+++ /dev/null
-
- COPYRIGHT NOTICE
-
-Copyright 1988, 1991, 1992, 1994, 1995, 1996 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
-
-
-The FASTA program package
-
-Introduction
-
- This documentation describes the version 2.0x of the FASTA
-program package (see W. R. Pearson and D. J. Lipman (1988),
-"Improved Tools for Biological Sequence Analysis", PNAS 85:2444-
-2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence
-Comparison with FASTP and FASTA" Methods in Enzymology 183:63-
-98). Version 2.0 modifies version 1.8 to include explicit
-statistical estimates for similarity scores based on the extreme
-value distribution. In addition, FASTA protein alignments now
-use the Smith-Waterman algorithm with no limitation on gap size.
-FASTA and SSEARCH now use the BLOSUM50 matrix by default, with
-options to change gap penalties on the command line. Version 1.7
-replaces rdf2 and rss with prdf and prss, which use the extreme-
-value distribution to calculate accurate probability estimates.
-
-
-Although there are a large number of programs in this package,
-they belong to four groups:
-
-
- Library search programs: FASTA, FASTX, TFASTA, TFASTX, SSEARCH
-
- Local homology programs: LFASTA, PLFASTA, LALIGN, PLALIGN, FLALIGN
-
- Statistical significance: PRDF, RELATE, PRSS, RANDSEQ
-
- Global alignment: ALIGN
-
-
-
-In addition, I have included several programs for protein
-sequence analysis, including a Kyte-Doolittle hydropathicity
-plotting program (GREASE, TGREASE), and a secondary structure
-prediction package (GARNIER).
-
- The FASTA sequence comparison programs on this disk are
-improved versions of the FASTP program, originally described in
-Science (Lipman and Pearson, (1985) Science 227:1435-1441). We
-have made several improvements. First, the library search
-programs use a more sensitive method for the initial comparison
-of two sequences which allows the scores of several similar
-regions to be combined. As a result, the results of a library
-search are now given with three scores, initn (the new initial
-score which may include several similar regions), init1 (the old
-fastp initial score from the best initial region), and opt (the
-old fastp optimized score allowing gaps in a 32 residue wide
-band).
-
- These programs have also been modified to become "universal"
-(hence FAST-A, for FASTA-All, as opposed to FAST-P (protein) or
-FAST-N (nucleotides)); by changing the environment variable
-SMATRIX, the programs can be used to search protein sequences,
-DNA sequences, or whatever you like. By default, FASTA, LFASTA,
-and the PRDF programs automatically recognize protein and DNA
-sequences. Sequences are first read as amino acids, and then
-converted to nucleotides if the sequence is greater than 85%
-A,C,G,T (the '-n' option can be used to indicate DNA sequences).
-TFASTA compares protein sequences to a translated DNA sequence.
-Alternative scoring matrices can also be used. In addition to
-the BLOSUM50 matrix for proteins, the PAM250 matrix or matrices
-based on simple identities or the genetic code can also be used
-for sequence comparisons or evaluation of significance. Several
-different protein sequence matrices have been included;
-instructions for constructing your own scoring matrix are
-included in the file FORMAT.DOC.
-
-
-The remainder of this document is divided into three sections:
-(1) a brief history of the changes to the FASTA package; (2) A
-guide to installing the programs and databases; (3) A guide to
-using the FASTA programs. The programs are very easy to use, so
-if you are using them on a machine that is administered by
-someone else, you may want to skip to section (3) to learn how to
-use the programs, and then read section (1) to look at some of
-the more recent changes. If you are installing the programs on
-your own machine, you will need to read section (2) carefully.
-
-
-1. Revision History
-
-1.1. Changes with version 2.0u
-
- Version 2.0u provides several major improvements over
-previous versions of FASTA (and SSEARCH). The most important is
-the incorporation of explicit statistical estimates and
-appropriate normalization of similarity scores. This improvement
-is discussed in more detail below in the section entitled
-Statistical Significance. In addition, all of the protein
-comparison programs now use the BLOSUM50 matrix, with gap
-penalties of -12, -2, by default. BLOSUM50 performs
-significantly better than the older PAM250 matrix. PAM250 can
-still be used with the command line option: -s 250. (DNA
-sequence comparisons use a more stringent gap penalty of -16, -4,
-which produces excellent statistical estimates when optimized
-scores are used. TFASTA uses -16, -4 as well.)
-
- The quality of the fit of the extreme value distribution to
-the actual distribution of similarity scores is summarized with
-the Kolmogorov-Smirnov statistic. The acceptance limits for this
-statistic can be found in many statistics books. In general,
-values <0.10 (N=30) indicate excellent agreement between the
-actual and theoretical distributions. If this statistic is >
-0.2, consider using a higher (more stringent) gap penalty, e.g.
