This document describes the WWW BLAST interface.
BLAST (Basic Local Alignment Search Tool) is the heuristic
search algorithm employed by the programs blastp, blastn,
blastx, tblastn, and tblastx; these programs ascribe signi-
ficance to their findings using the statistical methods of
Karlin and Altschul (1990, 1993) with a few enhancements.
The BLAST programs were tailored for sequence similarity
searching -- for example to identify homologs to a query
sequence. The programs are not generally useful for motif-
style searching. For a discussion of basic issues in simi-
larity searching of sequence databases, see Altschul et al.
(1994).
The five BLAST programs described here perform the following
tasks:
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 nucleo-
tide query sequence against the six-frame transla-
tions of a nucleotide sequence database.
BLAST Search parameters
The fundamental unit of BLAST algorithm output is the High-
scoring Segment Pair (HSP). An HSP consists of two sequence
fragments of arbitrary but equal length whose alignment is
locally maximal and for which the alignment score meets or
exceeds a threshold or cutoff score. A set of HSPs is thus
defined by two sequences, a scoring system, and a cutoff
score; this set may be empty if the cutoff score is suffi-
ciently high. In the programmatic implementations of the
BLAST algorithm described here, each HSP consists of a seg-
ment from the query sequence and one from a database
sequence. The sensitivity and speed of the programs can be
adjusted via the standard BLAST algorithm parameters W, T,
and X (Altschul et al., 1990); selectivity of the programs
can be adjusted via the cutoff score.
A Maximal-scoring Segment Pair (MSP) is defined by two
sequences and a scoring system and is the highest-scoring of
all possible segment pairs that can be produced from the two
sequences. The statistical methods of Karlin and Altschul
(1990, 1993) are applicable to determining the significance
of MSP scores in the limit of long sequences, under a random
sequence model that assumes independent and identically dis-
tributed choices for the residues at each position in the
sequences. In the programs described here, Karlin-Altschul
statistics have been extrapolated to the task of assessing
the significance of HSP scores obtained from comparisons of
potentially short, biological sequences.
The approach to similarity searching taken by the BLAST pro-
grams is first to look for similar segments (HSPs) between
the query sequence and a database sequence, then to evaluate
the statistical significance of any matches that were found,
and finally to report only those matches that satisfy a
user-selectable threshold of significance. Findings of mul-
tiple HSPs involving the query sequence and a single data-
base sequence may be treated statistically in a variety of
ways. By default the programs use "Sum" statistics (Karlin
and Altschul, 1993). As such, the statistical significance
ascribed to a set of HSPs may be higher than that ascribed
to any individual member of the set. Only when the ascribed
significance satisfies the user-selectable threshold (E
parameter) will the match be reported to the user.
The task of finding HSPs begins with identifying short words
of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned
with a word of the same length in a database sequence. T is
referred to as the neighborhood word score threshold
(Altschul et al., 1990). These initial neighborhood word
hits act as seeds for initiating searches to find longer
HSPs containing them. The word hits are extended in both
directions along each sequence for as far as the cumulative
alignment score can be increased. Extension of the word
hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below,
due to the accumulation of one or more negative-scoring
residue alignments; or the end of either sequence is
reached.
From Karlin and Altschul (1990), the principal equation
relating the score of an HSP to its expected frequency of
chance occurrence is:
E = K N exp(-Lambda S)
where E is the expected frequency of chance occurrence of an
HSP having score S (or one scoring higher); K and Lambda are
Karlin-Altschul parameters; N is the product of the query
and database sequence lengths, or the size of the search
space; and exp is the exponentiation function.
Lambda may be thought of as the expected increase in relia-
bility of an alignment associated with a unit increase in
alignment score. Reliability in this case is expressed in
units of information, such as bits or nats, with one nat
being equivalent to 1/log(2) (roughly 1.44) bits.
