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+.nr pp 11
+.nr sp 11
+.nr tp 11
+.nr fp 10
+.nr fi 0n
+.sz 11
+.if t \{
+.po 1i
+.he 'FASTA3.DOC''Release 3.4, Fall, 2003'
+.fo ''- % -''
+\}
+.if n \{
+.po 0
+.na
+.nh
+\}
+.ll 6.5i
+.ce
+\fBCOPYRIGHT NOTICE\fP
+.lp
+Copyright 1988, 1991, 1992, 1994, 1995, 1996, 1999 by William
+R. Pearson and the University of Virginia. All rights reserved. The
+FASTA program and documentation may not be sold or incorporated into a
+commercial product, in whole or in part, without written consent of
+William R. Pearson and the University of Virginia. For further
+information regarding permission for use or reproduction, please
+contact: David Hudson, Assistant Provost for Research, University of
+Virginia, P.O. Box 9025, Charlottesville, VA 22906-9025, (434)
+924-6853
+.uh "\s+2The FASTA program package\s0"
+.uh "Introduction"
+.pp
+This documentation describes the version 3 of the FASTA program
+package (see W. R. Pearson and D. J. Lipman (1988), "Improved Tools
+for Biological Sequence Analysis", PNAS 85:2444-2448 [.wrp881.]; W. R.
+Pearson (1996) "Effective protein sequence comparison"
+Meth. Enzymol. 266:227-258;[.wrp960.] Pearson et. al. (1997) Genomics
+46:24-36;[.wrp971.] Pearson, (1999) Meth. in Molecular Biology
+132:185-219.[.wrp000.] Version 3 of the FASTA packages contains many
+programs for searching DNA and protein databases and one program
+(prss3) for evaluating statistical significance from randomly shuffled
+sequences. Several additional analysis programs, including programs
+that produce local alignments, are available as part of version 2 of
+the FASTA package, which is still available.
+.pp
+This document is divided into three sections: (1) A summary overview of
+the programs in the FASTA3 package; (2) A guide to installing the
+programs and databases; (3) A guide to using the FASTA programs. The
+revision history of the programs can be found in the
+\fCreadme.v30..v34\fP, files. The programs are easy to use, so if
+you are using them on a machine that is administered by someone else,
+you can skip section (2) and focus on (1) and (3) to learn how to use
+the programsIf you are installing the programs on your own
+machine, you will need to read section (2) carefully.
+.sh 1 "An overview of the \f(CBFASTA\fP programs"
+.pp
+Although there are a large number of programs in this package, they
+belong to three groups: (1)
+"Conventional" Library search programs:
+FASTA3, FASTX3, FASTY3, TFASTA3, TFASTX3, TFASTY3, SSEARCH3;
+(2)
+Programs for searching with short fragments:
+FASTS3, FASTF3, TFASTS3, TFASTF3;
+(3)
+Statistical significance: PRSS3.
+Programs that start with \f(CBfast\fP search protein
+databases, while \f(CBtfast\fP programs search translated DNA databases.
+Table I gives a brief description of the programs.
+.lp
+.(z
+.TS
+center;
+c s
+c s
+= =
+l lw(5.5i).
+\d\fBTable I. Comparison programs in the FASTA3 package\fP\u
+
+\fCfasta3\fP T{
+Compare a protein sequence to a protein sequence
+database or a DNA sequence to a DNA sequence database using the FASTA
+algorithm.[.wrp881,wrp960.] Search speed and selectivity are
+controlled with the \fIktup\fP(wordsize) parameter. For protein
+comparisons, \fIktup\fP = 2 by default; \fIktup\fP =1 is more sensitive
+but slower. For DNA comparisons, \fIktup\fP=6 by default; \fIktup\fP=3 or
+\fIktup\fP=4 provides higher sensitivity; \fIktup\fP=1 should be used for
+oligonucleotides (DNA query lengths < 20).
+T}
+
+\fCssearch3\fP T{
+Compare a protein sequence to a protein sequence
+database or a DNA sequence to a DNA sequence database using the
+Smith-Waterman algorithm.[.wat815.] \fCssearch3\fP is about 10-times
+slower than FASTA3, but is more sensitive for full-length protein
+sequence comparison.
+T}
+
+\fCfastx3\fP/ \fCfasty3\fP T{
+Compare a DNA sequence to a protein
+sequence database, by comparing the translated DNA sequence in three
+frames and allowing gaps and frameshifts. \fCfastx3\fP uses a
+simpler, faster algorithm for alignments that allows frameshifts only
+between codons; \fCfasty3\fP is slower but produces better alignments
+with poor quality sequences because frameshifts are allowed within
+codons.
+T}
+
+\fCtfastx3\fP/ \fCtfasty3\fP T{
+Compare a protein sequence to a DNA sequence
+database, calculating similarities with frameshifts to the forward and
+reverse orientations.
+T}
+
+\fCtfasta3\fP T{
+Compare a protein sequence to a DNA sequence database, calculating
+similarities (without frameshifts) to the 3 forward and three reverse
+reading frames. \fCtfastx3\fP and \fCtfasty3\fP are preferred because
+they calculate similarity over frameshifts.
+T}
+
+\fCfastf3/tfastf3\fP T{
+Compares an ordered peptide mixture, as would be obtained by
+Edman degredation of a CNBr cleavage of a protein, against a protein
+(\fCfastf\fP) or DNA (\fCtfastf\fP) database.