--16, -4 rather than -12, -2. The default scoring matrix for DNA
-has been changed to score +5 for an identity and -4 for a
-mismatch. These are the same scores used by BLASTN.
-
- With explicit expectation calculations, the program now
-shows all scores and alignments with expectations less than 10.0
-(with optimized scores, 2.0 without optimization) when the "-Q"
-(quiet) mode is used. The expectation threshold can be changed
-with the "-E" option.
-
- Finally, the algorithm used to produce the final alignments
-of protein sequences is now a full Smith-Waterman, with unlimited
-gaps. (The older band-limited alignments are used for DNA
-sequences and TFASTA by default, because Smith-Waterman
-alignments are very slow for long sequences.) Both the optimized
-and Smith-Waterman scores are reported; if the Smith-Waterman
-score is higher, then additional gaps allowed a better alignment
-and similarity score to be calculated.
-
- FASTA searches now optimize similarity scores by default
-(this slows searches about 2-fold (worst case) for ktup=2). Thus,
-the meaning of the "-o" option has been reversed; "-o" now turns
-off optimization and reports results sorted by "initn" scores.
-Optimization significantly improves the sensitivity of FASTA, so
-that it almost matches Smith-Waterman. With version 2.0, the
-default band width used for optimized calculations can be varied
-with the "-y" option. For proteins with ktup=2, a width of 16
-(-y 16) is used; 16 is also used for DNA sequences. For proteins
-and ktup=1, a width of 32 is used. Searches that disable
-optimization with the "-o" option will work fine for sequences
-that share 25% or more identity in general, but to detect
-evolutionary relationships with 20% - 25% identity, the more
-sensitive default optimization is often required. Optimization
-is required for accurate statistical estimates with either
-protein or DNA sequences.
-
- The FASTA package now includes FASTX, a program that
-compares a DNA sequence to a protein sequence database by
-translating the DNA sequence in three frames (the reverse frames
-are selected with the -i option) and aligning the three-frame
-translation with the sequences in the protein database.
-Alignment scores allow frameshifts so that a cDNA or EST sequence
-with insertion/deletion errors can be aligned with its homologues
-from beginning to end.
-
- With release 20u6, there is also a TFASTX program, which is
-a replacement for TFASTA. TFASTA treats each of the six reading
-frames of a DNA library sequence as a different sequence; TFASTX
-compares a protein sequence against only two sequences from each
-DNA sequence - the forward and reverse orientation. For a given
-orientation, TFASTX calculates a similarity score for alignments
-that allow frameshifts, thus considering all possible reading
-frames.
-
- Another new program is included - randseq - which will
-produce a randomly shuffled (uniform or local shuffle) from an
-input sequence. This randomly shuffled sequence can be used to
-evaluate the statistical estimates produced by FASTA, SSEARCH, or
-BLAST.
-
-1.2. Changes with version 1.7
-Version 1.7 has been released to provide the PRDF and PRSS
-programs for shuffling sequences and estimating accurately the
-probabilities of the unshuffled-sequence scores.
-
-PRDF a version of RDF2 that uses calculates the probability
- of a similarity score more accurately by using a fit to
- an extreme value distribution. Code to fit the extreme
- value distribution parameters and the impetus to update
- RDF2 was provided by Phil Green, U. of Washington.
-
-PRSS a version of PRDF that uses a rigorous Smith-Waterman
- calculation to score similarities
-
-1.3. Changes with version 1.6
-
- FASTA version 1.6 uses a new method for calculating optimal
-scores in a band (the optimization or last step in the FASTA
-algorithm). In addition, it uses a linear-space method for
-calculating the actual alignments. FASTA v1.6 package includes
-several new programs:
-
-SSEARCH a program to search a sequence database using the
- rigorous Smith-Waterman algorithm (this program is
- about 100-fold slower than FASTA with ktup=2 (for
- proteins).
-
-LALIGN A rigorous local sequence alignment program that will
- display the N-best local alignments (N=10 by default).
-
-PLALIGN a version of lalign that plots the local alignments to
- a tektronix display.
-
-FLALIGN a version of lalign that plots the local alignments to
- a GCG Figure file.
-
- The LALIGN/PLALIGN/FLALIGN programs incorporate the "sim"
-algorithm described by Huang and Miller (1991) Adv. Appl. Math.
-12:337-357. The SSEARCH and PRSS programs incorporate algorithms
-described by Huang, Hardison, and Miller (1990) CABIOS 6:373-381.
-
- LFASTA and PLFASTA now calculate a different number of local
-similarities; they now behave more like LALIGN/PLALIGN. Since
-local alignments of identical sequences produce "mirror-image"
-alignments, lalign and lfasta consider only one-half of the
-potential alignments between sequences from identical file names.
