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BlastpgpBlastpgp performs gapped blastp searches and can be used to perform iterative searches in psi-blast and phi-blast mode. See the PSI-Blast and PHI-BLAST sections (below) for a description of this binary. The options may be obtained by executing 'blastpgp -'.
-T Produce HTML output [T/F]
default = F
-Q Output File for PSI-BLAST Matrix in ASCII [File Out] Optional
PSI-BlastThe blastpgp program can do an iterative search in which sequences found in one round of searching are used to build a score model for the next round of searching. In this usage, the program is called Position-Specific Iterated BLAST, or PSI-BLAST. As explained in the accompanying paper, the BLAST algorithm is not tied to a specific score matrix. Traditionally, it has been implemented using an AxA substitution matrix where A is the alphabet size. PSI-BLAST instead uses a QxA matrix, where Q is the length of the query sequence; at each position the cost of a letter depends on the position w.r.t. the query and the letter in the subject sequence. The position-specific matrix for round i+1 is built from a constrained multiple alignment among the query and the sequences found with sufficiently low e-value in round i. The top part of the output for each round distinguishes the sequences into: sequences found previously and used in the score model, and sequences not used in the score model. The output currently includes lots of diagnostics requested by users at NCBI. To skip quickly from the output of one round to the next, search for the string "producing", which is part of the header for each round and likely does not appear elsewhere in the output. PSI-BLAST "converges" and stops if all sequences found at round i+1 below the e-value threshold were already in the model at the beginning of the round. There are several blastpgp parameters specifically for PSI-BLAST:
-j is the maximum number of rounds (default 1; i.e., regular BLAST)
-h is the e-value threshold for including sequences in the
score matrix model (default 0.001)
-c is the "constant" used in the pseudocount formula specified in the
paper (default 10)
The -C and -R flags provide a "checkpointing" facility whereby a score model can be stored and later reused.
-C stores the query and frequency count ratio matrix in a
file
-R restarts from a file stored previously.
When using -R, it is required that the query specified on the command line match exactly the query in the restart file. Two additional arguments specify the format of the input/output checkpoint file:
-q Format of the input checkpoint file:
0: the default: a byte-encoded (not human readable) format
1: a text ASN.1 scoremat object
2: a binary ASN.1 scoremat object
-u Format of the output checkpoint file:
0: the default: a byte-encoded (not human readable) format
1: a text ASN.1 scoremat object
2: a binary ASN.1 scoremat object
Users who also develop their own sequence analysis software may wish to develop their own scoring systems. For this purpose the code in posit.c that writes out the checkpoint can be easily adapated to write out scoring systems derived by other algorithms in such a way that PSI-BLAST can read the files in later. The checkpoint structure is general in the sense that it can handle any position-specific matrix that fits in the Karlin-Altschul statistical framework for BLAST scoring. The -B flag provides a way to jump start PSI-BLAST from a master-slave multiple alignment computed outside PSI-BLAST. The multiple alignment must include the query sequence as one of the sequences, but it need not be the first sequence. The multiple alignment must be specified in a format that is derived from Clustal, but without some headers and trailers. See example below. The rules are also described by the following words. Suppose the multiple alignments has N sequences. It may be presented in 1 or more blocks, where each block presents a range of columns from the multiple alignment. E.g., the first block might have columns 1-60, the second block might have columns 61-95, the third block might have columns 96-128. Each block should have N rows, 1 row per sequence. The sequences should be in the same order in every block. Blocks are separated by 1 or more blank lines. Within a block there are no blank lines, and each line consists of 1 sequence identifier followed by some white space followed by characters (and gaps) for that sequence in the multiple alignment. In each column, all letters must be in upper case, or all letters must be in lower case. Upper case means that this column is to be given position-specific scores. Lower-case means to use the underlying matrix (specified by -M) for this column; e.g., if the query sequence has an 'l' residue in the column, then the standard scores for matching an L are used in the column. A sample usage would be:
blastpgp -i seq1 -B align1 -j 2 -d nr
where seq1 is the query
align1 is the alignment file
-j 2 indicates to do 2 rounds
-d nr indicates to use the nr database
The example files
seq1
align1
copied below were kindly supplied by L. Aravind from a paper he and Chris Ponting published in Protein Science: Aravind L, Ponting CP, Homologues of 26S proteasome subunits are regulators of transcription and translation, Protein Science 7(1998) 1250-1254. L. Aravind (aravind@ncbi.nlm.nih.gov) was the first user and helped define how -B should work. Y. Wolf (wolf@ncbi.nlm.nih.gov) helped design a more flexible input format for the alignments. If you like how -B works, let them know. If you do not like how -B works, complain to A. Schaffer(schaffer@helix.nih.gov) who did the implementation.
