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Manual Reference Pages  -  FASTA36 (1)


fasta36 - scan a protein or DNA sequence library for similar sequences

fastx36 - compare a DNA sequence to a protein sequence database, comparing the translated DNA sequence in forward and reverse frames.

tfastx36 - compare a protein sequence to a DNA sequence database, calculating similarities with frameshifts to the forward and reverse orientations.

fasty36 - compare a DNA sequence to a protein sequence database, comparing the translated DNA sequence in forward and reverse frames.

tfasty36 - compare a protein sequence to a DNA sequence database, calculating similarities with frameshifts to the forward and reverse orientations.

fasts36 - compare unordered peptides to a protein sequence database

fastm36 - compare ordered peptides (or short DNA sequences) to a protein (DNA) sequence database

tfasts36 - compare unordered peptides to a translated DNA sequence database

fastf36 - compare mixed peptides to a protein sequence database

tfastf36 - compare mixed peptides to a translated DNA sequence database

ssearch36 - compare a protein or DNA sequence to a sequence database using the Smith-Waterman algorithm.

ggsearch36 - compare a protein or DNA sequence to a sequence database using a global alignment (Needleman-Wunsch)

glsearch36 - compare a protein or DNA sequence to a sequence database with alignments that are global in the query and local in the database sequence (global-local).

lalign36 - produce multiple non-overlapping alignments for protein and DNA sequences using the Huang and Miller sim algorithm for the Waterman-Eggert algorithm.

prss36, prfx36 - discontinued; all the FASTA programs will estimate statistical significance using 500 shuffled sequence scores if two sequences are compared.


Running the FASTA programs
FASTA program options
Option summary:
Reading sequences from STDIN
Environment variables:


Release 3.6 of the FASTA package provides a modular set of sequence comparison programs that can run on conventional single processor computers or in parallel on multiprocessor computers. More than a dozen programs - fasta36, fastx36/tfastx36, fasty36/tfasty36, fasts36/tfasts36, fastm36, fastf36/tfastf36, ssearch36, ggsearch36, and glsearch36 - are currently available.

All the comparison programs share a set of basic command line options; additional options are available for individual comparison functions.

Threaded versions of the FASTA programs (built by default under Unix/Linux/MacOX) run in parallel on modern Linux and Unix multi-core or multi-processor computers. Accelerated versions of the Smith-Waterman algorithm are available for architectures with the Intel SSE2 or Altivec PowerPC architectures, which can speed-up Smith-Waterman calculations 10 - 20-fold.

In addition to the serial and threaded versions of the FASTA programs, MPI parallel versions are available as fasta36_mpi, ssearch36_mpi, fastx36_mpi, etc. The MPI parallel versions use the same command line options as the serial and threaded versions.

Running the FASTA programs

By default, the FASTA programs are no longer interactive; they are run from the command line by specifying the program, query.file, and library.file. Program options must preceed the query.file and library.file arguments:

   fasta36 -option1 -option2 -option3 query.file library.file > fasta.output

The "classic" interactive mode, which prompts for a query.file and library.file, is available with the -I option. Typing a program name without any arguments (ssearch36) provides a short help message; program_name -help provides a complete set of program options.

Program options MUST preceed the query.file and library.file arguments.

FASTA program options

The default scoring matrix and gap penalties used by each of the programs have been selected for high sensitivity searches with the various algorithms. The default program behavior can be modified by providing command line options before the query.file and library.file arguments. Command line options can also be used in interactive mode.

Command line arguments come in several classes.

(1) Commands that specify the comparison type. FASTA, FASTS, FASTM, SSEARCH, GGSEARCH, and GLSEARCH can compare either protein or DNA sequences, and attempt to recognize the comparison type by looking the residue composition. -n, -p specify DNA (nucleotide) or protein comparison, respectively. -U specifies RNA comparison.

(2) Commands that limit the set of sequences compared: -1, -3, -M.

(3) Commands that modify the scoring parameters: -f gap-open penalty\P, -g gap-extend penalty, -j inter-codon frame-shift, within-codon frameshift, -s scoring-matrix, -r match/mismatch score, -x X:X score.

(4) Commands that modify the algorithm (mostly FASTA and [T]FASTX/Y): -c, -w, -y, -o. The -S can be used to ignore lower-case (low complexity) residues during the initial score calculation.

(5) Commands that modify the output: -A, -b number, -C width, -d number, -L, -m 0-11,B, -w line-width, -W context-width, -o offset1,ofset2

(6) Commands that affect statistical estimates: -Z, -k.