-Thus
-
- lfasta mchu.aa mchu.aa
-
-Displays only two alignments, with earlier versions of the
-program, it would have displayed five, including the identity
-alignment. PLFASTA does display five alignments; when two
-identical filenames are given, it draws the identity alignment,
-calculates the two unique local alignments, draws them, and draws
-their mirror images. LFASTA/PLFASTA and LALIGN/PLALIGN use the
-filenames, rather than the actual sequences, to determine whether
-sequences are identical; you can "trick" the programs into
-behaving the old way by putting the same sequence in two
-different files.
-
-1.4. Changes with version 1.5
-
- FASTA version 1.5 includes a number of substantial revisions
-to improve the performance and sensitivity of the program. It is
-now possible to tell the program to optimize all of the initn
-scores greater than a threshold. The threshold is set at the
-same value as the old FASTA cutoff score. Alternatively, you can
-tell FASTA to sort the results by the init1, rather than the
-initn, score by using the -1 option. FASTA -1 ... will report
-the results the way the older FASTP program did.
-
- A new method has been provided for selecting libraries. In
-the past, one could enter the name of a sequence file to be
-searched or a single letter that would specify a library from the
-list included in the $FASTLIBS file. Now, you can specify a set
-of library files with a string of letters preceded by a '%'.
-Thus, if the FASTLIBS file has the lines:
-
- Genbank 70 primates$1P/seqlib/gbpri.seq 1
- Genbank 70 rodents$1R/seqlib/gbrod.seq 1
- Genbank 70 other mammals$1M/seqlib/gbmam.seq 1
- Genbank 70 vertebrates $1B/seqlib/gbvrt.seq 1
-
-Then the string: "%PRMB" would tell FASTA to search the four
-libraries listed above. The %PRMB string can be entered either
-on the command line or when the program asks for a filename or
-library letter.
-
- FASTA1.5 also provides additional flexibility for specifying
-the number of results and alignments to be displayed with the -Q
-(quiet) option. The -b number option allows you to specify the
-number of sequence scores to show when the search is finished.
-Thus
-
-
- FASTA -b 100 ...
-
-
-tells the program to display the top 100 sequence scores. In the
-past, if you displayed 100 scores (in -Q mode), you would also
-have store 100 alignments. The -d option allows you to limit the
-number of alignments shown. FASTA -b 100 -d 20 would show 100
-scores and 20 alignments.
-
- Finally, FASTA can provide a complete list of all of the
-sequences and scores calculated to a file with the -r (results)
-option. FASTA -r results.out ... creates a file with a list of
-scores for every sequence in the library. The list is not
-sorted, and only includes those scores calculated during the
-initial scan of the library.
-
-2. Installing the FASTA package
-
-2.1. Installing the programs
-
-2.1.1. Unix version
-
- The FASTA distribution comes with several makefile's that
-can be used to compile the FASTA programs. Over the years, as
-ATT Unix System 5 and BSD unix have converged, these files have
-become very similar. To begin with, I recommend using the
-standard Makefile. There are two values in the makefile that
-should be checked against the values used on your system: the HZ
-value, which is the frequency in ticks per second used by the
-times() system call, this value can usually be found by running:
-
- grep HZ /usr/include/sys/*
-
-and the functions available to return random numbers. If you
-have a rand48() function that returns a 32-bit random number, use
-it and use the lines:
-
- NRAND=nrand48
- RANFLG= -DRAND32
-
-If not, you will need to use the rand() function call and
-determine whether it returns a 16-bit or a 32-bit value. These
-functions are used by PRDF and PRSS. If you have problems
-compiling the programs, you may want to examine the makefile.unx
-and makefile.sun files, to look for differences. I have tried to
-use very standard unix functions in these programs, and they have
-been successfully compiled, with very small changes to the
-Makefile, on Sun's (Sun OS 4.1), IBM RS/6000's (AIX), and MIPS
-machines (under the BSD environment).
-
-2.1.2. IBM-PC/DOS version
-
- For the IBM-PC/DOS version, the FASTA source code disk
-contains the complete source code to all of the programs on the
-other disks. The programs were compiled with Borland's Turbo
-'C++', using Borland's MAKE utility. The graphics programs
-(PLFASTA, TGREASE) use the graphics device drivers supplied with
-the Turbo 'C' V2.0 package. Also included are the documentation
-files PROGRAMS.DOC and FORMAT.DOC. You do not need any of the
-files the source code disk to run the programs. The files on
-this disk are identical to the UNIX and VMS versions that run on
-larger machines. Also included is the code to compile
-ALIGN0.EXE. ALIGN0 is the same as ALIGN, but does not penalize
-for end-gaps.
-
- If you have the DOS or Macintosh version of the FASTA
-package, to install the programs you should:
-
- (1) Make a new directory (folder) for the FASTA programs.
- This need not be the same as the directory for your
- sequence databases.