The expectation E (range 0 to infinity) calculated for an
alignment between the query sequence and a database sequence
can be extrapolated to an expectation over the entire
database search, by converting the pairwise expectation to a
probability (range 0-1) and multiplying the result by the
ratio of the entire database size (expressed in residues) to
the length of the matching database sequence. In detail:
E_database = (1 - exp(-E)) D / d
where D is the size of the database; d is the length of the
matching database sequence; and the quantity (1 - exp(-E))
is the probability, P, corresponding to the expectation E
for the pairwise sequence comparison. Note that in the
limit of infinite E, P approaches 1; and in the limit as E
approaches 0, E and P approach equality. Due to inaccuracy
in the statistical methods as they are applied in the BLAST
programs, whenever E and P are less than about 0.05, the two
values can be practically treated as being equal.
In contrast to the random sequence model used by Karlin-
Altschul statistics, biological sequences are often short in
length -- an HSP may involve a relatively large fraction of
the query or database sequence, which reduces the effective
size of the 2-dimensional search space defined by the two
sequences. To obtain more accurate significance estimates,
the BLAST programs compute effective lengths for the query
and database sequences that are their real lengths minus the
expected length of the HSP, where the expected length for an
HSP is computed from its score. In no event is an effective
length for the query or database sequence permitted to go
below 1. Thus, the effective length of either the query or
the database sequence is computed according to the follow-
ing:
Length_eff = MAX( Length_real - Lambda S / H , 1)
where H is the relative entropy of the target and background
residue frequencies (Karlin and Altschul, 1990), one of the
statistics reported by the BLAST programs. H may be thought
of as the information expected to be obtained from each pair
of aligned residues in a real alignment that distinguishes
the alignment from a random one.
The default scoring matrix used by blastp, blastx, tblastn,
and tblastx is the BLOSUM62 matrix (Henikoff and Henikoff,
1992).
Several PAM (point accepted mutations per 100 residues)
amino acid scoring matrices are provided in the BLAST
software distribution, including the PAM40, PAM120, and
PAM250. While the BLOSUM62 matrix is a good general purpose
scoring matrix and is the default matrix used by the BLAST
programs, if one is restricted to using only PAM scoring
matrices, then the PAM120 is recommended for general protein
similarity searches (Altschul, 1991). The pam(1) program
can be used to produce PAM matrices of any desired iteration
from 2 to 511. Each matrix is most sensitive at finding
similarities at its particular PAM distance. For more
thorough searches, particularly when the mutational distance
between potential homologs is unknown and the significance
of their similarity may be only marginal, Altschul (1991,
1992) recommends performing at least three searches, one
each with the PAM40, PAM120 and PAM250 matrices.
In blastn, the M parameter sets the reward score for a pair
of matching residues; the N parameter sets the penalty score
for mismatching residues. M and N must be positive and
negative integers, respectively. The relative magnitudes of
M and N determines the number of nucleic acid PAMs (point
accepted mutations per 100 residues) for which they are most
sensitive at finding homologs. Higher ratios of M:N
correspond to increasing nucleic acid PAMs (increased diver-
gence). The default values for M and N, respectively 5 and
-4, having a ratio of 1.25, correspond to about 47 nucleic
acid PAMs, or about 58 amino acid PAMs; an M:N ratio of 1
corresponds to 30 nucleic acid PAMs or 38 amino acid PAMs.
At higher than about 40 nucleic acid PAMs, or 50 amino acid
PAMs, better sensitivity at detecting similarities between
coding regions is expected by performing comparisons at the
amino acid level (States et al., 1991), using conceptually
translated nucleotide sequences (re: blastx, tblastn, and
tblastx).
Independent of the values chosen for M and N, the default
wordlength W=11 used by blastn restricts the program to
finding sequences that share at least an 11-mer stretch of
100% identity with the query. Under the random sequence
model, stretches of 11 consecutive matching residues are
unlikely to occur merely by chance even between only
moderately diverged homologs. Thus, blastn with its default
parameter settings is poorly suited to finding anything but
very similar sequences. If better sensitivity is needed,
one should use a smaller value for W.