+T}
+
+\fCfasts3/tfasts3\fP T{
+Compares set of short peptide fragments, as would be obtained
+from mass-spec. analysis of a protein, against a
+protein (\fCfasts\fP) or DNA (\fCtfasts\fP) database.
+T}
+= =
+.TE
+.)z
+.sh 1 "Installing FASTA and the sequence databases"
+.sh 2 "Obtaining the libraries"
+.pp
+The FASTA program package does not include any protein or DNA sequence
+libraries. Protein databases are available on CD-ROM from the PIR and
+EMBL (see below), or via anonymouse FTP from many different sources.
+As this document is updated in the fall of 1999, no DNA databases are
+available on CD-ROM from the major sequence databases: Genbank at the
+National for Biotechnology Information (\fCwww.ncbi.nlm.nih.gov\fP and
+\fCftp://ncbi.nlm.nih.gov\fP) and EMBL at the European Bioinformatics
+Institute (\fCwww.ebi.ac.uk\fP). However, the databases are available
+via anonymous FTP from both sites.
+.sh 3 "The GENBANK DNA sequence library"
+.pp
+Because of the large size of DNA databases, you will probably want to
+keep DNA databases in only one, or possibly two, formats. The FASTA3
+programs that search DNA databases - \fCfasta3\fP, \fCtfastx/y3\fP,
+and \fCtfasta3\fP can read DNA databases in Genbank flatfile (not
+ASN.1), FASTA, GCG/compressed-binary, BLAST1.4 (\fCpressdb\fP), and
+BLAST2.0 (\fCformatdb\fP) formats, as well as EMBL format. If you are
+also running the GCG suite of sequence analysis programs, you should
+use GCG/compressed-binary format or BLAST2.0 format for your
+\fCfasta3\fP searches. If not, BLAST2.0 is a good choice. These
+files are considerably more compact than Genbank flat files, and are
+preferred. The NCBI does not provide software for converting from
+Genbank flat files to Blast2.0 DNA databases, but you can use the
+Blast \fCformatdb\fP program to convert ASN.1 formated Genbank files,
+which are available from the NCBI \fCftp\fP site.
+.pp
+The NCBI also provides the \fCnr\fP, \fCswissprot\fP, and several EST
+databases that are used by BLAST in FASTA format from:
+\fCftp://ncbi.nlm.nih.gov/blast/db\fP. These databases are updated
+nightly.
+.sh 3 "The NBRF protein sequence library"
+.pp
+You can obtain the PIR protein sequence database
+[.pir980.] from:
+.(l
+National Biomedical Research Foundation
+Georgetown University Medical Center
+3900 Reservoir Rd, N.W.
+Washington, D.C. 20007
+.)l
+or via ftp from \fCnbrf.georgetown.edu\fP or from the NCBI
+(\fCncbi.nlm.nih.gov/repository/PIR\fP). The data in the \fCascii\fP
+directory is in PIR Codata format, which is not widely used. I
+recommend the PIR/VMS format data (libtype=5) in the \fCvms\fP
+directory.
+.sh 3 "The EBI/EMBL CD-ROM libraries"
+.pp
+The European Bioinformatics Institute (EBI) distributes both the EMBL
+DNA database and the SwissProt database on CD-ROM,[.apw961.] and they
+are available from:
+.(l
+EMBL-Outstation European Bioinformatics Institute
+Wellcome Trust Genome Campus,
+Hinxton Hall
+Hinxton,
+Cambridge CB10 1SD
+United Kingdom
+Tel: +44 (0)1223 494444
+Fax: +44 (0)1223 494468
+Email: DATALIB@ebi.ac.uk
+.)l
+In addition, the SWISS-PROT protein sequence database is available via
+anonymous FTP from \fCftp://ftp.expasy.ch/databases/swiss-prot/\fP
+(also see \fCwww.expasy.ch\fP).
+.sh 2 "Finding the libraries: FASTLIBS"
+.pp
+The major problem that most new users of the FASTA package have is in
+setting up the program to find the databases and their library type.
+In general, if you cannot get \fCfasta3\fP to read a sequence
+database, it is likely that something is wrong with the \fCFASTLIBS\fP
+file. A common problem is that the database file is found, but either
+no sequences are read, or an incorrect number of entries is read.
+This is almost always because the library format (\fClibtype\fP) is
+incorrect. Note that a type 5 file (PIR/VMS format) can be read
+as a type 0 (default FASTA) format file, and the number of entries
+will be correct, but the sequence lengths will not.