-
- (2) Copy the files from the FASTA source disk to the new
- directory.
-
- (3) (DOS only) Edit your AUTOEXEC.BAT file to (a) modify your
- PATH command to include the FASTA directory and (b) add
- the line:
-
- set FASTLIBS=c:\yourfastadirectory\fastgbs
-
- On the Macintosh, you may need to edit the "environment"
- file and change the line that reads:
-
- FASTLIBS=fastgbs
-
- to indicate the full directory path for the fastgbs file,
- for example:
-
- FASTLIBS=Q105:FASTA:fastgbs
-
-
- (4) Finally, you will need to edit the fastgbs file. This is
- usually the most confusing part of the installation. An
- example of this file is shown below; to customize this
- file for your machine, you will need to change the file
- names from those provided in the fastgbs file to ones that
- reflect the directory names and file names you use on your
- machine. This is explained in more detail below. In
- addition, some entries in the fastgbs file refer to other
- files of file names. These files of file names (as
- opposed to actual database files) may also need to be
- edited.
-
-2.2. Installing the libraries
-
-2.2.1. The NBRF protein sequence library
-
- The FASTA program package does not include any protein or
-DNA sequence libraries. You can obtain the PIR protein sequence
-database from:
-
- National Biomedical Research Foundation
- Georgetown University Medical Center
- 3900 Reservoir Rd, N.W.
- Washington, D.C. 20007
-
-In addition, this database is available via anonymous ftp from
-the host "ftp.bchs.uh.edu". It is available in two formats, VMS
-and CODATA format. The "VMS" format (library type 5 below) can
-be searched much faster, can be easily reformatted for use by the
-"BLAST" rapid searching program, and is compatible with the
-Genetics Computer Group package of programs. The CODATA format
-is used by the EUGENE/MBIR computing package from Baylor (library
-type 2).
-
-2.2.2. The GENBANK DNA sequence library
-
- FASTA, and TFASTA search sequences from the GENBANK
-"flatfile" (not ASN.1) DNA sequence library in the flat-file
-format distributed by the National Center for Biotechnology
-Information and the PIR format used by EBI/EMBL. CD-ROMs can be
-obtained from:
-
- Genbank
- National Center for Biotechnology Information
- National Library of Medicine
- National Institutes of Health
- 8600 Rockville Pike
- Bethesda, MD 20894
-
-
- The GenBank DNA sequence library is also available via
-anonymous FTP from ncbi.nlm.nih.gov.
-
-2.2.3. The EBI/EMBL CD-ROM libraries
-
- The European Bioinformatics Institute (EBI) is now
-distributing the EMBL CD-ROM that contains both the complete EMBL
-DNA sequence database (which should be essentially identical to
-the GenBank DNA sequence database) and the SWISS-PROT protein
-sequence database. SWISS-PROT is derived from the NBRF Protein
-sequence database with additions from the EBI/EMBL DNA sequence
-database. This CD-ROM is a "best-buy," since it provides both
-DNA and protein sequence libraries. It is available from:
-
-
- European Bioinformatics Institute
- Hinxton Genome Campus, Hinxton Hall
- Hinxton, Cambridge CB10 1RQ,
- United Kingdom
- Tel: +44 1223 4944
- Fax: +44 1223 494468
- Email: DATALIB@ebi.ac.uk
-
-
-
- In addition, the SWISS-PROT protein sequence database is
-available via anonymous FTP from ncbi.nlm.nih.gov.
-
-2.3. Finding the libraries: FASTLIBS
-
- FASTA and TFASTA use the environment variable FASTLIBS to
-find the protein and DNA sequence libraries. The FASTLIBS
-variable contains the name of a file that has the actual
-filenames of the libraries. The FASTGBS file on is an example of
-a file that can be referred to by FASTLIBS. To use the FASTGBS
-file, type:
-
- setenv FASTLIBS /usr/lib/fasta/fastgbs (BSD UNIX/csh)
- or
- export FASTLIBS=/usr/lib/fasta/fastgbs (SysV UNIX/ksh)
-
-Then edit the FASTGBS 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
-/usr/lib/aabank.lib and your Genbank DNA sequence library is kept
-in the directory: /usr/lib/genbank, then fastgbs might contain:
-
- 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$1M@/usr/lib/genbank/gmammal.nam
- ^ 1 ^^^^ 4 ^ ^
- 23 (5)
-
-The first line of this file says that there is a copy of the NBRF
-protein sequence database (which is a protein database) that can
-be selected by typing "P" on the command line or when the
-database menu is presented in the file /usr/lib/seq/aabank.lib.
-
- Note that there are 4 or 5 fields in the lines in fastgbs.
-The first field is the description of the library which will be
-displayed by FASTA; it ends with a '$'. 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.
