fasta3x.me

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

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</seqdb/genbank
gbpri1.seq 1
gbpri2.seq 1
gbpri3.seq 1
gbpri4.seq 1
gbrod.seq 1
gbmam.seq 1
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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:
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/usr/lib/genbank/gbpri.seq
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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:
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</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
\&#
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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.
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.pp
Test the setup by running FASTA.  Enter the sequence
file '\fCmgstm1.aa\fP' when the program requests it (this file is
included with the programs).  The program should then ask you to
select a protein sequence library.  Alternatively, if you run the
TFASTA program and use the mgstm1.aa query sequence, the program
should show you a selection of DNA sequence libraries.
Once the fastgbs file has been set up correctly, you can
set FASTLIBS=fastgbs in your AUTOEXEC.BAT file, and you will not need to
remember where the libraries are kept or how they are named.
.ne 8
.sh 1 "Using the FASTA Package"
.sh 2 "Overview"
.pp
The FASTA sequence comparison programs all require similar
information, the name of a query sequence file, a library file, and
the \fIktup\fP parameter.  All of the programs can accept arguments
on the command line, or they will prompt for the file names and
\fIktup\fP value.
.lp
To use FASTA, simply type:
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\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)
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The program can also be run by typing
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FASTA test.aa /lib/bigfile.lib \fIktup\fP (1 or 2)
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.lp
Included with the package are several test files.
To check to make certain that everything is working, you can try:
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fasta musplfm.aa prot_test.lib
and
tfastx mgstm1.aa gst.nlib
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.sh 2 "Sequence files"
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The \fCfasta3\fP programs know about three kinds of sequence files:
(1) plain sequence files - files that contain nothing but
sequence residues - can only be used as query sequences. (2) FASTA
format files.  These are the same as plain sequence files, each
sequence is preceded by a comment line with a '>' in the first
column. (3) distributed sequence libraries (this is a broad class that
includes the NBRF/PIR VMS and blocked ascii formats, Genbank flat-file
format, EMBL flat-file format, and Intelligenetics format.  All of the
files that you create should be of type (1) or (2).  FASTA format
files (ones with a '>' and comment before the sequence) are preferred,
because they can be used as query or library sequence files by all of
the programs.
.pp
I have included several sample test files, \fC*.aa\fP and \fC*.seq\fP
as well as two small sequence libraries, \fCprot_test.lib\fP and
\fCgst.nlib\fP.  The first line may begin with a '>' by a comment.
Spaces and tabs (and anything else that is not an amino-acid code) are
ignored.
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Library files should have the form:
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>Sequence name and identifier
A F A S Y T .... actual sequence.
F S S       .... second line of sequence.
>Next sequence name and identifier
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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.
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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.
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A different format is required to specify the ordered peptide mixture for \fCfastf3/tfastf3\fP. For example:
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>mgstm1
MGCEN,
MIDYP,
MLLAY,
MLLGY
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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.
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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.
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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"
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Command line options are available to change the scoring parameters
and output display. \fBCommand line options must preceed other program
arguments, such as the query and library file names.\fP
.sh 2 "Command line options"
.ip "-a"
(fasta3, ssearch3 only) show both sequences in their entirety.
.ip "-A"
force Smith-Waterman alignments for fasta3 DNA sequences.  By default,
only fasta3 protein sequence comparisons use Smith-Waterman alignments.
.ip "-B"
Show normalized score as a z-score, rather than a bit-score in the list
of best scores.
.ip "-b #"
Number of sequence scores to be shown on output.  In the absence of
this option, fasta (and tfasta and ssearch) display all library
sequences obtaining similarity scores with expectations less than
10.0 if optimized score are used, or 2.0 if they are not. The -b
option can limit the display further, but it will not cause additional
sequences to be displayed.
.ip "-c #"
Threshold score for optimization (OPTCUT).  Set "-c 1" to
optimize every sequence in a database.
.ip "-E #"
Limit the number of scores and alignments shown based on the
expected number of scores.  Used to override the expectation value of 10.0
used by default.  When used with -Q, -E 2.0 will show all library sequences
with scores with an expectation value <= 2.0.
.ip "-d #"
Maximum number of alignments to be displayed.  Ignored if "-Q" is not
used.
.ip "-f"
Penalty for the first residue in a gap (-12 by default for proteins,
-16 for DNA, -15 for FAST[XY]/TFAST[XY]).
.ip "-F #"
Limit the number of scores and alignments shown based on the expected
number of scores. "-E #" sets the highest E()-value shown; "-F #" sets
the lowest E()-value. Thus, "-F 0.0001" will not show any matches or
alignments with E() < 0.0001.  This allows one to skip over close
relationships in searches for more distant relationships.
.ip "-g"
Penalty for additional residues in a gap (-2 by default for proteins,
-4 for DNA, -3 for FAST[XY]/TFAST[XY]).
.ip "-h"
Penalty for frameshift (fastx3/y3, tfastx3/y3 only).
.ip "-H"
Omit histogram.
.ip "-i"
Invert (reverse complement) the query sequence if it is DNA.  For
tfasta3/x3/y3, search the reverse complement of the library sequence
only.
.ip "-j #"
Penalty for frameshift within a codon (fasty3/tfasty3 only).
.ip "-l file"
Location of library menu file (FASTLIBS).
.ip "-L"
Display more information about the library sequence in the alignment.
.ip "-M low-high"
Range of amino acid sequence lengths to be included in the search.
.ip "-m #"
Specify alignment type: 0, 1, 2, 3, 4, 5, 6, 9, 10
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    \-m 0        \-m 1          \-m 2          \-m 3        \-m 4
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MWRTCGPPYT   MWRTCGPPYT    MWRTCGPPYT                 MWRTCGPPYT
::..:: :::     xx  X       ..KS..Y...    MWKSCGYPYT   ----------
MWKSCGYPYT   MWKSCGYPYT
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