+.pp
+All the search programs in the FASTA3 package use the environment
+variable \fCFASTLIBS\fP to find the protein and DNA sequence libraries. The
+\fCFASTLIBS\fP variable contains the name of a file that has the actual
+filenames of the libraries. The \fCfastlibs\fP file included with the
+distribution on is an example of a file that can be referred to by
+FASTLIBS. To use the \fCfastlibs\fP file, type:
+.(l
+\fCsetenv FASTLIBS /usr/lib/fasta/fastgbs\fP (BSD UNIX/csh)
+or
+\fCexport FASTLIBS=/usr/lib/fasta/fastgbs\fP (SysV UNIX/ksh)
+.)l
+Then edit the \fCfastlibs\fP file to indicate where the protein and DNA
+sequence libraries can be found. If you have a hard disk and your
+protein sequence library is kept in the file \fC/usr/lib/aabank.lib\fP and
+your Genbank DNA sequence library is kept in the directory:
+\fC/usr/lib/genbank\fP, then \fCfastgbs\fP might contain:
+.ne 8
+.(l
+.ft C
+NBRF Protein$0P/usr/lib/seq/aabank.lib 0
+SWISS PROT 10$0S/usr/lib/vmspir/swiss.seq 5
+GB Primate$1P@/usr/lib/genbank/gpri.nam
+GB Rodent$1R@/usr/lib/genbank/grod.nam
+GB Mammal$1M@/usr/lib/genbank/gmammal.nam
+^ 1 ^^^^ 4 ^ ^
+ 23 (5)
+.ft R
+.)l
+The first line of this file says that there is a copy of the NBRF
+protein sequence database (which is a protein database) that can be
+selected by typing "P" on the command line or when the database menu
+is presented in the file \fC/usr/lib/seq/aabank.lib\fP.
+.pp
+Note that there are 4 or 5 fields in the lines in \fCfastgbs\fP. The first
+field is the description of the library which will be displayed by
+FASTA; it ends with a '$'. The second field (1 character), is a 0 if
+the library is a protein library and 1 if it is a DNA library. The
+third field (1 character) is the character to be typed to select the
+library.
+.pp
+The fourth field is the name of the library file. In the example
+above, the \fC/usr/lib/seq/aabank.lib\fP file contains the entire
+protein sequence library. However the DNA library file names are
+preceded by a '@', because these files (\fCgpri.nam, grod.nam,
+gmammal.nam\fP) do not contain the sequences; instead they contain the names
+of the files which contain the sequences. This is done because the
+GENBANK DNA database is broken down in to a large number of smaller
+files. In order to search the entire primate database, you must
+search more than a dozen files.
+.pp
+In addition, an optional fifth field can be used to specify the format
+of the library file. Alternatively, you can specify the library
+format in a file of file names (a file preceded by an '@'). This
+field must be separated from the file name by a space character ('\ ')
+from the filename. In the example above, the \fCaabank.lib\fP file is
+in Pearson/FASTA format, while the \fCswiss.seq\fP file is in PIR/VMS format
+(from the EMBL CD-ROM). Currently, FASTA can read the following formats:
+.(l I
+.ft C
+0 Pearson/FASTA (>SEQID - comment/sequence)
+1 Uncompressed Genbank (LOCUS/DEFINITION/ORIGIN)
+2 NBRF CODATA (ENTRY/SEQUENCE)
+3 EMBL/SWISS-PROT (ID/DE/SQ)
+4 Intelligenetics (;comment/SEQID/sequence)
+5 NBRF/PIR VMS (>P1;SEQID/comment/sequence)
+6 GCG (version 8.0) Unix Protein and DNA (compressed)
+11 NCBI Blast1.3.2 format (unix only)
+12 NCBI Blast2.0 format (unix only, fasta32t08 or later)
+.ft R
+.)l
+In particular, this version will work with the EMBL and PIR VMS
+formats that are distributed on the EMBL CD-ROM. The latter format
+(PIR VMS) is much faster to search than EMBL format. This release
+also works with the protein and DNA database formats created for the
+BLASTP and BLASTN programs by SETDB and PRESSDB and with the new NCBI
+search format. If a library format is not specified, for example,
+because you are just comparing two sequences, Pearson/FASTA (format 0)
+is used by default. To specify a library type on the command line,
+add it to the library filename and surround the filename and library
+type in quotes:
+.(l
+.ft C
+fasta3 query.file "/seqdb/genbank/gbpri1.seq 1"
+.ft P
+.)l
+.pp
+You can specify a group of library files by putting a '@' symbol
+before a file that contains a list of file names to be searched. For
+example, if @gmam.nam is in the fastgbs file, the file "gmam.nam"
+might contain the lines:
+.(l
+.ft C
+</seqdb/genbank
+gbpri1.seq 1
+gbpri2.seq 1
+gbpri3.seq 1
+gbpri4.seq 1
+gbrod.seq 1
+gbmam.seq 1
+.ft R
+.)l
+In this case, the line beginning with a '<' indicates the directory
+the files will be found in. The remaining lines name the actual
+sequence files. So the first sequence file to be searched would be:
+.(l
+.ft C
+/usr/lib/genbank/gbpri.seq
+.ft R
+.)l
+The notation "\fC<PIRNAQ:\fP" might be used under the VAX/VMS operating
+system. Under UNIX, the trailing '/' is left off, so the library
+directory might be written as "\fC</usr/seqlib\fP".
+.pp
+The FASTA programs can search a database composed of different files
+in different sequence formats. For example, you may wish to search
+the Genbank files (in GenBank flat file format) and the EMBL DNA
+sequence database on CD-ROM. To do this, you simply list the names
+and filetypes of the files to be searched in a file of filenames. For
+example, to search the mammalian portion of Genbank, the unannotated
+portion of Genbank, and the unannotated portion of the EMBL library,
+you could use the file:
+.(l I
+.ft C
+</usr/lib/DNA
+gbpri.seq 1
+\&# (this '#' causes the program to display the size of the library)
+gbrod.seq 1
+\&...
