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RPS Blast: Reversed Position Specific Blast
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RPS-BLAST (Reverse PSI-BLAST) searches a query sequence against a database
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of profiles. This is the opposite of PSI-BLAST that searches a profile
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against a database of sequences, hence the 'Reverse'. RPS-BLAST
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uses a BLAST-like algorithm, finding single- or double-word hits
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and then performing an ungapped extension on these candidate matches.
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If a sufficiently high-scoring ungapped alignment is produced, a gapped
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extension is performed and those (gapped) alignments with sufficiently
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low expect value are reported. This procedure is in contrast to IMPALA
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that performs a Smith-Waterman calculation between the query and
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each profile, rather than using a word-hit approach to identify
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matches that should be extended.
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RPS-BLAST uses a BLAST database, but also has some other files that
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contain a precomputed lookup table for the profiles to allow the search
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to proceed faster. Unfortunately it was not possible to make this
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lookup table architecture independent (like the BLAST databases themselves)
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and one cannot take a RPS-BLAST databases prepared on a big-endian
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system (e.g., Solaris Sparc) and run it on a small-endian system
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(e.g., NT). The RPS-BLAST database must be prepared again for the small-endian
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The CD-Search databases for RPS-BLAST can be found at:
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ftp://ftp.ncbi.nih.gov/pub/mmdb/cdd/
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It is necessary to untar the archive and run copymat and formatdb.
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It is not necessary to run makemat on the databases from this
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RPS-BLAST was coded by Sergei Shavirin with some help from Tom Madden.
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RPS-BLAST reuses some of the IMPALA code for precomputing the lookup tables
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and all of the IMPALA code for evaluating the statistical significance of a match.
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1. Binary files used in RPS Blast:
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The following binary files are used to setup and run RPS Blast:
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makemat : primary profile preprocessor
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(converts a collection of binary profiles, created by the -C option
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of PSI-BLAST, into portable ASCII form);
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copymat : secondary profile preprocessor
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(converts ASCII matrices, produced by the primary preprocessor,
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into database that can be read into memory quickly);
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formatdb : general BLAST database formatter.
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rpsblast : search program (searches a database of score
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matrices, prepared by copymat, producing BLAST-like output).
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2. Conversion of profiles into searchable database
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*Note*: if you are starting with *.mtx files obtained from the NCBI FTP site or
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another source you should skip the steps listed in 2.1.
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2.1. Primary preprocessing
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Prepare the following files:
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i. a collection of PSI-BLAST-generated profiles with arbitrary
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names and suffix .chk;
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ii. a collection of "profile master sequences", associated with
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the profiles, each in a separate file with arbitrary name and a 3 character
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suffix starting with c;
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the sequences can have deflines; they need not be sequences in nr or
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in any other sequence database; if the sequences have deflines, then
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the deflines must be unique.
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iii. a list of profile file names, one per line, named
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<database_name>.pn;
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iv. a list of master sequence file names, one per line, in the same
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order as a list of profile names, named
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<database_name>.sn;
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The following files will be created:
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a. a collection of ASCII files, corresponding to each of the
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original profiles, named
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<profile_name>.mtx;
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b. a list of ASCII matrix files, named
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<database_name>.mn;
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c. ASCII file with auxiliary information, named
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<database_name>.aux;
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Arguments to makemat:
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-P database name (required)
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-G Cost to open a gap (optional)
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-E Cost to extend a gap (optional)
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-U Underlying amino acid scoring matrix (optional)
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-d Underlying sequence database used to create profiles (optional)
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-z Effective size of sequence database given by -d
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default = current size of -d option
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Note: It may make sense to use -z without -d when the
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profiles were created with an older, smaller version of an
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-S Scaling factor for matrix outputs to avoid round-off problems
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default = PRO_DEFAULT_SCALING_UP (currently defined as 100)
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Use 1.0 to have no scaling
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Output scores will be scaled back down to a unit scale to make
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them look more like BLAST scores, but we found working with a larger
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scale to help with roundoff problems.