Option summary:

-1 Sort by "init1" score (obsolete)
-3 ([t]fast[x,y] only) use only forward frame translations
-a Displays the full length (included unaligned regions) of both sequences with fasta36, ssearch36, glsearch36, and fasts36.
-A (fasta36 only) For DNA:DNA, force Smith-Waterman alignment for
  output. Smith-Waterman is the default for FASTA protein alignment and [t]fast[x,y], but not for DNA comparisons with FASTA. For protein:protein, use band-alignment algorithm.
-b # number of best scores/descriptions to show (must be < expectation cutoff if -E is given). By default, this option is no longer used; all scores better than the expectation (E()) cutoff are listed. To guarantee the display of # descriptions/scores, use -b =#, i.e. -b =100 ensures that 100 descriptions/scores will be displayed. To guarantee at least 1 description, but possibly many more (limited by -E e_cut), use -b >1.
-c "E-opt E-join"
  threshold for gap joining (E-join) and band optimization (E-opt) in FASTA and [T]FASTX/Y. FASTA36 now uses BLAST-like statistical thresholds for joining and band optimization. The default statistical thresholds for protein and translated comparisons are E-opt=0.2, E-join=0.5; for DNA, E-join = 0.1 and E-opt= 0.02. The actual number of joins and optimizations is reported after the E-join and E-opt scoring parameters. Statistical thresholds improves search speed 2 - 3X, and provides much more accurate statistical estimates for matrices other than BLOSUM50. The "classic" joining/optimization thresholds that were the default in fasta35 and earlier programs are available using -c O (upper case O), possibly followed a value > 1.0 to set the optcut optimization threshold.
-C # length of name abbreviation in alignments, default = 6. Must be less than 20.
-d # number of best alignments to show ( must be < expectation (-E) cutoff and <= the -b description limit).
-D turn on debugging mode. Enables checks on sequence alphabet that cause problems with tfastx36, tfasty36 (only available after compile time option). Also preserves temp files with -e option.
  Run a script to expand the set of sequences displayed/aligned based on the results of the initial search. When the -e option is used, after the initial scan and statistics calculation, but before the "Best scores" are shown, with a single argument, the name of a file that contains the accession information (the text on the fasta description line between > and the first space) and the E()-value for the sequence. then uses this information to send a library of additional sequences to stdout. These additional sequences are included in the list of high-scoring sequences (if their scores are significant) and aligned. The additional sequences do not change the statistics or database size.
-E e_cut e_cut_r
  expectation value upper limit for score and alignment display. Defaults are 10.0 for FASTA36 and SSEARCH36 protein searches, 5.0 for translated DNA/protein comparisons, and 2.0 for DNA/DNA searches. FASTA version 36 now reports additional alignments between the query and the library sequence, the second value sets the threshold for the subsequent alignments. If not given, the threshold is e_cut/10.0. If given and value > 1.0, e_cut_r = e_cut / value; for value < 1.0, e_cut_r = value; If e_cut_r < 0, then the additional alignment option is disabled.
-f # penalty for opening a gap.
-F # expectation value lower limit for score and alignment display. -F 1e-6 prevents library sequences with E()-values lower than 1e-6 from being displayed. This allows the use to focus on more distant relationships.
-g # penalty for additional residues in a gap
-h Show short help message.
-help Show long help message, with all options.
-H show histogram (with fasta-36.3.4, the histogram is not shown by default).
-i (fasta DNA, [t]fastx[x,y]) compare against only the reverse complement of the library sequence.
-I interactive mode; prompt for query filename, library.
-j # # ([t]fast[x,y] only) penalty for a frameshift between two codons, ([t]fasty only) penalty for a frameshift within a codon.
-J (lalign36 only) show identity alignment.
-k specify number of shuffles for statistical parameter estimation (default=500).
-l str specify FASTLIBS file
-L report long sequence description in alignments (up to 200 characters).
-m 0,1,2,3,4,5,6,8,9,10,11,B,BB,"F# out.file" alignment display
  options. -m 0, 1, 2, 3 display different types of alignments. -m 4 provides an alignment "map" on the query. -m 5 combines the alignment map and a -m 0 alignment. -m 6 provides an HTML output.
-m 8 seeks to mimic BLAST -m 8 tabular output. Only query and
  library sequence names, and identity, mismatch, starts/stops, E()-values, and bit scores are displayed. -m 8C mimics BLAST tabular format with comment lines. -m 8 formats do not show alignments.
-m 9 does not change the alignment output, but provides
  alignment coordinate and percent identity information with the best scores report. -m 9c adds encoded alignment information to the -m 9; -m 9C adds encoded alignment information as a CIGAR formatted string. To accomodate frameshifts, the CIGAR format has been supplemented with F (forward) and R (reverse). -m 9i provides only percent identity and alignment length information with the best scores. With current versions of the FASTA programs, independent -m options can be combined; e.g. -m 1 -m 9c -m 6.