-
- The fourth field is the name of the library file. In the
-example above, the /usr/lib/seq/aabank.lib file contains the
-entire protein sequence library. However the DNA library file
-names are preceded by a '@', because these files (gpri.nam,
-grod.nam, gmammal.nam) 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.
-
- 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
-aabank.lib file is in Pearson/FASTA format, while the swiss.seq
-file is in PIR/VMS format (from the EMBL CD-ROM). Currently,
-FASTA can read the following formats:
-
- 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)
-
-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
-change this default, you may set the LIBTYPE environment variable
-to a number. For example,
-
- setenv LIBTYPE 1
-
-would cause the program to use the GenBank LOCUS format by
-default for libraries (or the second sequence file), but the
-Pearson/FASTA format would still be used for the query sequence.
-
- 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:
-
- </usr/lib/genbank
- gbpri.seq 1
- gbrod.seq 1
- gbmam.seq 1
-
-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:
-
- /usr/lib/genbank/gbpri.seq
-
-The notation "<PIRNAQ:" might be used under the VAX/VMS operating
-system. Under UNIX, the trailing '/' is left off, so the library
-directory might be written as "</usr/seqlib".
-
- With version 1.4 of the FASTA package, the FASTA and TFASTA
-programs can search a library 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:
-
- </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
- #
-
- You do not need to include library format numbers if you
- only use the Pearson/FASTA version of the PIR protein se-
- quence library. If no library type is specified, the
- program assumes that type 0 is being used (unless you
- have set LIBTYPE).
-
-Support for the old compressed GenBank files, which have not been
-distributed for more than four years, has been removed from
-programs in the FASTA package.
-
-
- Test the setup by running FASTA. Enter the sequence file
-'MUSPLFM.AA' 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 MUSPLFM.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.
-
- FASTA and TFASTA must open a large number of files when
-searching and reporting the results of a GENBANK floppy disk
-format library search. You may have problems with the large
-number of files under DOS on IBM-PC's (Unix and VMS users will
-not have these problems). If you are going to search the GENBANK
-floppy disk format DNA sequence library under DOS, you should add
-the line:
-
- FILES=16
-
-to your CONFIG.SYS file. (Typically this is already done for
-programs like Windows or WordPerfect.)
-
-3. Using the FASTA Package
-
-3.1. Overview
-
- The FASTA sequence comparison programs all require similar
-information, the name of a query sequence file, a library file,
-and the ktup parameter. All of the programs can accept arguments
-on the command line, or they will prompt for the file names and
-ktup value.
-
-To use FASTA, simply type:
-
- FASTA
- and you will be prompted for :
- 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.
- For a 200 aa sequence against a 10,000,000 aa
- library, the program takes about 30 min with
- ktup = 2, 150 min with ktup = 1, on a 12 Mhz 286
- IBM-PC.
-
-
-The program can also be run by typing
-
- FASTA test.aa /lib/bigfile.lib ktup (1 or 2)
-
-
-Included with the package are the test files, MUSPLFM.AA,
-LCBO.AA, MCHU.AA and BOVPRL.SEQ. To check to make certain that
-everything is working, you can try:
-
- fasta musplfm.aa lcbo.aa
- and
- tfasta musplfm.aa bovprl.seq
-
-To test the local similarity programs LFASTA and PLFASTA, try:
-
- lfasta mchu.aa mchu.aa
- and
- plfasta mchu.aa mchu.aa (use this only on an IBM-PC with graphics
- or on a Tektronix terminal under UNIX or VMS)
-
-MCHU (calmodulin) has four duplicated calcium binding sites that
-are clearly detected by LFASTA. For a more complicated example,
-try MWRTC1.aa, myosin heavy chain.
-
-3.2. Sequence files
-
- The FASTA programs know about three kinds of sequence files
-(four under VMS): (1) plain sequence files that can only be used
-as query sequences or for LFASTA, PRDF, and ALIGN. (2) Standard
-library 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). Type (2) files (ones with a be used as query or library
-sequence files by all of the programs.
-
- I have included several sample test files, *.AA. The first
-line may begin with a '>' or ';' followed by a comment. The
-text after ';' in other lines will be ignored. Spaces and
-tabs (and anything else that is not an amino-acid code) are
-ignored.
-
- Library files should have the form:
-
- >Sequence name and identifier
- A F A S Y T .... actual sequence.
- F S S .... second line of sequence.
- >Next sequence name and identifier
-
-This is often referred to as "FASTA" or "Pearson" 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.
-
- 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.
-
-Program Summary
-
-3.3. Sequence search programs
-
-FASTA universal sequence comparison. Defaults to comparing
- protein sequences; if the sequences are > 85% A+C+G+T
- or the -n option is used, a DNA sequence is assumed.