seq1
----
> 26SPS9_Hs
IHAAEEKDWKTAYSYFYEAFEGYDSIDSPKAITSLKYMLLCKIMLNTPEDVQALVSGKLALRYAGRQTEA
LKCVAQASKNRSLADFEKALTDYRAELRDDPIISTHLAKLYDNLLEQNLIRVIEPFSRVQIEHISSLIKL
SKADVERKLSQMILDKKFHGILDQGEGVLIIFDEPP
align1
------
26SPS9_Hs IHAAEEKDWKTAYSYFYEAFEGYdsidspkaitslkymllckimlntpedvqalvsgk
lalryagrqtealkcvaqasknr
F57B9_Ce LHAADEKDFKTAFSYFYEAFEGYdsvdekvsaltalkymllckvmldlpdevnsllsa
klalkyngsdldamkaiaaaaqk
YDL097c_Sc ILHCEDKDYKTAFSYFFESFESYhnltthnsyekacqvlkymllskimlnliddvkni
lnakytketyqsrgidamkavae
YMJ5_Ce LYSAEERDYKTSFSYFYEAFEGFasigdkinatsalkymilckimlneteqlagllaa
keivayqkspriiairsmadafr
FUS6_ARATH KNYIRTRDYCTTTKHIIHMCMNAilvsiemgqfthvtsyvnkaeqnpetlepmvnakl
rcasglahlelkkyklaarkfld
COS41.8_Ci SLDYKLKTYLTIARLYLEDEDPVqaemyinrasllqnetadeqlqihykvcyarvldy
rrkfleaaqrynelsyksaihet
644879 KCYSRARDYCTSAKHVINMCLNVikvsvylqnwshvlsyvskaestpeiaeqrgerds
qtqailtklkcaaglaelaarky
YPR108w_Sc IHCLAVRNFKEAAKLLVDSLATFtsieltsyesiatyasvtglftlertdlkskvids
pellslisttaalqsissltisl
eif-3p110_Hs SKAMKMGDWKTCHSFIINEKMNGkvw--------------------------------
T23D8.4_Ce SKAMLNGDWKKCQDYIVNDKMNQkvw--------------------------------
YD95_Sp IYLMSIRNFSGAADLLLDCMSTFsstellpyydvvryavisgaisldrvdvktkivds
pevlavlpqnesmssleacinsl
KIAA0107_Hs LYCVAIRDFKQAAELFLDTVSTFtsyelmdyktfvtytvyvsmialerpdlrekvikg
aeilevlhslpavrqylfslyec
F49C12.8_Hs LYRMSVRDFAGAADLFLEAVPTFgsyelmtyenlilytvitttfaldrpdlrtkvirc
nevqeqltggglngtlipvreyl
Int-6_Mm KFQYECGNYSGAAEYLYFFRVLVpatdrnalsslwgklaseilmqnwdaamedltrlk
etidnnsvssplqslqqrtwlih
26SPS9_Hs sladfekaltdy----------------------------------------------
F57B9_Ce rslkdfqvafgsf---------------------------------------------
YDL097c_Sc aynnrslldfntalkqy-----------------------------------------
YMJ5_Ce krslkdfvkalaeh--------------------------------------------
FUS6_ARATH vnpelgnsyneviapqdiatygglcalasfdrselkqkvidninfrnflelvpdvrel
indfyssryascleylasl------------------
COS41.8_Ci eqtkalekalncailapagqqrsrmlatlfkdercqllpsfgilekmfldriiksdem
eefar--------------------------------
644879 kqaakclllasfdhcdfpellspsnvaiygglcalatfdrqelqrnvissssfklfle
lepqvrdiifkfyeskyasclkmldem----------
YPR108w_Sc yasdyasyfpyllety------------------------------------------
eif-3p110_Hs ----------------------------------------------------------
T23D8.4_Ce ----------------------------------------------------------
YD95_Sp ylcdysgffrtladve------------------------------------------
KIAA0107_Hs rysvffqslavv----------------------------------------------
F49C12.8_Hs esyydchydrffiqlaale---------------------------------------
Int-6_Mm wslfvffnhpkgrdniidlflyqpqylnaiqtmcphilrylttavitnkdvrkrrqvl
kdlvkviqqesytykdpitefveclyvnfdfdgaqkk
26SPS9_Hs ----RAELRDDPIISTHLAKLYDNLLEQNLIRVIEPFSRVQIEHISSLIKLSKADVER
KLSQMILDKKFHGILDQGEGVLIIFDEPP
F57B9_Ce ----PQELQMDPVVRKHFHSLSERMLEKDLCRIIEPYSFVQIEHVAQQIGIDRSKVEK
KLSQMILDQKLSGSLDQGEGMLIVFEIAV
YDL097c_Sc ----EKELMGDELTRSHFNALYDTLLESNLCKIIEPFECVEISHISKIIGLDTQQVEG
KLSQMILDKIFYGVLDQGNGWLYVYETPN
YMJ5_Ce ----KIELVEDKVVAVHSQNLERNMLEKEISRVIEPYSEIELSYIARVIGMTVPPVER
AIARMILDKKLMGSIDQHGDTVVVYPKAD
FUS6_ARATH ----KSNLLLDIHLHDHVDTLYDQIRKKALIQYTLPFVSVDLSRMADAFKTSVSGLEK
ELEALITDNQIQARIDSHNKILYARHADQ
COS41.8_Ci ----QLMPHQKAITADGSNILHRAVTEHNLLSASKLYNNIRFTELGALLEIPHQMAEK
VASQMICESRMKGHIDQIDGIVFFERRET
644879 ----KDNLLLDMYLAPHVRTLYTQIRNRALIQYFSPYVSADMHRMAAAFNTTVAALED
ELTQLILEGLISARVDSHSKILYARDVDQ
YPR108w_Sc ----ANVLIPCKYLNRHADFFVREMRRKVYAQLLESYKTLSLKSMASAFGVSVAFLDN
DLGKFIPNKQLNCVIDRVNGIVETNRPDN
eif-3p110_Hs ----DLFPEADKVRTMLVRKIQEESLRTYLFTYSSVYDSISMETLSDMFELDLPTVHS
IISKMIINEELMASLDQPTQTVVMHRTEP
T23D8.4_Ce ----NLFHNAETVKGMVVRRIQEESLRTYLLTYSTVYATVSLKKLADLFELSKKDVHS
IISKMIIQEELSATLDEPTDCLIMHRVEP
YD95_Sp ----VNHLKCDQFLVAHYRYYVREMRRRAYAQLLESYRALSIDSMAASFGVSVDYIDR