For the blastn program, it may be easy to see how multiply-
ing both M and N by some large number will yield proportion-
ally larger alignment scores with their statistical signifi-
cance remaining unchanged. This scale-independence of the
statistical significance estimates from blastn has its ana-
log in the scoring matrices used by the other BLAST pro-
grams: multiplying all elements in a scoring matrix by an
arbitrary factor will proportionally alter the alignment
scores but will not alter their statistical significance
(assuming numerical precision is maintained). From this it
should be clear that raw alignment scores are meaningless
without specific knowledge of the scoring matrix that was
used.
Regardless of the scoring scheme employed, two stringent
criteria must be met in order to be able to calculate the
Karlin-Altschul parameters Lambda and K. First, given the
residue composition for the query sequence and the residue
composition assumed for the database, the alignment score
expected for any randomly selected pair of residues (one
from the query sequence and one from the database) must be
negative. Second, given the sequence residue compositions
and the scoring scheme, a positive score must be possible to
achieve. For instance, the match reward score of blastn
must have a positive value; and given the assumption made by
blastn that the 4 nucleotides A, C, G and T are represented
at equal 25% frequencies in the database, a wide range of
value combinations for M and N are precluded from use --
namely those combinations where the magnitude of the ratio
M:N is greater than or equal to 3.
The parameter C can be set to a positive integer to select
the genetic code that will be used by blastx and tblastx to
translate the query sequence. The -dbgcode parameter can be
used to select an alternate genetic code for translation of
the database by the programs tblastn and tblastx. In each
case, the default genetic code is the so-called "Standard"
or "Universal" genetic code. To obtain a listing of the
genetic codes available and their associated numerical iden-
tifiers, invoke blastx or tblastx with the command line
parameter C=list. Note: the numerical identifiers used here
for genetic codes parallel those defined in the NCBI
software Toolbox; hence some numerical values will be
skipped as genetic codes are updated.
The list of genetic codes available and their associated
values for the parameters C and -dbgcode are:
1 Standard or Universal
2 Vertebrate Mitochondrial
3 Yeast Mitochondrial
4 Mold, Protozoan, Coelenterate Mitochondrial and
Mycoplasma/Spiroplasma
5 Invertebrate Mitochondrial
6 Ciliate Macronuclear
9 Echinodermate Mitochondrial
10 Alternative Ciliate Macronuclear
11 Eubacterial
12 Alternative Yeast
13 Ascidian Mitochondrial
14 Flatworm Mitochondrial
The Expect and P-values reported for HSPs are dependent on
several factors including: the scoring system employed, the
residue composition of the query sequence, an assumed resi-
due composition for a typical database sequence, the length
of the query sequence, and the total length of the database.
HSP scores from different program invocations are appropri-
ate for comparison even if the databases searched are of
different lengths, as long as the other factors mentioned
here do not vary. For example, alignment scores from
searches with the default BLOSUM62 matrix should not be
directly compared with scores obtained with the PAM120
matrix; and scores produced using two versions of the same
PAM matrix, each created to different scales (see above),
can not be meaningfully compared without conversion to the
same scale.
Some isolation from the many factors involved in assessing
the statistical significance of HSPs can be attained by
observing the information content reported (in bits) for the
alignments. While the information content of an HSP may
change when different scoring systems are used (e.g., with
different PAM matrices), the number of bits reported for an
HSP will at least be independent of the scale to which the
scoring matrix was generated. (In practice, this statement
is not quite true, because the alignment scores used by the
BLAST programs are integers that lack much precision). In
other words, when conveying the statistical significance of
an alignment, the alignment score itself is not useful
unless the specific scoring matrix that was employed is also
provided, but the informativeness of an alignment is a mean-
ingful statistic that can be used to ascribe statistical
significance (a P-value) to the match independently of
specific knowledge about the scoring matrix.
The BLAST programs all provide information in roughly the
same format. First comes (A) an introduction to the pro-
gram; (B) a histogram of expectations (see above) if one was
requested; (C) a series of one-line descriptions of matching
database sequences; (D) the actual sequence alignments; and
finally the parameters and other statistics gathered during
the search.
Sample blastp output from comparing pir|A01243|DXCH against
the SWISS-PROT database is presented below.