-
-FASTX Search a protein sequence library using amino acid
- sequence comparison to the forward three frames of a
- translated DNA query sequence. (The reverse frames are
- specified with the -i option.) Alignment scores allow
- frameshifts; the final alignment uses a Smith-Waterman
- type alignment routine (no limit on gaps) that allows
- frameshifts.
-
-TFASTA Search DNA library for a protein sequence by
- translating the DNA sequence to protein in all six
- frames (three forward frames with the -3 command line
- option). TFASTA with ktup=2 is about as fast as a DNA
- FASTA with ktup=4, and is substantially more sensitive.
- (also reads the GENBANK library)
-
-TFASTX Search DNA library for a protein sequence by
- translating the DNA sequence to protein in all six
- frames (three forward frames with the -3 command line
- option) calculating similarity scores that allow
- frameshifts. TFASTX produces an optimal Smith-Waterman
- alignment of the query and translated-library sequence.
-
-SSEARCH Universal sequence comparison using the Smith-Waterman
- algorithm ( T. F. Smith and M. S. Waterman (1981) J.
- Mol. Biol. 147:195-197). This program uses code
- developed by Huang and Miller (X. Huang, R. C.
- Hardison, W. Miller (1990) CABIOS 6:373-381) for
- calculating the local similarity score and code from
- the ALIGN program (see below) for calculating the local
- alignment. SSEARCH is about 50-times slower than FASTA
- with ktup=2 (for proteins).
-
-ALIGN optimal global alignment of two sequences with no
- short-cuts. This program is a slightly modified
- version of one taken from E. Myers and W. Miller. The
- algorithm is described in E. Myers and W. Miller,
- "Optimal Alignments in Linear Space" (CABIOS (1988)
- 4:11-17).
-
-3.4. Local similarity programs
-
-LFASTA local similarity searches showing local alignments.
- The algorithm used to calculate the local alignment in
- a band has been improved (Chao, Pearson, and Miller,
- submitted).
-
-PLFASTA local similarity searches with plot output (on the IBM,
- this program requires that the environment variable
- BGIDIR be set).
-
-PCLFASTA (unix only) local similarity searches with plot output
- using pic commands.
-
-LALIGN Calculates the N-best local alignments using a rigorous
- algorithm. (N=10 by default.) The algorithm was
- developed by Huang and Miller (X. Huang and W. Miller
- (1991) Adv. Appl. Math. 12:337-357), which is a
- linear-space version of an algorithm described by M. S.
- Waterman and M. Eggert (J. Mol. Biol. 197:723-728).
- Like SSEARCH, LALIGN is rigorous, but also very slow.
-
-PLALIGN A version of LALIGN that plots its output to a screen
- or to a Tektronix terminal emulator.
-
-3.5. Statistical Significance
-
- With version 2.0 of the FASTA program distribution, FASTA,
-TFASTA, and SSEARCH now provide estimates of statistical
-significance for library searches. Work by Altschul, Arratia,
-Karlin, Mott, Waterman, and others (see Altschul et al. (1994)
-Nature Genetics 6:119 for an excellent review) suggests that
-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. FASTA and SSEARCH 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. 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 of FASTA
-and SSEARCH uses a slightly different strategy for fitting the
-data than those originally provided by Dr. Green.
-
- 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 FASTA and SSEARCH. For FASTA, 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 SSEARCH Smith-
-Waterman scores typically agrees closely with the actual
-distribution of "z-scores." The agreement with unoptimized
-scores, ktup=2, 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.
-
- The statistical routines assume that the library contains a
-large sample of unrelated sequences. If this is not the case,
-then the expectation values are meaningless. Likewise, if there
-are fewer than 20 sequences in the library, the statistical
-calculations are not done.
-
- 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. 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. The programs
-below, PRDF and PRSS, can address this problem by calculating
-similarity scores for random sequences with the same length and
-amino acid composition.
-
- If optimization is not used ("-o"), E-values for DNA
-sequences overestimate the significance of the scores that are
-obtained and unrelated sequences frequently have E()-values <
-0.0005. With optimization, the agreement between E()-value
-compares favorably with protein sequence comparison. This is in
-part due to the use of more stringent gap penalties for DNA
-sequence comparison, -16, -4 rather than -12, -2. With the
-latter penalties, many unrelated sequences appear to have
-significant similarity. Nevertheless, since protein sequence
-comparison is much more sensitive, DNA sequence comparison should
-not be used to identify sequences that encode protein. Even with
-ktup=6, optimization rarely increases run-times more than 50%
-with mRNA-size query sequences. Optimization should be used
-whenever possible.
-
- Similar comments apply to TFASTA, where higher gap
-penalties (-16,-4) are required for accurate statistical
-estimates. Because TFASTA produces so many artificial "coding"
-sequences with atypical amino acid compositions, the statistical
-estimates with TFASTA are often over estimates. With optimized
-scores, ktup=1, and gap penalties of -16, -4, unrelated sequences
-will sometimes have E() values of 0.1. If initn scores are used,
-unrelated sequences may have have E() values < 0.01.