DLASFIPDNKLNCVIDRVNGVVFTNRPDE
KIAA0107_Hs ----EQEMKKDWLFAPHYRYYVREMRIHAYSQLLESYRSLTLGYMAEAFGVGVEFIDQ
ELSRFIAAGRLHCKIDKVNEIVETNRPDS
F49C12.8_Hs ----SERFKFDRYLSPHFNYYSRGMRHRAYEQFLTPYKTVRIDMMAKDFGVSRAFIDR
ELHRLIATGQLQCRIDAVNGVIEVNHRDS
Int-6_Mm lrecESVLVNDFFLVACLEDFIENARLFIFETFCRIHQCISINMLADKLNMTPEEAER
WIVNLIRNARLDAKIDSKLGHVVMGNNAV
PHI-BlastPHI-BLAST (Pattern-Hit Initiated BLAST) is a search program that combines matching of regular expressions with local alignments surrounding the match. The most important features of the program have been incorporated into the BLAST software framework partly for user convenience and partly so that PHI-BLAST may be combined seamlessly with PSI-BLAST. Other features that do not fit into the BLAST framework will be released later as a separate program and/or separate Web page query options. One very restrictive way to identify protein motifs is by regular expressions that must contain each instance of the motif. The PROSITE database is a compilation of restricted regular expressions that describe protein motifs. Given a protein sequence S and a regular expression pattern P occurring in S, PHI-BLAST helps answer the question: What other protein sequences both contain an occurrence of P and are homologous to S in the vicinity of the pattern occurrences? PHI-BLAST may be preferable to just searching for pattern occurrences because it filters out those cases where the pattern occurrence is probably random and not indicative of homology. PHI-BLAST may be preferable to other flavors of BLAST because it is faster and because it allows the user to express a rigid pattern occurrence requirement. The pattern search methods in PHI-BLAST are based on the algorithms in: R. Baeza-Yates and G. Gonnet, Communications of the ACM 35(1992), pp. 74-82. S. Wu and U. Manber, Communications of the ACM 35(1992), pp. 83-91. The calculation of local alignments is done using a method very similar to (and much of the same code as) gapped BLAST. However, the method of evaluating statistical significance is different, and is described below. In the stand-alone mode the typical PHI-BLAST usage looks like: blastpgp -i -k -p patseedp where -i is followed by the file containing the query in FASTA format where -k is followed by the file containing the pattern in a syntax given below and "patseedp" indicates the mode of usage, not representing any file. The syntax for the query sequence is FASTA format as for all other BLAST queries. The syntax for patterns follows the rules of PROSITE and is documented in detail below. The specified pattern is not required to be in the PROSITE list. Most of the other BLAST flags can be used with PHI-BLAST. One important exception is that PHI-BLAST requires gapped alignments (i.e. forbids -g F in the flags) because ungapped alignments do not make sense for almost all patterns in PROSITE. There is a second mode of PHI-BLAST usage that is important when the specified pattern occurs more than 1 time in the query. In this case, the user may be interested in restricting the search for local alignments to a subset of the pattern occurrences. This can be done with a search that looks like: blastpgp -i -k -p seedp in which case the use of the "seedp" option requires the user to specify the location(s) of the interesting pattern occurrence(s) in the pattern file. The syntax for how to specify pattern occurrences is below. When there are multiple pattern occurrences in the query it may be important to decide how many are of interest because the E-value for matches is effectively multiplied by the number of interesting pattern occurrences. The PHI-BLAST