+gbmam.seq 1
+\&...
+gbuna.seq 1
+\&...
+unanno.seq 5
+\&#
+.ft R
+.)l
+.(l I F
+You do not need to include library format numbers if you only use the
+Pearson/FASTA version of the PIR protein sequence library. If no
+library type is specified, the program assumes that type 0 is being
+used.
+.)l
+.pp
+Test the setup by running FASTA. Enter the sequence
+file '\fCmgstm1.aa\fP' when the program requests it (this file is
+included with the programs). The program should then ask you to
+select a protein sequence library. Alternatively, if you run the
+TFASTA program and use the mgstm1.aa query sequence, the program
+should show you a selection of DNA sequence libraries.
+Once the fastgbs file has been set up correctly, you can
+set FASTLIBS=fastgbs in your AUTOEXEC.BAT file, and you will not need to
+remember where the libraries are kept or how they are named.
+.ne 8
+.sh 1 "Using the FASTA Package"
+.sh 2 "Overview"
+.pp
+The FASTA sequence comparison programs all require similar
+information, the name of a query sequence file, a library file, and
+the \fIktup\fP parameter. All of the programs can accept arguments
+on the command line, or they will prompt for the file names and
+\fIktup\fP value.
+.lp
+To use FASTA, simply type:
+.(l
+.ft C
+\f(CBFASTA\fP
+and you will be prompted for :
+.in +0.5i
+the name of the test sequence file
+the name of the library file
+and whether you want ktup = 1 or 2. (or 1 to 6 for DNA sequences)
+(ktup of 2 is about 5 times faster than ktup = 1)
+.ft R
+.)l
+The program can also be run by typing
+.(l
+.ft C
+FASTA test.aa /lib/bigfile.lib \fIktup\fP (1 or 2)
+.ft R
+.)l
+.lp
+Included with the package are several test files.
+To check to make certain that everything is working, you can try:
+.(l
+.ft C
+fasta musplfm.aa prot_test.lib
+and
+tfastx mgstm1.aa gst.nlib
+.ft R
+.)l
+.sh 2 "Sequence files"
+.pp
+The \fCfasta3\fP programs know about three kinds of sequence files:
+(1) plain sequence files - files that contain nothing but
+sequence residues - can only be used as query sequences. (2) FASTA
+format files. These are the same as plain sequence files, each
+sequence is preceded by a comment line with a '>' in the first
+column. (3) distributed sequence libraries (this is a broad class that
+includes the NBRF/PIR VMS and blocked ascii formats, Genbank flat-file
+format, EMBL flat-file format, and Intelligenetics format. All of the
+files that you create should be of type (1) or (2). FASTA format
+files (ones with a '>' and comment before the sequence) are preferred,
+because they can be used as query or library sequence files by all of
+the programs.
+.pp
+I have included several sample test files, \fC*.aa\fP and \fC*.seq\fP
+as well as two small sequence libraries, \fCprot_test.lib\fP and
+\fCgst.nlib\fP. The first line may begin with a '>' by a comment.
+Spaces and tabs (and anything else that is not an amino-acid code) are
+ignored.
+.pp
+Library files should have the form:
+.(l
+.ft C
+>Sequence name and identifier
+A F A S Y T .... actual sequence.
+F S S .... second line of sequence.
+>Next sequence name and identifier
+.ft R
+.)l
+This is often referred to as "FASTA" or format. You can
+build your own library by concatenating several sequence files. Just
+be sure that each sequence is preceded by a line beginning with a '>'
+with a sequence name.
+.pp
+The test file should not have lines longer than 120 characters, and
+sequences entered with word processors should use a document
+mode, with normal carriage returns at the end of lines.
+.pp
+A different format is required to specify the ordered peptide mixture for \fCfastf3/tfastf3\fP. For example:
+.(l I
+.ft C
+>mgstm1
+MGCEN,
+MIDYP,
+MLLAY,
+MLLGY
+.ft P
+.)l
+indicates \fCm\fP in the first position of all three peptides (as
+from CNBr), \fCG, I, L\fP (twice) in the second position (first cycle),
+\fCC,D,L\fP (twice) in the third position, etc. The commas (\fC,\fP)
+are required to indicate the number of fragments in the mixture, but
+there should be no comma after the last residue.
+.pp
+For the \fCfasts3/tfasts3\fP program, the format is the same, except that there
+is no requirement for the peptides to be the same length.
+.sh 1 "Statistical Significance"
+.pp
+All the programs in the FASTA3 package attempt to calculate accurate
+estimates of the statistical significance of a match. For
+\fCfasta3\fP, \fCssearch3\fP, and \fCfastx3/y3\fP, these estimates are
+very accurate.[.wrp971,wrp981.]. Altschul et al. [.alt940.] provides
+an excellent review of the statistics of local similarity scores.