A. Program Introduction
The introductory output provides the program name (BLASTP in
this case), the version number (1.4.6MP in this case), the
date the program source code last changed substantially
(June 13, 1994), the date the program was built (Sept. 22,
1994), and a description of the query sequence and database
to be searched. These may all be important pieces of infor-
mation if a bug is suspected or if reproducibility of
results is important.
The "Searching..." indicator indicates progress that the
program made in searching the database. A complete database
search will yield 50 periods (.), or one period per database
sequence, whichever number is smaller. When searching a
database consisting of 50 sequences or more, if fewer than
50 periods are displayed and the program aborted for some
reason, dividing the number of periods by 0.5 will yield the
approximate percentage (0-100%) of the database that was
searched before the program died. If the program had diffi-
culty making progress through the database, one or more
asterisks (*) may be interspersed between the periods at
one-minute intervals.
B. Histogram of Expectations
Shown in the output below is a histogram of the lowest (most
significant) Expect values obtained with each database
sequence. This information is useful in determining the
numbers of database sequences that achieved a particular
level of statistical significance. It indicates the number
of database matches that would be reportable at various set-
tings for the expectation threshold (E parameter).
C. One-line Summaries
The one-line sequence descriptions and summaries of results
are useful for identifying biologically interesting database
matches and correlating this interest with the statistical
significance estimates. Unless otherwise requested, the
database sequences are sorted by increasing P-value (proba-
bility). Identifiers for the database sequences appear in
the first column; then come brief descriptions of each
sequence, which may need to be truncated in order to fit in
the available space. The "High Score" column contains the
score of the highest-scoring HSP found with each database
sequence. The "P(N)" column contains the lowest P-value
ascribed to any set of HSPs for each database sequence; and
the "N" column displays the number of HSPs in the set which
was ascribed the lowest P-value. The P-values are a func-
tion of N, as used in Karlin-Altschul "Sum" statistics or
Poisson statistics, to treat situations where multiple HSPs
are found. It should be noted that the highest-scoring HSP
whose score is reported in the "High Score" column is not
necessarily a member of the set of HSPs which yields the
lowest P-value; the highest-scoring HSP may be excluded from
this set on the basis of consistency rules governing the
grouping of HSPs (see the -consistency option). Numbers of
the form "7.7e-160" are in scientific notation. In this
particular example, the number being represented is 7.7
times 10 to the minus 160th power. which is astronomically
close to zero.
D. Alignments
Alignments found with the BLAST algorithm are ungapped.
Several statistics are used to describe each HSP: the raw
alignment Score; the raw score converted to bits of informa-
tion by multiplying by Lambda (see the Statistics output);
the number of times one might Expect to see such a match (or
a better one) merely by chance; the P-value (probability in
the range 0-1) of observing such a match; the number and
fraction of total residues in the HSP which are identical;
the number and fraction of residues for which the alignment
scores have positive values. When Sum statistics have been
used to calculate the Expect and P-values, the P-value is
qualified with the word "Sum" and the N parameter used in
the Sum statistics is provided in parentheses to indicate
the number of HSPs in the set; when Poisson statistics have
been used to calculate the Expect and P-values, the P-value
is qualified with the word "Poisson". Between the two lines
of Query and Subject (database) sequence is a line indicat-
ing the specific residues which are identical, as well as
those which are non-identical but nevertheless have positive
alignment scores defined in the scoring matrix that was used
(the BLOSUM62 matrix in this case). Identical letters or
residues, when paired with each other, are not highlighted
if their alignment score is negative or zero. Examples of
this would be an X juxtaposed with an X in two amino acid
sequences, or an N juxtaposed with another N in two nucleo-
tide sequences. Such ambiguous residue-residue pairings may
be uninformative and thus lend no support to the overall
alignment being either real or random; however, the informa-
tiveness of these pairings is left up to the user of the
BLAST programs to decide, because any values desired can be
specified in a scoring matrix of the user's own making.
