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.he 'FASTA3.DOC''Release 3.3, November 1999'
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\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, (804)
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.[.wrp990.]  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..v33\fP, files. The programs are very 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(6.0i).
\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
fasta3 query.file "/seqdb/genbank/gbpri1.seq 1"
.)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.
.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)
.)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 (four
under VMS): (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 #"
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 "-f"
Penalty for the first residue in a gap (-12 by default for proteins,
-16 for DNA, -15 for FAST[XY]/TFAST[XY]).
.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
.ft C
  -m 0          -m 1          -m 2          -m 3        -m 4

MWRTCGPPYT   MWRTCGPPYT    MWRTCGPPYT                 MWRTCGPPYT
::..:: :::     xx  X       ..KS..Y...    MWKSCGYPYT   ----------
MWKSCGYPYT   MWKSCGYPYT
.ft R
.)l
.lp
In addition \fC -m 10\fP is a new, parseable format for use
with other programs.  See the file"readme.v20u4" for a more complete
description. 
.lp
\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. \fC-m 9\fP
provides coordinates and scores rather than alignments.
.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 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 "-w #"
Line length (width) = number (<200)  60 residues by default.
.ip "-W #"
Context width (amount of sequence shown before alignment starts and
after alignment ends).  By default, one-half line (30 residues) of
unaligned sequence is shown before and after the aligned region in
\fCfasta3\fP and \fCssearch3\fP.  To see more upstream or downstream
context, use \fC-W #\fP.  Context is not shown with translated
alignments (\fCfastx3\fP or \fCtfastx3\fP, etc.).
.ip "-x #"
Specify the penalty for a match to an '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 "-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).  If DNA sequences are to be read
from stdin, the '-n' option must be used, as fasta cannot check for
DNA queries when stdin is used.
.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"
.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
Box 440, Jordan Hall
U. of Virginia
Charlottesville, VA

wrp@virginia.EDU

.sh 1 "References"
.[]
