RPS Blast: Reversed Position Specific Blast RPS-BLAST (Reverse PSI-BLAST) searches a query sequence against a database of profiles. This is the opposite of PSI-BLAST that searches a profile against a database of sequences, hence the 'Reverse'. RPS-BLAST uses a BLAST-like algorithm, finding single- or double-word hits and then performing an ungapped extension on these candidate matches. If a sufficiently high-scoring ungapped alignment is produced, a gapped extension is performed and those (gapped) alignments with sufficiently low expect value are reported. This procedure is in contrast to IMPALA that performs a Smith-Waterman calculation between the query and each profile, rather than using a word-hit approach to identify matches that should be extended. RPS-BLAST uses a BLAST database, but also has some other files that contain a precomputed lookup table for the profiles to allow the search to proceed faster. Unfortunately it was not possible to make this lookup table architecture independent (like the BLAST databases themselves) and one cannot take a RPS-BLAST databases prepared on a big-endian system (e.g., Solaris Sparc) and run it on a small-endian system (e.g., NT). The RPS-BLAST database must be prepared again for the small-endian system. The CD-Search databases for RPS-BLAST can be found at: ftp://ftp.ncbi.nih.gov/pub/mmdb/cdd/ It is necessary to untar the archive and run copymat and formatdb. It is not necessary to run makemat on the databases from this directory. RPS-BLAST was coded by Sergei Shavirin with some help from Tom Madden. RPS-BLAST reuses some of the IMPALA code for precomputing the lookup tables and all of the IMPALA code for evaluating the statistical significance of a match. 1. Binary files used in RPS Blast: The following binary files are used to setup and run RPS Blast: makemat : primary profile preprocessor (converts a collection of binary profiles, created by the -C option of PSI-BLAST, into portable ASCII form); copymat : secondary profile preprocessor (converts ASCII matrices, produced by the primary preprocessor, into database that can be read into memory quickly); formatdb : general BLAST database formatter. rpsblast : search program (searches a database of score matrices, prepared by copymat, producing BLAST-like output). 2. Conversion of profiles into searchable database *Note*: if you are starting with *.mtx files obtained from the NCBI FTP site or another source you should skip the steps listed in 2.1. 2.1. Primary preprocessing Prepare the following files: i. a collection of PSI-BLAST-generated profiles with arbitrary names and suffix .chk; ii. a collection of "profile master sequences", associated with the profiles, each in a separate file with arbitrary name and a 3 character suffix starting with c; the sequences can have deflines; they need not be sequences in nr or in any other sequence database; if the sequences have deflines, then the deflines must be unique. iii. a list of profile file names, one per line, named <database_name>.pn; iv. a list of master sequence file names, one per line, in the same order as a list of profile names, named <database_name>.sn; The following files will be created: a. a collection of ASCII files, corresponding to each of the original profiles, named <profile_name>.mtx; b. a list of ASCII matrix files, named <database_name>.mn; c. ASCII file with auxiliary information, named <database_name>.aux; Arguments to makemat: -P database name (required) -G Cost to open a gap (optional) default = 11 -E Cost to extend a gap (optional) default = 1 -U Underlying amino acid scoring matrix (optional) default = BLOSUM62 -d Underlying sequence database used to create profiles (optional) default = nr -z Effective size of sequence database given by -d default = current size of -d option Note: It may make sense to use -z without -d when the profiles were created with an older, smaller version of an existing database -S Scaling factor for matrix outputs to avoid round-off problems default = PRO_DEFAULT_SCALING_UP (currently defined as 100) Use 1.0 to have no scaling Output scores will be scaled back down to a unit scale to make them look more like BLAST scores, but we found working with a larger scale to help with roundoff problems. -H get help (overrides all other arguments) Note: It is not enforced that the values of -G and -E passed to makemat were actually used in making the checkpoints. However, the values fed in to makemat are propagated to copymat and rpsblast. ATTENTION: It is strongly recommended to use -S 1 - the scaling factor should be set to 1 for rpsblast at this point in time. 2.2. Secondary preprocessing Prepare the following files: i. a collection of ASCII files, corresponding to each of the original profiles, named <profile_name>.mtx (created by makemat); ii. a collection of "profile master sequences", associated with the profiles, each in a separate file with arbitrary name and a 3 character suffix starting with c. iii. a list of ASCII_matrix files, named <database_name>.mn (created by makemat); iv. a list of master sequence file names, one per line, in the same order as a list of matrix names, named <database_name>.sn; v. ASCII file with auxiliary information, named <database_name>.aux (created by makemat); The files input to copymatices are in ASCII format and thus portable between machines with different encodings for machine-readable files The following files will be created: a. a huge binary file, containing all profile matrices, named <database_name>.rps; b. a huge binary file, containing lookup table for the Blast search corresponding to matrixes named <database_name>.loo c. File containing concatenation of all FASTA "profile master sequences". named <database_name> (without extention) Arguments to copymat -P database name (required) -H get help (overrides all other arguments) -r format data for RPS Blast ATTENTION: "-r" parameter have to be set to TRUE to format data for RPS Blast at this step. NOTE: copymat requires a fair amount of memory as it first constructs the the lookup table in memory before writing it to disk. Users have found that they require a machine with at least 500 Meg of memory for this task. 2.3 Creating of BLAST database from <database_name> file containing all "profile master sequences". "formatdb" program should be run to create regular BLAST database of all "profile master sequences": formatdb -i <database_name> -o T 3. Search Arguments to RPS Blast -i query sequence file (required) -d RPS BLAST Database [File In] -p if query sequence protein (if FALSE 6 frame franslation will be conducted as in blastx program) -P 0 for multiple hits 1-pass, 1 for single hit 1-pass [Integer] default = 0 -o output file (optional) default = stdout -e Expectation value threshold (E), (optional, same as for BLAST) default = 10 -m alignment view (optional, same as for BLAST) -z effective length of database (optional) -1 = length given via -z option to makemat default (0) implies length is actual length of profile library adjusted for end effects APPENDIX: A. Documentation of the .mtx file format Format of the .mtx file: L = Length of SEQ SEQ = Sequence ka#-* = Karlin/Altschul parameters, block #. There are three blocks, each containing four floating point numbers on separate lines. pX-Y = The position specific scores as integers. The first element of this file format is [L]. This is the sequence length. The second line contains the sequence itself, in NCBI AA notation. After this, there are three KA blocks (four lines of floating point numbers each), then the positional scores. The positional scores are arranged in a grid. Each line contains 26 elements, corresponding to the 26 elements in the NCBI AA encoding, and there are L lines where L is the previously mentioned sequence length. Using the symbols mentioned above, it looks something like this: <L> <SEQ> <ka1-1> <ka1-2> <ka1-3> <ka1-4> <ka2-1> <ka2-2> <ka2-3> <ka2-4> <ka3-1> <ka3-2> <ka3-3> <ka3-4> <p1-1> <p1-2> <p1-3> ... <p1-26> <p2-1> <p2-2> <p2-3> ... <p2-26> ... <pL-1> <pL-2> <pL-3> ... <pL-26> One can find the explanation for the three blocks of KA-parameters in makemat's source code, lines 188-190: putMatrixKbp(checkFile, compactSearch->kbp_gap_std, scaleScores, 1/scalingFactor); putMatrixKbp(checkFile, compactSearch->kbp_gap_psi, scaleScores, 1/scalingFactor); putMatrixKbp(checkFile, sbp->kbp_ideal, scaleScores, 1/scalingFactor); Thus, the first KA block is the standard score, the second is for PSI-Blast, and the third is the ideal score.