+Local sequence similarity scores follow the extreme value
+distribution, so that P(s > x) = 1 - exp(-exp(-lambda(x-u)) where u =
+ln(Kmn)/lambda and m,m are the lengths of the query and library
+sequence. This formula can be rewritten as: 1 - exp(-Kmn exp(-lambda
+x), which shows that the average score for an unrelated library
+sequence increases with the logarithm of the length of the library
+sequence. The \fCfasta3\fP programs use simple linear regression
+against the the log of the library sequence length to calculate a
+normalized "z-score" with mean 50, regardless of library sequence
+length, and variance 10. (Several other estimation methods are
+available with the \fC\-z\fP option.) These z-scores can then be used
+with the extreme value distribution and the poisson distribution (to
+account for the fact that each library sequence comparison is an
+independent test) to calculate the number of library sequences to
+obtain a score greater than or equal to the score obtained in the
+search. The original idea and routines to do the linear regression on
+library sequence length were provided Phil Green, U. Washington. This
+version uses a slightly different strategy for fitting the data than
+those originally provided by Dr. Green.
+.pp
+The expected number of sequences is plotted in the histogram using an
+"*". Since the parameters for the extreme value distribution are not
+calculated directly from the distribution of similarity scores, the
+pattern of "*'s" in the histogram gives a qualitative view of how well
+the statistical theory fits the similarity scores calculated by the
+programs. For \fCfasta3\fP, if optimized scores are calculated for
+each sequence in the database (the default), the agreement between the
+actual distribution of "z-scores" and the expected distribution based
+on the length dependence of the score and the extreme value
+distribution is usually very good. Likewise, the distribution of
+\fCssearch3\fP Smith-Waterman scores typically agrees closely with the
+<actual distribution of "z-scores." The agreement with unoptimized
+scores, \fIktup=2\fP, is often not very good, with too many high
+scoring sequences and too few low scoring sequences compared with the
+predicted relationship between sequence length and similarity score.
+In those cases, the expectation values may be overestimates.
+.pp
+With version 33t01, all the FASTA programs also report a "bit" score,
+which is equivalent to the bit score reported by BLAST2. The
+FASTA33/BLAST2 bit score is calculated as: (lambda*S - ln K)/ln 2,
+where S is the raw similarity score, lambda and K are statistical
+parameters estimated from the distribution of unrelated sequence
+similarity scores. The statistical signficance of a given bit score
+depends on the lengths of the query and library sequences and the size
+of the library, but a 1 bit increase in score corresponds to a 2-fold
+reduction in expectation; a 10-bit increase implies 1000-fold lower
+expectation, etc.
+.pp
+The statistical routines assume that the library contains a large
+sample of unrelated sequences. If this is not true, then statistical
+parameters can be estimated by using the \fC\-z 11\-15\fP, options.
+\fC\-z\fP options greater than 10 calculate a shuffled similarity score
+for each library sequence, in addition to the unshuffled score, and
+estimate the statistical parameters from the scores of the shuffled
+sequences. If there are fewer than 20 sequences in the library, the
+statistical calculations are not done.
+.pp
+For protein searches, library sequences with E() values < 0.01 for
+searches of a 10,000 entry protein database are almost always
+homologous. Frequently sequences with E()-values from 1 - 10 are
+related as well, but unrelated sequences ( 1 \- 10 per search) will
+have scores in this renage as well. Remember, however, that these E()
+values also reflect differences between the amino acid composition of
+the query sequence and that of the "average" library sequence. Thus,
+when searches are done with query sequences with "biased" amino-acid
+composition, unrelated sequences may have "significant" scores because
+of sequence bias. \fCPRSS3\fP can address this problem by calculating
+similarity scores for random sequences with the same length and amino
+acid composition.
+.sh 1 "Options"
+.pp
+Command line options are available to change the scoring parameters
+and output display. \fBCommand line options must preceed other program
+arguments, such as the query and library file names.\fP
+.sh 2 "Command line options"
+.ip "-a"
+(fasta3, ssearch3 only) show both sequences in their entirety.
+.ip "-A"
+force Smith-Waterman alignments for fasta3 DNA sequences. By default,
+only fasta3 protein sequence comparisons use Smith-Waterman alignments.
+.ip "-B"
+Show normalized score as a z-score, rather than a bit-score in the list
+of best scores.
+.ip "-b #"
+Number of sequence scores to be shown on output. In the absence of
+this option, fasta (and tfasta and ssearch) display all library
+sequences obtaining similarity scores with expectations less than
+10.0 if optimized score are used, or 2.0 if they are not. The -b
+option can limit the display further, but it will not cause additional
+sequences to be displayed.
+.ip "-c #"
+Threshold score for optimization (OPTCUT). Set "-c 1" to
+optimize every sequence in a database.
+.ip "-E #"
+Limit the number of scores and alignments shown based on the
+expected number of scores. Used to override the expectation value of 10.0
+used by default. When used with -Q, -E 2.0 will show all library sequences
+with scores with an expectation value <= 2.0.
+.ip "-d #"
+Maximum number of alignments to be displayed. Ignored if "-Q" is not
+used.
+.ip "-f"
+Penalty for the first residue in a gap (-12 by default for proteins,
+-16 for DNA, -15 for FAST[XY]/TFAST[XY]).
+.ip "-F #"
+Limit the number of scores and alignments shown based on the expected
+number of scores. "-E #" sets the highest E()-value shown; "-F #" sets
+the lowest E()-value. Thus, "-F 0.0001" will not show any matches or
+alignments with E() < 0.0001. This allows one to skip over close
+relationships in searches for more distant relationships.
+.ip "-g"
+Penalty for additional residues in a gap (-2 by default for proteins,
+-4 for DNA, -3 for FAST[XY]/TFAST[XY]).
+.ip "-h"
+Penalty for frameshift (fastx3/y3, tfastx3/y3 only).