BLASTP 1.4.6MP [13-Jun-94] [Build 13:58:36 Sep 22 1994]
Reference: Altschul, Stephen F., Warren Gish, Webb Miller, Eugene W. Myers,
and David J. Lipman (1990). Basic local alignment search tool. J. Mol. Biol.
215:403-10.
Query = pir|A01243|DXCH 232 Gene X protein - Chicken (fragment)
(232 letters)
Database: SWISS-PROT Release 29.0
38,303 sequences; 13,464,008 total letters.
Searching..................................................done
Observed Numbers of Database Sequences Satisfying
Various EXPECTation Thresholds (E parameter values)
Histogram units: = 31 Sequences : less than 31 sequences
EXPECTation Threshold
(E parameter)
|
V Observed Counts-->
10000 4863 1861 |============================================================
6310 3002 782 |=========================
3980 2220 812 |==========================
2510 1408 303 |=========
1580 1105 393 |============
1000 712 179 |=====
631 533 161 |=====
398 372 80 |==
251 292 73 |==
158 219 50 |=
100 169 32 |=
63.1 137 18 |:
39.8 119 9 |:
25.1 110 6 |:
15.8 104 9 |:
>>>>>>>>>>>>>>>>>>>>> Expect = 10.0, Observed = 95 <<<<<<<<<<<<<<<<<
10.0 95 4 |:
6.31 91 3 |:
3.98 88 1 |:
2.51 87 3 |:
1.58 84 0 |
1.00 84 2 |:
Smallest
Sum
High Probability
Sequences producing High-scoring Segment Pairs: Score P(N) N
sp|P01013|OVAX_CHICK GENE X PROTEIN (OVALBUMIN-RELATED) (... 1191 7.7e-160 1
sp|P01014|OVAY_CHICK GENE Y PROTEIN (OVALBUMIN-RELATED). 949 7.0e-127 1
sp|P01012|OVAL_CHICK OVALBUMIN (PLAKALBUMIN). 645 3.4e-100 2
sp|P19104|OVAL_COTJA OVALBUMIN. 626 1.2e-96 2
sp|P05619|ILEU_HORSE LEUKOCYTE ELASTASE INHIBITOR (LEI). 216 3.7e-71 3
sp|P80229|ILEU_PIG LEUKOCYTE ELASTASE INHIBITOR (LEI) (... 325 4.0e-71 2
sp|P29508|SCCA_HUMAN SQUAMOUS CELL CARCINOMA ANTIGEN (SCC... 439 3.5e-70 2
sp|P30740|ILEU_HUMAN LEUKOCYTE ELASTASE INHIBITOR (LEI) (... 211 1.3e-66 3
sp|P05120|PAI2_HUMAN PLASMINOGEN ACTIVATOR INHIBITOR-2, P... 176 1.8e-65 4
sp|P35237|PTI_HUMAN PLACENTAL THROMBIN INHIBITOR. 473 1.3e-61 1
sp|P29524|PAI2_RAT PLASMINOGEN ACTIVATOR INHIBITOR-2, T... 183 9.4e-61 4
sp|P12388|PAI2_MOUSE PLASMINOGEN ACTIVATOR INHIBITOR-2, M... 179 1.8e-60 4
sp|P36952|MASP_HUMAN MASPIN PRECURSOR. 198 2.6e-58 4
sp|P32261|ANT3_MOUSE ANTITHROMBIN-III PRECURSOR (ATIII). 142 4.0e-48 5
sp|P01008|ANT3_HUMAN ANTITHROMBIN-III PRECURSOR (ATIII). 122 7.5e-48 5
WARNING: Descriptions of 80 database sequences were not reported due to the
limiting value of parameter V = 15.
... alignments with the top 8 database sequences deleted ...
>sp|P05120|PAI2_HUMAN PLASMINOGEN ACTIVATOR INHIBITOR-2, PLACENTAL (PAI-2)
(MONOCYTE ARG- SERPIN).