Web page supports only the "patseedp" option. PHI-BLAST is integrated with PSI-BLAST. In the command-line mode, PSI-BLAST can be invoked by using the -j option, as usual. When this is done as: blastpgp -i -k -p patseedp -jthen the first round of searching uses PHI-BLAST and all subsequent rounds use PSI-BLAST. In the Web page setting, the user must explicitly invoke one round at a time, and the PHI-BLAST Web page provides the option to initiate a PSI-BLAST round with the PHI-BLAST results. To describe a combined usage, use the term "PHI-PSI-BLAST" (Pattern-Hit Initiated, Position-Specific Iterated BLAST). Determining statistical significance. When a query sequence Q matches a database sequence D in PHI-BLAST, it is useful to subdivide Q and D into 3 disjoint pieces
Qleft Qpattern Qright
Dleft Dpattern Dright
The substrings Qpattern and Dpattern contain the pattern specified in the pattern file. The pieces Qpattern and Dpattern are aligned and that alignment is displayed as part of the PHI-BLAST output, but the score for that alignment is mostly ignored. The "reduced" score r of an alignment is the sum of the scores obtained by aligning Qleft with Dleft and by aligning Qright with Dright.
The expected number of alignments with a reduced score >= x
is given by:
CN(Lambda*x + 1)e^(-Lambda *x)
where:
C and Lambda are "constants" depending on the score matrix and the
gap costs.
N is (number of occurrences of pattern in database) * (number of
occurrences of pattern in Q)
e is the base of the natural logarithm.
It is important to understand that this method of computing the statistical significance of a PHI-BLAST alignment is mathematically different from the method used for BLAST and PSI-BLAST alignments. However, both methods provide E-values, so they the E_values are displayed with a similar output syntax. Rules for pattern syntax for PHI-BLAST. The syntax for patterns in PHI-BLAST follows the conventions of PROSITE. When using the stand-alone program, it is permissible to have multiple patterns in a file separated by a blank line between patterns. When using the Web-page only one pattern is allowed per query.
Valid protein characters for PHI-BLAST patterns:
ABCDEFGHIKLMNPQRSTVWXYZU
Valid DNA characters for PHI-BLAST patterns:
ACGT
Other useful delimiters:
[ ] means any one of the characters enclosed in the brackets
e.g., [LFYT] means one occurrence of L or F or Y or T
- means nothing (this is a spacer character used by PROSITE)
x with nothing following means any residue
x(5) means 5 positions in which any residue is allowed (and
similarly for any other single number in parentheses after x)
x(2,4) means 2 to 4 positions where any residue is allowed,
and similarly for any other two numbers separated by a comma;
the first number should be < the second number.
> can occur only at the end of a pattern and means nothing
it may occur before a period
(another spacer used by PROSITE)
. may be used at the end of the pattern and means nothing
When using the stand-alone program, the pattern should
be in a file, with the first line starting:
ID
followed by 2 spaces and a text string giving the pattern a name.
There should also be a line starting
PA
followed by 2 spaces followed by the pattern description.
All other PROSITE codes in the first two columns are allowed,
but only the HI code, described below is relevant to PHI-BLAST.
Here is an example from PROSITE.
ID CNMP_BINDING_2; PATTERN.
AC PS00889;
DT OCT-1993 (CREATED); OCT-1993 (DATA UPDATE); NOV-1995 (INFO UPDATE).
DE Cyclic nucleotide-binding domain signature 2.
PA [LIVMF]-G-E-x-[GAS]-[LIVM]-x(5,11)-R-[STAQ]-A-x-[LIVMA]-x-[STACV].
