BLAST-A faster alternative BLAST (Basic Local Alignment Search Tool) is a heuristic method to find the highest scoring locally optimal alignments between a query sequence and a database. Previous versions of BLAST did not allow gapped alignments, but BLAST2 (from the HGMP-RC telnet and www menus) does. A gapped BLAST search allows gaps (deletions and insertions) to be introduced into the alignments that are returned. Allowing gaps means that similar regions are not broken into several segments. The scoring of these gapped alignments tends to reflect biological relationships more closely. The BLAST algorithm and family of programs rely on work on the statistics of ungapped sequence alignments by Karlin and Altschul. The statistics allow us to estimate the probability of obtaining an alignment (MSP - Maximal Segment Pair) with a particular score. The BLAST algorithm permits nearly all MSP's above a cutoff to be located efficiently in a database. The algorithm operates in three steps: For a given word length w (usually 3 for proteins) and score matrix, a list is created of all words (w-mers) that can score greater than T. The database is searched, using the list of w-mers to find the corresponding w-mers in the database (hits). Each hit is extended to determine if an MSP that includes the w-mer, scores greater than S (S is the preset threshold score for an MSP). Since pair score matrices typically include negative values, extension of the initial w-mer hit may increase or decrease the score. Accordingly, a parameter X defines how great an extension will be tried in an attempt to raise the score above S. A low value for T reduces the possibility of missing MSPs with the required S score, however lower T values also increase the size of the hit list generated in step 2 and hence the execution time and memory required. In practice, the BLASTP program used for protein searches sets compromise values of T and X to balance the processor requirements and sensitivity. BLAST is unlikely to be as sensitive for all protein searches as a full dynamic programming algorithm. However, the underlying statistics provide a direct estimate of the significance of any match found. The program was developed at the NCBI and benefits from strong technical support and continuing refinement. For example, filters have recently been developed to automatically exclude regions of the query sequence that have low compositional complexity or short periodicity internal repeats. The presence of such sequences can yield extremely large numbers of statistically significant but biologically uninteresting MSPs. For example, searching with a sequence that contains a long section of hydrophobic residues will find many proteins with transmembrane helices. Blast Family of Programs The BLAST family of programs allows all combinations of DNA or protein query sequences with searches against DNA or protein databases. (Most of the time use of these is transparent, behind an interface.) blastp: compares an amino acid query sequence against a protein sequence database. blastn: compares a nucleotide query sequence against a nucleotide sequence database. blastx: compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. tblastn: compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands). tblastx: compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The default matrix for all protein-protein comparisons is BLOSUM62. For more information on the BLAST algorithm, read our BLAST FAQ, or the preprint from the "Protein Sequence Alignment and Database Scanning" chapter in: Protein Structure prediction - a practical approach, edited by M. J. E. Sternberg, IRL Press at Oxford University Press, 1996, ISBN 0 19 963496 3. Please see the book for the final version! You could also find further reading on BLAST at the NCBI www site. How do I Interpret Blast Results? The BLAST report consists of a number of sections. The descriptions below are for a BLASTP comparison, but the format for the other programs is analogous. Header The BLAST report starts with some header information that lists the type of program (here BLASTP), the version (here 2.0.1), and a release date. Also listed are a reference to the BLAST program, the query definition line, and summary of the database used. BLASTP 2.0.1 [Aug-20-1997] 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= gi|129295|sp|P01013|OVAX_CHICK gene X protein - chicken (fragment) (232 letters) Database: Non-redundant SwissProt sequences 59,576 sequences; 21,219,450 total letters List One-line descriptions of the database matches found are presented next. These include a database sequence identifier, the corresponding definition line, as well as the score (in bits) and the statistical significance ('E value') for this match (please see the section on statistics for an explanation of bits and significance). Consider the output below, from a gapped blastp comparison of SwissProt accession P01013 against the SwissProt database. High E Sequences producing significant alignments: Score Value sp|P01013|OVAX_CHICK GENE X PROTEIN (OVALBUMIN-RELATED) 442 e-124 sp|P01014|OVAY_CHICK GENE Y PROTEIN (OVALBUMIN-RELATED) 353 9e-98 sp|P01012|OVAL_CHICK OVALBUMIN (PLAKALBUMIN) (ALLERGEN GAL D II) 278 5e-75 sp|P19104|OVAL_COTJA OVALBUMIN 268 5e-72 sp|P48595|BOMA_HUMAN BOMAPIN (PROTEASE INHIBITOR 10) 199 2e-51 sp|P29508|SCC1_HUMAN SQUAMOUS CELL CARCINOMA ANTIGEN 1 (SCCA-1) ... 