-
-PRDF improved version of RDF program that includes accurate
- probability estimates for all three scoring methods
- (includes local or window shuffle routine)
-
-PRSS A version of PRDF that uses the rigorous Smith-Waterman
- calculation used by SSEARCH.
-
-RANDSEQ produces a randomly shuffled sequence from a query
- sequence.
-
-RELATE significance program described by Dayhoff (Atlas of
- Protein Sequence and Structure, Vol. 5, Supplement 3).
- Each chunk of 25 residues in one sequence is compared
- to every 25 residue fragment of the second sequence.
- Sequences which are genuinely related will have a large
- number of scores greater than 3 standard deviations
- above the mean score of all of the comparisons.
-
-3.6. Other analysis programs
-
-AACOMP calculate the amino acid composition and molecular
- weight of a sequence.
-
-BESTSCOR calculate the best self-comparison score.
-
-GREASE Kyte-Doolittle hydropathicity profile
-
-TGREASE graphic plot of Kyte-Doolittle profile
-
-FROMGB convert from GenBank LOCUS format (also used by the
- IBI-Pustell programs) to Pearson/FASTA format.
-
-GARNIER A secondary structure prediction program using the
- method of Garnier, Osgusthorpe, and Robson, J. Mol.
- Biol., (1978) 120:97-120.
-
-3.7. Options
-
- These programs have a number of output options, which are
-invoked by the environment variables LINLEN, SHOWALL, and MARKX.
-Alternatively, these values can be controlled by command line
-options. The number of sequence residues per output line is now
-adjustable by setting the environment variable LINLEN, or the
-command line option -w. LINLEN is normally 60, to change it set
-LINLEN=80 before running the program or add -w 80 to the command
-line. LINLEN can be set up to 200. SHOWALL (-a) determines
-whether all, or just a portion, of the aligned sequences are
-displayed. Previously, FASTP would show the entire length of
-both sequences in an alignment while FASTN would only show the
-portions of the two sequences that overlapped. Now the default is
-to show only the overlap between the two sequences, to show
-complete sequences, set SHOWALL=1, or use the -a option on the
-command line.
-
- The differences between the two aligned sequences can be
-highlighted in three different ways by changing the environment
-variable MARKX or the -m option. Normally (MARKX=0) the program
-uses ':' do denote identities and '.' to denote conservative
-replacements. If MARKX=1, the program will not mark identities;
-instead conservative replacements are denoted by a 'x' and non-
-conservative substitutions by a 'X'. If MARKX=2, the residues in
-the second sequence are only shown if they are different from the
-first. MARKX=3 displays the aligned library sequences without the
-query sequence; these can be used to build a primitive multiple
-alignment. MARKX=4 provides a graphical display of the
-boundaries of the alignments. Thus the five options are:
-
-
- MARKX=0 MARKX=1 MARKX=2 MARKX=3 MARKX=4
-
- MWRTCGPPYT MWRTCGPPYT MWRTCGPPYT MWRTCGPPYT
- ::..:: ::: xx X ..KS..Y... MWKSCGYPYT ----------
- MWKSCGYPYT MWKSCGYPYT
-
-
-(fasta20u4, Feb. 1996) In addition MARKX=10 is a new, parseable
-format for use with other programs. See the file"readme.v20u4"
-for a more complete description.
-
-3.8. Command line options
-
- It is now possible to specify several options on the
-command line, instead of using environment variables. The
-command line options are preceded by a dash; the following
-options are available:
-
--a same as showall=1
-
--A force Smith-Waterman alignments for DNA sequences and
- TFASA. By default, only FASTA protein sequence
- comparisons use Smith-Waterman alignments.
-
--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.
-
--c # Threshold score for optimization (OPTCUT). Set "-c 1"
- to optimize every sequence in a database. (This slows
- the program down about 5-fold).
-
--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.
-
--d # Number of alignments to be reported by default. (Used
- in conjunction with -Q). No longer necessary, see "-b"
- above.
-
--f Penalty for the first residue in a gap (-12 by default
- for proteins, -16 for DNA or for TFASTA).
-
--g Penalty for additional residues in a gap (-2 by default
- for proteins, -4 for DNA and TFASTA ).
-
--h Penalty for frameshift (FASTX, TFASTX only).
-
--H Omit histogram.
-
--i Invert (reverse complement) the query sequence if it is
- DNA. For TFASTX, search the reverse complement of the
- library sequence only.
-
--k # Threshold for joining init1 segments to build an initn
- score (GAPCUT).
-
--l file Location of library menu file (FASTLIBS).
-
--L Display more information about the library sequence in
- the alignment.
