding all of the exact match BLOSUM values. 2) The ten highest scoring diagonals for each query-database pair are then rescored using BLOSUM similarities as well as identities and ends are trimmed to maximize the score. The best of each of these is called the Init1 score. 3) Next the program looks around to see if nearby off-diagonal Init1 alignments can be combined by incorporating gaps. If so, a new score, Initn, is calculated by summing up all the contributing Init1 scores, penalizing gaps with a penalty for each. 4) The program then constructs an optimal local alignment for all Initn pairs with scores better than some set threshold using a variation of dynamic programming in a band. A sixteen residue band centered at the highest Init1 region is used by default with peptides. The score generated from this step called opt. The Fast algorithm, still continued 5) Next, Fast uses a simple linear regression against the natural log of the search set sequence length to calculate a normalized z-score for the sequence pair. Note that this is not the same Monte Carlo style Z score described earlier, and can not be directly compared to one. 6) Finally, it compares the distribution of these z-scores to the actual extreme-value distribution of the search. Using this distribution, the program estimates the number of sequences that would be expected to have, purely by chance, a z-score greater than or equal to the z-score obtained in the search. This is reported as the Expectation value. 7) If the user requests pair-wise alignments in the output, then the program uses full Smith-Waterman local dynamic programming, not restricted to a band, to produce its final alignments. 12

13 Let s see em in action over what we did in lab I ll demonstrate with my favorite example, elongation factor 1α protein from Giardia lamblia, named EF1A_Giala in the Swiss-Prot database, but we have to use the accession code, Q08046, for most sequence similarity search servers to find the sequence. Let s see how they work and how quickly we get results back. To begin we ll go to the most widely used (and abused!) biocomputing program on earth: NCBI s BLAST Connect to NCBI s BLAST page with any Web browser: There is a wealth of information there, including a wonderful tutorial and several very good essays for teaching yourself way more about BLAST than this lecture can ever hope for. How about a Web-based FastA? We ll go to Bill Pearson s site itself at the University of Virginia (though others are available): This site can perform all the various FastA implementations on a variety of databases as well as allow you to change search parameters. It s also remarkably fast! Because of all this, it s a great site for teaching yourself all about the many nuances of sequence comparison. 13

14 Note to self: Contrast that with how you did it in lab with GCG s BLAST (TBLASTX), FastA (FastX) and the SeqLab GUI. But remember, one convenient advantage of using GCG s FastA is you can take advantage of its flexible database search syntax, being able to use any valid GCG sequence specification. OK, what about even more powerful ways to do it? Using Moab on the HPC for sequence similarity searching! MPI parallel processing with either BLAST or FastA. In general you ll want to build your own custom database of those sequences you are interested in. Remember, the smaller the database, the more sensitive the search statistics will be. BLAST databases need to be partitioned by mpiformatdb; FastA ones do not. You can use GCG routines to help build your database (LookUp and SeqConv+). And then model your MOAB script off the examples I provide on the HPC Software Bioinformatics Suite Web pages. Finally Why do I keep diss ing DNA for searches and alignment? All database similarity searching and sequence alignment, regardless of the algorithm used, is far more sensitive at the amino acid level than at the DNA level. This is because proteins have twenty match criteria versus DNA s four, and those four DNA bases can generally only be identical, not similar, to each other; and many DNA base changes (especially third position changes) do not change the encoded protein. All of these factors drastically increase the noise level of a DNA against DNA search, and give protein searches a much greater look-back time, typically doubling it. Therefore, whenever dealing with coding sequence, it is always prudent to search at the protein level! 14

15 References and a Comment: The better you understand the chemical, physical, and biological systems involved, the better your chance of success in analyzing them. Certain strategies are inherently more appropriate to others in certain circumstances. Making these types of subjective, discriminatory decisions and utilizing all of the available options so that you can generate the most practical data for evaluation are two of the most important take-home messages that I can offer! Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic Local Alignment Tool. Journal of Molecular Biology 215, Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a New Generation of Protein Database Search Programs. Nucleic Acids Research 25, Genetics Computer Group (GCG) (Copyright ) Program Manual for the Wisconsin Package, Version 10.3, Accelrys, Inc. A Pharmocopeia Company, San Diego, California, U.S.A. Gribskov, M. and Devereux, J., editors (1992) Sequence Analysis Primer. W.H. Freeman and Company, New York, New York, U.S.A. Henikoff, S. and Henikoff, J.G. (1992) Amino Acid Substitution Matrices from Protein Blocks. Proceedings of the National Academy of Sciences U.S.A. 89, Needleman, S.B. and Wunsch, C.D. (1970) A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins. Journal of Molecular Biology 48, Pearson, W.R. and Lipman, D.J. (1988) Improved Tools for Biological Sequence Analysis. Proceedings of the National Academy of Sciences U.S.A. 85, Schwartz, R.M. and Dayhoff, M.O. (1979) Matrices for Detecting Distant Relationships. In Atlas of Protein Sequences and Structure, (M.O. Dayhoff editor) 5, Suppl. 3, , National Biomedical Research Foundation, Washington D.C., U.S.A. Smith, T.F. and Waterman, M.S. (1981) Comparison of Bio-Sequences. Advances in Applied Mathematics 2, Wilbur, W.J. and Lipman, D.J. (1983) Rapid Similarity Searches of Nucleic Acid and Protein Data Banks. Proceedings of the National Academy of Sciences U.S.A. 80,