NR /RELEASE=32,49340;
NR /TOTAL=57(36); /POSITIVE=57(36); /UNKNOWN=0(0); /FALSE_POS=0(0);
NR /FALSE_NEG=1; /PARTIAL=1;
CC /TAXO-RANGE=??EP?; /MAX-REPEAT=2;
The line starting
ID
gives the pattern a name.
The lines starting
AC, DT, DE, NR, NR, CC
are relevant to PROSITE users, but irrelevant to PHI-BLAST.
These lines are tolerated, but ignored by PHI-BLAST.
The line starting
PA
describes the pattern as:
one of LIVMF
followed by
G
followed by
E
followed by
any single character
followed by
one of GAS
followed by
one of LIVM
followed by
any 5 to 11 characters
followed by
R
followed by
one of STAQ
followed by
A
followed by
any single character
followed by
one of LIVMA
followed by
any single character
followed by
one of STACV
In this case the pattern ends with a period.
It can end with nothing after the last specifying symbol
or any number of > signs or periods or combination thereof.
Here is another example, illustrating the use of an HI line.
ID ER_TARGET; PATTERN.
PA [KRHQSA]-[DENQ]-E-L>.
HI (19 22)
HI (201 204)
In this example, the HI lines specify that the pattern occurs twice, once from positions 19 through 22 in the sequence and once from positions 201 through 204 in the sequence. These specifications are relevant when stand-alone PHI-BLAST is used with the seedp option, in which the interesting occurrences of the pattern in the sequence are specified. In this case the HI lines specify which occurrence(s) of the pattern should be used to find good alignments. In general, the seedp option is more useful than the standard patternp option ONLY when the pattern occurs K > 1 times in the sequence AND the user is interested in matching to J < K of those occurrences. Then using the HI lines enables the user to specify which occurrences are of interest. Additional functionality related to PHI-BLAST. PHI-BLAST takes as input both a sequence and a query containing that sequence and searches a sequence database for other sequences containing the same pattern and having a good alignment. One may be interested in asking two related, simpler questions: 1. Given a sequence and a database of patterns, which patterns occur in the sequence and where? 2. Given a pattern and a sequence database, which sequences contain the pattern and where? These queries can be answered wih software closely related to PHI-BLAST, but they do not fit into the output framework of BLAST because the answers are simple lists without alignments and with no notion of statistical significance. The NCBI toolbox includes another program, currently called seedtop to answer the two queries above. Query 1 can be asked with: seedtop -i -k -p patmatchp Query 2 can be asked with: seedtop -d -k -p patternp The -k argument is used similarly in all queries and the file format is always the same. The standard pattern database is PROSITE, but others (or a subset) can be used. There are plans afoot to offer the patmatchp query (number 1) on the PHI-BLAST web page or in its vicinity, but this would be restricted to having PROSITE as the pattern database. Documentation for PSI-TBLASTN PSI-BLASTN is a variant of blastall that searches a protein query sequence against a nucleotide sequence database using a position specific matrix created by PSI-BLAST. The nucleotide sequence database is dynamically translated in all reading frames during PSI-TBLASTN search. Using a position specific matrix may enable finding more distantly related sequences.
Programs:
blastpgp [takes a protein query and perform PSI-BLAST search to
creates a position specific matrix using a protein
database]
blastall [reads position specific matrix and performs PSI-TBLASTN
search]
Usage: A user would typically run blastpgp to create and save a position specific matrix, followed by a run of blastall for PSI-TBLASTN search. blastpgp must be executed with -C option followed by a file name to save position specific score matrix. blastall with "-p psitblastn" option executes PSI-TBLASTSN search, and -R option followed by a file name specifying the file that contains position specific score matrix. All other options that apply when using "blastall -p tblastn ..." also apply when using "blastall -p psitblastn ...", but there are some restrictions to parameters: 1) The query must be the same as the one used in blastpgp for creating a position specific matrix. 2) By default, blastpgp has filtering off (-F F) and blastall has filtering on (-F T). To ensure consistent usage of the blastpgp/psitblastn combination, the -F option should be explicitly set in one or the other run. Example: One may run PSI-BLST to create and save a position specific score matrix as follows:
blastpgp -d nr -i ff.chd -j 2 -C ff.chd.ckp
Position specific score matrix is saved in ff.chd.ckp. Then, using this matrix, one may run PSI-TBLASTN search:
blastall -i ff.chd -d yeast -p psitblastn -R ff.chd.ckp
Note that this allows the score matrix to be constructed using one database (nr in the example) and then used to search a second database (yeast in the example). Even if the two database names are the same, blastpgp uses the protein version while "blastall -p psitblastn" uses the DNA version. |