-
--m # MARKX = # (0, 1, 2, 3, 4, 10)
-
--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.
-
--O Send copy of results to "filename." Helpful for
- environments without STDOUT.
-
--o Turn off default optimization of all scores greater
- than OPTCUT. Sort results by "initn" scores.
-
--Q,-q Quiet - does not prompt for any input. Writes scores
- and alignments to the terminal or standard output file.
-
--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 (see W. R. Pearson (1991) Genomics 11:635-
- 650.)
-
--s file SMATRIX is read from file. Several SMATRIX files are
- provided with the standard distribution. For protein
- sequences: codaa.mat - based on minimum mutation
- matrix; idnaa.mat - identity matrix; pam250.mat - the
- PAM250 matrix developed by Dayhoff et al (Atlas of
- Protein Sequence and Structure, vol. 5, suppl. 3,
- 1978); pam120.mat - a PAM120 matrix. The default
- scoring matrix is BLOSUM50, PAM250 is available with
- "-s 250", BLOSUM62 ("-s BL62") is also available.
-
--v (LINEVAL) values used for line styles in plfasta
-
--w # Line length (width) = number (<200)
-
--x 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:
-
- fasta -x "-500 -300" seq1.nt seq2.nt
-
- If the -x option is not used, FASTA assumes numbering
- starts with 1. This option will not work properly with
- the translated library sequence with tfasta. (You
- should double check to be certain the negative
- numbering works properly.)
-
--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.
-
--z Turn off statistical calculations.
-
--1 sort output by init1 score (as FASTP used to do).
-
--3 (TFASTA, TFASTX only) translate only three forward
- frames
-
-
-For example:
-
- fasta -w 80 -a seq1.aa seq.aa
-
-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.
-
- Not all of these options are appropriate for all of the
-programs. The options above are used by FASTA and TFASTA. RELATE
-uses the -s option, ALIGN uses the -w, -m, and -s options, and
-the PRDF program uses -c, -f, -k, and -s.
-
-4. Environment variable summary
-
- Environment variables allow you to set search parameters
-that will be used frequently when you run a program; for example,
-if you prefer to use the PAM250 scoring matrix, you might "set
-SMATRIX=250." Command line parameters, if used, always override
-environment variable settings. The following environment
-variables are used by this program:
-
-AABANK the file name of the default sequence library.
-
-FASTLIBS the location of the file which contains the list of
- library files to be searched.
-
-GAPCUT threshold used for joining init1 regions in the second
- step of FASTA. Normally set based on sequence length
- and ktup.
-
-LIBTYPE used to specify the format of the library sequence for
- FASTA and TFASTA.
-
-LINLEN output line length - can go up to 200
-
-LINEVAL used by plfasta to determine the relationship between
- line style and similarity score (-v). This should be a
- string of three numbers, e.g. "200 100 50"
-
-MARKX symbol for denoting matches, mismatches. Note that this
- symbol is only used across the optimized local region;
- sequences that are outside this region are not marked.
-
-OPTCUT Set the threshold to be used for optimization in a band
- around the best initial region. Normally the OPTCUT
- value is calculated from the length of the sequence and
- the ktup value (for a 200 residue sequence, it is about
- 28). If OPTCUT=1, every sequence in the database will
- be optimized. This is the most sensitive option.
-
-PAMFACT This version of fasta uses a more sensitive method for
- identifying initial regions. Instead of using a
- constant factor (fact) for each match in a ktup, it
- uses the scoring matrix (PAM) scores. While this works
- well for protein sequences, it has not been as
- carefully tested for DNA sequences, so by default, this
- modification is used for proteins but not for DNA.
- Setting the PAMFACT environment variable to 1 forces
- the option on; PAMFACT=0 turns it off.
-
-SHOWALL on output, show the complete sequence instead of just
- the overlap of the two aligned sequences.
-
-SMATRIX alternative scoring matrix file.
-
-TEKPLOT (IBM-PC only, Unix and VMS versions generate Tektronix
- graphics by default) Generate Tektronix output.
- Normally, PLFASTA and TGREASE plot graphs using the
- Turbo C graphics library. Unfortunately, often these
- plots cannot be printed out without special programs.
- However, if you set TEKPLOT=1, tektronix graphics
- commands will be used. Tektronix commands can be used
- together with the PLOTDEV program, available from
- Microplot Systems. They no lonter sell this program,
- but it can be downloaded from
- http://iquest.com/~microplt/index1.html. PLOTDEV also
- allows you to print out graphics on the screen.
-
-As always, please inform me of bugs as soon as possible.
-
-William R. Pearson
-Department of Biochemistry
-Box 440, Jordan Hall
-U. of Virginia
-Charlottesville, VA
-
-wrp@virginia.EDU
git://source.jalview.org / jabaws.git / blobdiff