+.ip "-H"
+Omit histogram.
+.ip "-i"
+Invert (reverse complement) the query sequence if it is DNA. For
+tfasta3/x3/y3, search the reverse complement of the library sequence
+only.
+.ip "-j #"
+Penalty for frameshift within a codon (fasty3/tfasty3 only).
+.ip "-l file"
+Location of library menu file (FASTLIBS).
+.ip "-L"
+Display more information about the library sequence in the alignment.
+.ip "-M low-high"
+Range of amino acid sequence lengths to be included in the search.
+.ip "-m #"
+Specify alignment type: 0, 1, 2, 3, 4, 5, 6, 9, 10
+.(l I
+.ft C
+ \-m 0 \-m 1 \-m 2 \-m 3 \-m 4
+.ft C
+MWRTCGPPYT MWRTCGPPYT MWRTCGPPYT MWRTCGPPYT
+::..:: ::: xx X ..KS..Y... MWKSCGYPYT ----------
+MWKSCGYPYT MWKSCGYPYT
+.ft P
+.)l
+.ip
+\fC\-m 5\fP provides a combination of \fC\-m 4\fP and
+\fC\-m 0. \fC\-m 6 provides \fC\-m 5\fP plus HTML formatting.
+.ip "-m 9"
+provides coordinates and scores with the best score information.
+A simple "\fC -m 9\fP extends the normal best score information:
+.(l
+.ft C
+The best scores are: opt bits E(14548)
+XURTG4 glutathione transferase (EC 2.5.1.18) 4 - ( 219) 1248 291.7 1.1e-79
+.ft P
+.)l
+to include the additional information (on the same line, separated by
+a <tab>):
+.(l
+.ft C
+%_id %_gid sw alen an0 ax0 pn0 px0 an1 ax1 pn1 px1 gapq gapl fs
+0.771 0.771 1248 218 1 218 1 218 1 218 1 219 0 0 0
+.ft P
+.)l
+\fC -m 9c\fP provides additional information: an encoded alignment string. Thus:
+.(l I
+.ft C
+ 10 20 30 40 50 60 70
+GT8.7 NVRGLTHPIRMLLEYTDSSYDEKRYTMGDAPDFDRSQWLNEKFKL--GLDFPNLPYL-IDGSHKITQ
+ :.:: . :: :: . .::: : .: ::.: .: : ..:.. ::: :..:
+XURTG NARGRMECIRWLLAAAGVEFDEK---------FIQSPEDLEKLKKDGNLMFDQVPMVEIDG-MKLAQ
+ 20 30 40 50 60
+.ft P
+.)l
+would be encoded:
+.(l
+.ft C
+=23+9=13-2=10-1=3+1=5
+.ft P
+.)l
+The alignment encoding is with repect to the alignment, not the
+sequences. The coordinate of the alignment is given earlier in the
+"\fC -m 9c\fP" line.
+.ip "-m 10"
+\fC\-m 10\fP is a new, parseable format for use
+with other programs. See the file "readme.v20u4" for a more complete
+description.
+.ip
+As of version "fa34t23b2", it has become possible to combine independent
+"\fC\-m\fP" options. Thus, one can use "\fC\-m 1 -m 6 -m 9\fP".
+.ip "-M low\-high"
+Include library sequences (proteins only) with lengths between low and
+high.
+.ip "-n"
+Force the query sequence to be treated as a DNA sequence. This is
+particularly useful for query sequences that contain a large number of
+ambiguous residues, e.g. transcription factor binding sites.
+.ip "-O"
+Send copy of results to "filename." Helpful for environments without
+STDOUT (mostly for the Macintosh).
+.ip "-o "
+Turn off default optimization of all scores greater than OPTCUT. Sort
+results by "initn" scores (reduces the accuracy of statistical
+estimates).
+.ip "-p"
+Force query to be treated as protein sequence.
+.ip "-Q,-q"
+Quiet - does not prompt for any input. Writes scores and alignments
+to the terminal or standard output file.
+.ip "-r"
+Specify match/mismatch scores for DNA comparisons. The default is
+"+5/-4". "+3/-2" can perform better in some cases.
+.ip "-R file"
+Save a results summary line for every sequence in the sequence
+library. The summary line includes the sequence identifier,
+superfamily number (if available) position
+in the library, and the similarity scores calculated. This option can
+be used to evaluate the sensitivity and selectivity of different
+search strategies.[.wrp951,wrp981.]
+.ip "-s file"
+Specify the scoring matrix file. \fCfasta3\fP uses the same scoring
+matrices as Blast1.4/2.0. Several scoring matrix files are included
+in the standard distribution. For protein sequences: \fCcodaa.mat\fP
+- based on minimum mutation matrix; \fCidnaa.mat\fP - identity matrix;
+\fCpam250.mat\fP - the PAM250 matrix developed by Dayhoff et
+al.;[.day787.] \fCpam120.mat\fP - a PAM120 matrix. The default
+scoring matrix is BLOSUM50 ("-s BL50"). Other matrices available from
+within the program are: PAM250/"-s P250", PAM120/"-s P120", PAM40/"-s
+P40", PAM20/"-s P20", MDM10 - MDM40/"-s M10 \- M40" (MDM are modern
+PAM matrices from Jones et al.,[.tay925.]), BLOSUM50, 62, and 80/"-s
+BL50", "-s BL62", "-s BL80".
