fasta3x.doc

序列对齐 Compare a protein sequence to a protein sequence database or a DNA sequence to a DNA sequenc

(Updated December, 2003)


                        COPYRIGHT NOTICE

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, [phone]

The FASTA program package

Introduction

     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 (Pearson and Lipman, 1988); W. R.  Pearson (1996)
"Effective protein sequence comparison" Meth. Enzymol.
266:227-258 (Pearson, 1996); Pearson et. al. (1997) Genomics
46:24-36 (Zhang et al., 1997);  Pearson, (1999) Meth. in
Molecular Biology 132:185-219 (Pearson, 2000).  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.

     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 readme.v30..v34, 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.

1.  An overview of the FASTA programs

     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 fast search
protein databases, while tfast programs search translated DNA
databases.  Table I gives a brief description of the programs.


            Table I. Comparison programs in the FASTA3 package

---------------------------------------------------------------------------
fasta3             Compare  a  protein  sequence  to  a  protein  sequence
                   database  or  a DNA sequence to a DNA sequence database
                   using the FASTA algorithm  (Pearson  and  Lipman, 1988,
                   Pearson, 1996).   Search speed and selectivity are con-
                   trolled with the ktup(wordsize) parameter.  For protein
                   comparisons,  ktup = 2 by default; ktup =1 is more sen-
                   sitive but slower.  For DNA comparisons, ktup=6 by  de-
                   fault;  ktup=3  or  ktup=4 provides higher sensitivity;
                   ktup=1 should be used for oligonucleotides  (DNA  query
                   lengths < 20).

ssearch3           Compare  a  protein  sequence  to  a  protein  sequence
                   database or a DNA sequence to a DNA  sequence  database
                   using  the  Smith-Waterman  algorithm (Smith and Water-
                   man, 1981).  ssearch3 is  about  10-times  slower  than
                   FASTA3,  but  is more sensitive for full-length protein
                   sequence comparison.

fastx3/ fasty3     Compare a DNA sequence to a protein sequence  database,
                   by  comparing  the  translated  DNA  sequence  in three
                   frames and allowing gaps and frameshifts.  fastx3  uses
                   a  simpler, faster algorithm for alignments that allows
                   frameshifts only between codons; fasty3 is  slower  but
                   produces  better alignments with poor quality sequences
                   because frameshifts are allowed within codons.

tfastx3/ tfasty3   Compare a protein sequence to a DNA sequence  database,
                   calculating  similarities  with frameshifts to the for-
                   ward and reverse orientations.

tfasta3            Compare a protein sequence to a DNA sequence  database,
                   calculating similarities (without frameshifts) to the 3
                   forward and three reverse reading frames.  tfastx3  and
                   tfasty3 are preferred because they calculate similarity
                   over frameshifts.

fastf3/tfastf3     Compares an ordered peptide mixture, as  would  be  ob-
                   tained  by  Edman  degredation  of a CNBr cleavage of a
                   protein, against a  protein  (fastf)  or  DNA  (tfastf)
                   database.

fasts3/tfasts3     Compares  set  of  short peptide fragments, as would be
                   obtained from mass-spec. analysis of a protein, against
                   a protein (fasts) or DNA (tfasts) database.
---------------------------------------------------------------------------

2.  Installing FASTA and the sequence databases

2.1.  Obtaining the libraries

     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 (www.ncbi.nlm.nih.gov and ftp://ncbi.nlm.nih.gov) and
EMBL at the European Bioinformatics Institute (www.ebi.ac.uk).
However, the databases are available via anonymous FTP from both
sites.

2.1.1.  The GENBANK DNA sequence library

     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 - fasta3,
tfastx/y3, and tfasta3 can read DNA databases in Genbank flatfile
(not ASN.1), FASTA, GCG/compressed-binary, BLAST1.4 (pressdb),
and BLAST2.0 (formatdb) 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 fasta3 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 formatdb program to convert ASN.1 formated Genbank
files, which are available from the NCBI ftp site.

     The NCBI also provides the nr, swissprot, and several EST
databases that are used by BLAST in FASTA format from:
ftp://ncbi.nlm.nih.gov/blast/db.  These databases are updated
nightly.

2.1.2.  The NBRF protein sequence library

     You can obtain the PIR protein sequence database (Barker et
al., 1998) from:

    National  Biomedical Research Foundation
    Georgetown  University  Medical  Center
    3900 Reservoir Rd, N.W.
    Washington, D.C. 20007

or via ftp from nbrf.georgetown.edu or from the NCBI
(ncbi.nlm.nih.gov/repository/PIR). The data in the ascii
directory is in PIR Codata format, which is not widely used.  I
recommend the PIR/VMS format data (libtype=5) in the vms
directory.

2.1.3.  The EBI/EMBL CD-ROM libraries

     The European Bioinformatics Institute (EBI) distributes both
the EMBL DNA database and the SwissProt database on CD-ROM
(Bairoch and Apweiler, 1996), and they are available from:

    EMBL-Outstation  European Bioinformatics Institute
    Wellcome Trust Genome Campus,
    Hinxton Hall
    Hinxton,
    Cambridge CB10 1SD
    United Kingdom
    Tel: [phone]
    Fax: [phone]
    Email: [e-mail]

In addition, the SWISS-PROT protein sequence database is
available via anonymous FTP from
ftp://ftp.expasy.ch/databases/swiss-prot/ (also see
www.expasy.ch).

2.2.  Finding the libraries: FASTLIBS

     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 fasta3 to read a
sequence database, it is likely that something is wrong with the
FASTLIBS 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 (libtype) 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.

     All the search programs in the FASTA3 package 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
fastlibs file included with the distribution on is an example of
a file that can be referred to by FASTLIBS. To use the fastlibs
file, type:

    setenv FASTLIBS /usr/lib/fasta/fastgbs (BSD UNIX/csh)
    or
    export FASTLIBS=/usr/lib/fasta/fastgbs (SysV UNIX/ksh)

Then edit the fastlibs 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)
    12 NCBI Blast2.0 format  (unix only, fasta32t08 or later)

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:

    fasta3 query.file "/seqdb/genbank/gbpri1.seq 1"

     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:

    </seqdb/genbank
    gbpri1.seq 1
    gbpri2.seq 1
    gbpri3.seq 1
    gbpri4.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".

     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:

    </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