198 5e-51 sp|P80229|ILEU_PIG LEUKOCYTE ELASTASE INHIBITOR (LEI) (LEUCOCYTE... 197 1e-50 sp|P48594|SCC2_HUMAN SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2) ... 196 2e-50 sp|P50453|PTI9_HUMAN CYTOPLASMIC ANTIPROTEINASE 3 (CAP3) (PROTEA... 195 6e-50 sp|P05619|ILEU_HORSE LEUKOCYTE ELASTASE INHIBITOR (LEI) 193 2e-49 The first match, in this case, is the actual query sequence. The identifiers shown here are all from SwissProt, so they all have 'sp' in the first field, followed by the accession, and then a Locus name. The syntax of these identifiers is discussed in more detail in the appendices of ftp://ncbi.nlm.nih.gov/blast/db/README The definition lines are taken from the definition line in the database, with three dots (...) indicating that the definition line was too long for the space available (see entry P29508). Ungapped alignments and results from blastx and tblastn will have an additional column ('N'), displaying the number of different segment pairs used to produce the alignment, according to the Karlin-Altschul statistics. Alignments Each alignment is preceded by the sequence identifier, the full definition line and the length of the database sequence. The next lines display the score (both in bits and the raw score) and the statistical significance of the match, followed by the number of identities and positive matches according to the scoring system (e.g., BLOSUM62) and, if applicable, the number of gaps in the alignment. Finally the actual alignment is shown, with the query on top and the database match labelled as 'Sbjct'. Between the two sequences the residue is shown if it is identical and a '+' if it is a conservative substitution. One or more dashes, ¡§-¡§, indicate insertions or deletions. The example below is the third sequence listed in the one-line descriptions above. >sp|P01012|OVAL_CHICK OVALBUMIN (PLAKALBUMIN) (ALLERGEN GAL D II) Length = 386 Score = 278 bits (744), Expect = 5e-75 Identities = 149/231 (64%), Positives = 182/231 (78%), Gaps = 2/231 (0%) Query 2 IKDLLVSSSTDLDTTLVLVNAIYFKGMWKTAFNAEDTREMPFHVTKQESKPVQMMCMNNS 61 I+++L SS D T +VLVNAI FKG+W+ AF EDT+ MPF VT+QESKPVQMM Sbjct 158 IRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGL 217 Query 62 FNVATLPAEKMKILELPFASGDLSMLVLLPDEVSDLERIEKTINFEKLTEWTNPNTMEKR 121 F VA++ +EKMKILELPFASG +SMLVLLPDEVS LE++E INFEKLTEWT+ N ME+R Sbjct 218 FRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEER 277 Query 122 RVKVYLPQMKIEEKYNLTSVLMALGMTDLFIPSANLTGISSAESLKISQAVHGAFMELSE 181 ++KVYLP+MK+EEKYNLTSVLMA+G+TD+F SANL+GISSAESLKISQAVH A E++E Sbjct 278 KIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINE 337 Query 182 DGIEMAGSTGVIEDIKHSPESEQFRADHPFLFLIKHNPTNTIVYFGRYWSP 232 G E+ GS + + SE+FRADHPFLF IKH TN +++FGR SP Sbjct 338 AGREVVGSAEA--GVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP 386 Summary The last section lists specifics about the database searched as well as statistical and search parameters used: Database: Non-redundant SwissProt sequences Posted date: Aug 14, 1997 9:52 AM Number of letters in database: 21,219,450 Number of sequences in database: 59,576 Lambda K H 0.317 0.132 0.377 Gapped Lambda K H 0.255 0.0350 0.190 Matrix: BLOSUM62 Gap Penalties: Existence: 10, Extension: 1 Number of Hits to DB: 8938654 Number of Sequences: 59576 Number of extensions: 335248 Number of successful extensions: 1188 Number of sequences better than 10: 116 Number of HSP's better than 10.0 without gapping: 106 Number of HSP's successfully gapped in prelim test: 10 Number of HSP's that attempted gapping in prelim test: 868 Number of HSP's gapped (non-prelim): 120 length of query: 232 length of database: 21219450 effective HSP length: 52 effective length of query: 180 effective length of database: 18121498 effective search space: -1033097656 T: 11 A: 40 X1: 16 ( 7.3 bits) X2: 40 (14.7 bits) X3: 67 (24.6 bits) S1: 41 (21.7 bits) S2: 64 (28.4 bits) Blast Statistics and Scores We can judge the results of a BLAST search by two numbers. One is the 'bit' score, which is defined as: S' (bits) = [lambda x S (raw) - ln K] / ln 2 where lambda and K are Karlin-Altschul parameters. The expression of the score in terms of bits makes it independent of the scoring system used (i.e., the matrix). The Expect value estimates the statistical significance of the match, specifying the number of matches with a given score that are expected in a search of a database of this size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences. An Expect value of two would indicate that two matches with this score are expected purely by chance. The Expect value is the most intuitive way to rank results or compare the results of one query run against two different databases. The Expect value is used as a convenient way to create a significance threshold for reporting results. When the Expect value is increased from the default value of 10, a larger list with more low-scoring hits can be reported.