+.ip "-S"
+Treat lower-case characters in the query or library sequences as
+"low-complexity" ("seg"-ed) residues. Traditionally, the "seg"
+program [.woo935.] is used to remove low complexity regions in DNA
+sequences by replacing the residues with an "X". When the "-S" option
+is used, the FASTA33 (and later) programs provide a potentially more
+informative approach. With "-S", lower case characters in the query
+or database sequences are treated as "X"'s during the initial scan,
+but are treated as normal residues during the final alignment display.
+Since statistical significance is calculated from the similarity score
+calculated during the library search, when the lower case residues are
+"X"'s, low complexity regions will not produce statistically
+significant matches. However, if a significant alignment contains low
+complexity regions, their alignmen is shown. With "-S", lower case
+characters may be included in the alignment to indicate low complexity
+regions, and the final alignment score may be higher than the score
+obtained during the search.
+.ip
+The \fCpseg\fP program can be used to produce databases (or query
+sequences) with lower case residues indicating low complexity regions
+using the command:
+.(l I
+\fCpseg database.fasta -z 1 -q > database.lc_seg\fP
+.)l
+(\fCseg\fP can also be used with some post processing, see readme.v33tx.)
+.ip
+The \fC-S\fP option should always be used with \fCFASTX/Y\fCP and
+\fCTFASTX/Y\fP because out of frame translations often generate
+low-complexity protein sequences. However, only lower case characters
+in the protein sequence (or protein database) are masked; lower case
+DNA sequences are translated into upper case protein sequences, and
+not treated as low complexity by the translated alignment programs.
+.ip "-t #"
+Translation table - tfasta3, fastx3, tfastx3, fasty3, and
+tfasty3 now support the BLAST tranlation tables. See
+\fChttp://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi\fP.
+.ip
+In addition, "\-t t" or "\-t t#" turns on the addition of an implicit termination
+codon to a protein:translated DNA match. That is, each protein
+sequence implicitly ends with "*", which matches the termination codes
+for the appropriate genetic code. "\-t t#" sets implicit termination
+and a different genetic code.
+.ip "-U"
+Treat the query sequence an RNA sequence. In addition to selecting a
+DNA/RNA alphabet, this option causes changes to the scoring matrix so
+that 'G:A' , 'T:C' or 'U:C' are scored as 'G:G'.
+.ip "-V str"
+It is now possible to specify some annotation characters that can be
+included (and will be ignored), in the query sequence file. Thus, One
+might have a file with: \fC"ACVS*ITRLFT?"\fP, where "*" and "?" are
+used to indicate phosphorylation. By giving the option \fC\-V '*?'\fP,
+those characters in the query will be moved to an "annotation string",
+and alignments that include the annotated residues will be highlighted
+with the appropriate character above the sequence (on the number line).
+.ip "-w #"
+Line length (width) = number (<200)
+.ip "-W #"
+ context length (default is 1/2 of line width -w) for alignment,
+like fasta and ssearch, that provide additional sequence context.
+.ip "-x #match,#mismatch"
+Specify the penalty for a match to an 'X', and mismatch to 'X',
+independently of the PAM matrix. Particularly useful for
+\fCfastx3/fasty3\fP, where termination codons are encoded as 'X'.
+.ip "-X \"off1 off2\""
+Specifies offsets for the beginning of the query and library sequence.
+For example, if you are comparing upstream regions for two genes, and
+the first sequence contains 500 nt of upstream sequence while the
+second contains 300 nt of upstream sequence, you might try:
+.(l I
+\fCfasta -X "-500 -300" seq1.nt seq2.nt\fP
+.)l
+If the -X option is not used, FASTA assumes numbering starts with 1.
+(You should double check to be certain the negative numbering works
+properly.)
+.ip "-y"
+Set the width of the band used for calculating "optimized" scores.
+For proteins and ktup=2, the width is 16. For proteins with ktup=1,
+the width is 32 by default. For DNA the width is 16.
+.ip "-z -1,0,1,2,3,4,5"
+\fC\-z -1\fP turns off statistical calculations. \fCz 0\fP estimates
+the significance of the match from the mean and standard deviation of
+the library scores, without correcting for library sequence length.
+\fC\-z 1\fP (the default) uses a weighted regression of average score
+vs library sequence length; \fC\-z 2\fP uses maximum likelihood
+estimates of
+.if t \(*l
+.if n Lambda
+and \fIK\fP; \fC\-z 3\fP uses Altschul-Gish
+parameters;[.alt960.] \fC\-z 4 \- 5\fP uses two variations on the
+\fC\-z 1\fP strategy. \fC\-z 1\fP and \fC\-z 2\fP are the best methods,
+in general.
+.ip "-z 11,12,14,15"
+estimate the statistical parameters from shuffled copies of each
+library sequence. This doubles the time required for a search, but
+allows accurate statistics to be estimated for libraries comprised of
+a single protein family.
+.ip "-Z db_size"
+set the apparent size of the database to be used when calculating
+expectation E() values. If you searched a database with 1,000
+sequences, but would like to have the E()-values calculated in the
+context of a 100,000 sequence database, use '-Z 100000'.
+.ip "-1"
+sort output by init1 score (for compatibility with FASTP - do not
+use).