Length = 415
Score = 176 (80.2 bits), Expect = 1.8e-65, Sum P(4) = 1.8e-65
Identities = 38/89 (42%), Positives = 50/89 (56%)
Query: 1 QIKDLLVSSSTDLDTTLVLVNAIYFKGMWKTAFNAEDTREMPFHVTKQESKPVQMMCMNN 60
+I +LL S D DT +VLVNA+YFKG WKT F + PF V + PVQMM +
Sbjct: 180 KIPNLLPEGSVDGDTRMVLVNAVYFKGKWKTPFEKKLNGLYPFRVNSAQRTPVQMMYLRE 239
Query: 61 SFNVATLPAEKMKILELPFASGDLSMLVL 89
N+ + K +ILELP+A L+L
Sbjct: 240 KLNIGYIEDLKAQILELPYAGDVSMFLLL 268
Score = 165 (75.2 bits), Expect = 1.8e-65, Sum P(4) = 1.8e-65
Identities = 33/78 (42%), Positives = 47/78 (60%)
Query: 155 ANLTGISSAESLKISQAVHGAFMELSEDGIEMAGSTGVIEDIKHSPESEQFRADHPFLFL 214
AN +G+S L +S+ H A ++++E+G E A TG + + QF ADHPFLFL
Sbjct: 338 ANFSGMSERNDLFLSEVFHQAMVDVNEEGTEAAAGTGGVMTGRTGHGGPQFVADHPFLFL 397
Query: 215 IKHNPTNTIVYFGRYWSP 232
I H T I++FGR+ SP
Sbjct: 398 IMHKITKCILFFGRFCSP 415
Score = 144 (65.6 bits), Expect = 1.8e-65, Sum P(4) = 1.8e-65
Identities = 26/62 (41%), Positives = 41/62 (66%)
Query: 90 LPDEVSDLERIEKTINFEKLTEWTNPNTMEKRRVKVYLPQMKIEEKYNLTSVLMALGMTD 149
+ D + LE +E I ++KL +WT+ + M + V+VY+PQ K+EE Y L S+L ++GM D
Sbjct: 272 IADVSTGLELLESEITYDKLNKWTSKDKMAEDEVEVYIPQFKLEEHYELRSILRSMGMED 331
Query: 150 LF 151
F
Sbjct: 332 AF 333
Score = 61 (27.8 bits), Expect = 1.8e-65, Sum P(4) = 1.8e-65
Identities = 10/17 (58%), Positives = 16/17 (94%)
Query: 81 SGDLSMLVLLPDEVSDL 97
+GD+SM +LLPDE++D+
Sbjct: 259 AGDVSMFLLLPDEIADV 275
WARNING: HSPs involving 86 database sequences were not reported due to the
limiting value of parameter B = 9.
Parameters:
V=15
B=9
H=1
-ctxfactor=1.00
E=10
Query ----- As Used ----- ----- Computed ----
Frame MatID Matrix name Lambda K H Lambda K H
+0 0 BLOSUM62 0.316 0.132 0.370 same same same
Query
Frame MatID Length Eff.Length E S W T X E2 S2
+0 0 232 232 10. 57 3 11 22 0.22 33
Statistics:
Query Expected Observed HSPs HSPs
Frame MatID High Score High Score Reportable Reported
+0 0 62 (28.2 bits) 1191 (542.5 bits) 330 24
Query Neighborhd Word Excluded Failed Successful Overlaps
Frame MatID Words Hits Hits Extensions Extensions Excluded
+0 0 4988 5661199 1146395 4504598 10187 13
Database: SWISS-PROT Release 29.0
Release date: June 1994
Posted date: 1:29 PM EDT Jul 28, 1994
# of letters in database: 13,464,008
# of sequences in database: 38,303
# of database sequences satisfying E: 95
No. of states in DFA: 561 (55 KB)
Total size of DFA: 110 KB (128 KB)
Time to generate neighborhood: 0.03u 0.01s 0.04t Real: 00:00:00
No. of processors used: 8
Time to search database: 32.27u 0.78s 33.05t Real: 00:00:04
Total cpu time: 32.33u 0.91s 33.24t Real: 00:00:05
WARNINGS ISSUED: 2
This work is in the public domain.
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