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-H get help (overrides all other arguments)
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Note: It is not enforced that the values of -G and -E passed to makemat
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were actually used in making the checkpoints. However, the values fed
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in to makemat are propagated to copymat and rpsblast.
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ATTENTION: It is strongly recommended to use -S 1 - the scaling factor
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should be set to 1 for rpsblast at this point in time.
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2.2. Secondary preprocessing
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Prepare the following files:
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i. a collection of ASCII files, corresponding to each of the
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original profiles, named
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<profile_name>.mtx
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(created by makemat);
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ii. a collection of "profile master sequences", associated with
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the profiles, each in a separate file with arbitrary name and a 3 character
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suffix starting with c.
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iii. a list of ASCII_matrix files, named
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<database_name>.mn
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(created by makemat);
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iv. a list of master sequence file names, one per
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line, in the same order as a list of matrix names, named
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<database_name>.sn;
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v. ASCII file with auxiliary information, named
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<database_name>.aux
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(created by makemat);
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The files input to copymatices are in ASCII format and thus portable
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between machines with different encodings for machine-readable files
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The following files will be created:
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a. a huge binary file, containing all profile matrices, named
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<database_name>.rps;
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b. a huge binary file, containing lookup table for the Blast search
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corresponding to matrixes named <database_name>.loo
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c. File containing concatenation of all FASTA "profile master sequences".
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named <database_name> (without extention)
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-P database name (required)
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-H get help (overrides all other arguments)
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-r format data for RPS Blast
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ATTENTION: "-r" parameter have to be set to TRUE to format data for
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RPS Blast at this step.
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NOTE: copymat requires a fair amount of memory as it first constructs
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the the lookup table in memory before writing it to disk. Users have
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found that they require a machine with at least 500 Meg of memory for this
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2.3 Creating of BLAST database from <database_name> file containing
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all "profile master sequences".
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"formatdb" program should be run to create regular BLAST database of all
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"profile master sequences":
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formatdb -i <database_name> -o T
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Arguments to RPS Blast
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-i query sequence file (required)
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-d RPS BLAST Database [File In]
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-p if query sequence protein (if FALSE 6 frame franslation will be
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conducted as in blastx program)
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-P 0 for multiple hits 1-pass, 1 for single hit 1-pass [Integer]
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-o output file (optional)
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-e Expectation value threshold (E), (optional, same as for BLAST)
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-m alignment view (optional, same as for BLAST)
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-z effective length of database (optional)
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-1 = length given via -z option to makemat
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default (0) implies length is actual length of profile library
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adjusted for end effects
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A. Documentation of the .mtx file format
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Format of the .mtx file:
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ka#-* = Karlin/Altschul parameters, block #. There are three blocks,
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each containing four floating point numbers on separate lines.
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pX-Y = The position specific scores as integers.
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The first element of this file format is [L]. This is the sequence
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length. The second line contains the sequence itself, in NCBI AA
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notation. After this, there are three KA blocks (four lines of
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floating point numbers each), then the positional scores.
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The positional scores are arranged in a grid. Each line contains 26
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elements, corresponding to the 26 elements in the NCBI AA encoding,
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and there are L lines where L is the previously mentioned sequence
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Using the symbols mentioned above, it looks something like this:
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<p1-1> <p1-2> <p1-3> ... <p1-26>
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<p2-1> <p2-2> <p2-3> ... <p2-26>
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<pL-1> <pL-2> <pL-3> ... <pL-26>
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One can find the explanation for the three blocks of KA-parameters in
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makemat's source code, lines 188-190:
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putMatrixKbp(checkFile, compactSearch->kbp_gap_std[0], scaleScores, 1/scalingFactor);
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putMatrixKbp(checkFile, compactSearch->kbp_gap_psi[0], scaleScores, 1/scalingFactor);
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putMatrixKbp(checkFile, sbp->kbp_ideal, scaleScores, 1/scalingFactor);
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Thus, the first KA block is the standard score, the second is for
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PSI-Blast, and the third is the ideal score.
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