+.ip "-3"
+translate only three forward frames
+.sp
+.lp
+For example:
+.(l
+\fCfasta -w 80 -a seq1.aa seq.aa\fP
+.)l
+would compare the sequence in seq1.aa to that in seq2.aa and display the
+results with 80 residues on an output line, showing all of the residues
+in both sequences. Be sure to enter the options before entering the file
+names, or just enter the options on the command line, and the program will
+prompt for the file names.
+.sp
+.pp
+(November, 1997) In addition, it is now possible to provide the fasta
+programs with the query sequence (fasta, fasty, ssearch, tfastx), or
+two sequences (prss, lalign, plalign) from the unix "stdin" stream. This
+makes it much easier to set up FASTA or PRSS WWW pages. To specify
+that stdin be used, rather than a file, the file name should be
+specified as '-' or '@' (the latter file name makes it possible to
+specify a subset of the sequence).
+Thus:
+.(l
+cat query.aa | fasta -q @:25-75 s
+.)l
+would take residues 25-75 from query.aa and search the 's' library
+(see the discussion of FASTLIBS).
+.sh 2 "Environment variables"
+.pp
+Because the current version of the program allows the user to set
+virtually every option on the command line (except the \fIktup\fP,
+which must be set as the third command line argument), only the
+\fCFASTLIBS\fP environment variable is routinely used.
+.ip "FASTLIBS"
+specifies the location of the file which contains the list of library
+descriptions, locations, and library types (see section on finding
+library files).
+.sh 1 "Frequently Asked Questions (FAQs)"
+.np
+\fIWhich program should I use?\fP See Table I.
+.np
+\fIHow do I search with both DNA strands with\fP \fCfasta3\fP \fIand\fP
+\fCfastx3\fP? With version 32 of the FASTA program package, all
+searches that use DNA queries (e.g. \fCfasta3\fP, \fCfastx3/y3\fP)
+examine both strands. To revert to earlier FASTA behavior - only
+looking at the forward or reverse strand - use \fC\-3\fP to search only
+the forward strand and \fC\-i -3\fP to search only the reverse strand.
+.np
+\fIWhen I search Genbank - the program reports:\fP \fC0 residues in 0
+sequences\fP. This typically happens because the program does not
+know that you are searching a Genbank flatfile database and is looking
+for a FASTA format database. Be certain to specify the library type
+("1" for Genbank flatfile) with the database name.
+.np
+What is the difference between \fCfastx3\fP and \fCfasty3\fP (or
+\fCtfastx3\fP and \fCtfasty3\fP). \fC[t]fastx3\fP uses a simpler
+codon based model for alignments that does not allow frameshifts in
+some codon positions (see ref. [.wrp971.]). \fCtfastx3\fP is about
+30% faster, but \fCtfasty3\fP can produce higher quality alignments in
+some cases.
+.np
+\fIWhen I run\fP \fCfasta3 -q\fP, I don't see any (or very little)
+output, but I get lots of scores when I run interactively. With the
+\fC\-Q\fP option, the number of high scores displayed is limited by the
+\fC\-E #\fP cutoff, which is 10.0 for protein comparisons, 2.0 for DNA
+comparisons, and 5.0 for translated DNA:protein comparisons. In
+interactive mode (without \fC\-Q\fP), by default you see 20 high
+scores, regardless of \fCE()\fP value.
+.np
+\fIWhat is ktup\fP \- All of the programs with \fCfast\fP in their
+name use a computer science method called a lookup table to speed the
+search. For proteins with \fIktup\fP=2, this means that the program
+does not look at any sequence alignment that does not involve matching
+two identical residues in both sequences. Likewise with DNA and
+\fIktup\fP = 6, the initial alignment of the sequences looks for 6
+identical adjacent nucleotides in both sequences. Because it is less
+likely that two identical amino-acids will line up by chance in two
+unrelated proteins, this speeds up the comparison. But very distantly
+related sequences may never have two identical residues in a row but
+will have single aligned identities. In this case, \fIktup\fP = 1 may
+find alignments that \fIktup\fP=2 misses.
+.np
+\fISometimes, in the list of best scores, the same sequence is shown
+twice with exactly the same score. Sometimes, the sequence is there
+twice, but the scores are slightly different.\fP When any of the
+\fCfasta3\fP programs searches a long sequence, it breaks the sequence
+up into \fIoverlapping\fP pieces. The length of the piece depends on
+the length of the query and the particular program being used (it can
+also be controlled with the -N #### option). Since the pieces overlap
+by the length of the query sequence (or 3*query_length for fastx/y3
+and tfasta/x/y3), if the highest scoring alignment is at the end of
+one piece, it will be scored again at the beginning of the next piece.
+If the alignment is not be completely included in the overlap region,
+one of the pieces will give a higher score than the other. These
+duplications can be detected by looking at the coordinates of the
+alignment. If either the beginning or end coordinate is identical in
+two alignments, the alignments are at least partially duplicates.
+.lp
+As always, please inform me of bugs as soon as possible.
+.sp
+.nf
+William R. Pearson
+Department of Biochemistry
+Jordan Hall Box 800733
+U. of Virginia
+Charlottesville, VA
+
+wrp@virginia.EDU
+
+.sh 1 "References"
+.[]
git://source.jalview.org / jabaws.git / blobdiff