-m 11 provides lav format output from lalign36. It does not
  currently affect other alignment algorithms. The lav2ps and lav2svg programs can be used to convert lav format output to postscript/SVG alignment "dot-plots".
-m B provides BLAST-like alignments. Alignments are labeled as
  "Query" and "Sbjct", with coordinates on the same line as the sequences, and BLAST-like symbols for matches and mismatches. -m BB extends BLAST similarity to all the output, providing an output that closely mimics BLAST output.
-m "F# out.file" allows one search to write different alignment
  formats to different files. The ’F’ indicates separate file output; the ’#’ is the output format (1-6,8,9,10,11,B,BB, multiple compatible formats can be combined separated by commas -’,’).
-M #-# molecular weight (residue) cutoffs. -M "101-200" examines only library sequences that are 101-200 residues long.
-n force query to nucleotide sequence
-N # break long library sequences into blocks of # residues. Useful for bacterial genomes, which have only one sequence entry. -N 2000 works well for well for bacterial genomes. (This option was required when FASTA only provided one alignment between the query and library sequence. It is not as useful, now that multiple alignments are available.)
-o "#,#" offsets query, library sequence for numbering alignments
-O file send output to file.
-p force query to protein alphabet.
-P pssm_file
  (ssearch36, ggsearch36, glsearch36 only). Provide blastpgp checkpoint file as the PSSM for searching. Two PSSM file formats are available, which must be provided with the filename. ’pssm_file 0’ uses a binary format that is machine specific; ’pssm_file 1’ uses the "blastpgp -u 1 -C pssm_file" ASN.1 binary format (preferred).
-q/-Q quiet option; do not prompt for input (on by default)
-r "+n/-m"
  (DNA only) values for match/mismatch for DNA comparisons. +n is used for the maximum positive value and -m is used for the maximum negative value. Values between max and min, are rescaled, but residue pairs having the value -1 continue to be -1.
-R file save all scores to statistics file (previously -r file)
-s name specify substitution matrix. BLOSUM50 is used by default; PAM250, PAM120, and BLOSUM62 can be specified by setting -s P120, P250, or BL62. Additional scoring matrices include: BLOSUM80 (BL80), and MDM10, MDM20, MDM40 (Jones, Taylor, and Thornton, 1992 CABIOS 8:275-282; specified as -s MD10, -s MD20, -s MD40), OPTIMA5 (-s OPT5, Kann and Goldstein, (2002) Proteins 48:367-376), and VTML160 (-s VT160, Mueller and Vingron (2002) J. Comp. Biol. 19:8-13). Each scoring matrix has associated default gap penalties. The BLOSUM62 scoring matrix and -11/-1 gap penalties can be specified with -s BP62.
Alternatively, a BLASTP format scoring matrix file can be specified, e.g. -s matrix.filename. DNA scoring matrices can also be specified with the "-r" option.
With fasta36.3, variable scoring matrices can be specified by preceeding the scoring matrix abbreviation with ’?’, e.g. -s ’?BP62’. Variable scoring matrices allow the FASTA programs to choose an alternative scoring matrix with higher information content (bit score/position) when short queries are used. For example, a 90 nucleotide FASTX query can produce only a 30 amino-acid alignment, so a scoring matrix with 1.33 bits/position is required to produce a 40 bit score. The FASTA programs include BLOSUM50 (0.49 bits/pos) and BLOSUM62 (0.58 bits/pos) but can range to MD10 (3.44 bits/position). The variable scoring matrix option searches down the list of scoring matrices to find one with information content high enough to produce a 40 bit alignment score.
-S treat lower case letters in the query or database as low complexity regions that are equivalent to ’X’ during the initial database scan, but are treated as normal residues for the final alignment display. Statistical estimates are based on the ’X’ed out sequence used during the initial search. Protein databases (and query sequences) can be generated in the appropriate format using John Wooton’s "pseg" program, available from Once you have compiled the "pseg" program, use the command:
pseg database.fasta -z 1 -q > database.lc_seg
-t # Translation table - [t]fastx36 and [t]fasty36 support the BLAST tranlation tables. See
-T # (threaded, parallel only) number of threads or workers to use (on Linux/MacOS/Unix, the default is to use as many processors as are available; on Windows systems, 2 processors are used).
-U Do RNA sequence comparisons: treat ’T’ as ’U’, allow G:U base pairs (by scoring "G-A" and "T-C" as score(G:G)-3). Search only one strand.
-V "?$%*" Allow special annotation characters in query sequence. These characters will be displayed in the alignments on the coordinate number line.
-w # line width for similarity score, sequence alignment, output.
-W # context length (default is 1/2 of line width -w) for alignment,
  like fasta and ssearch, that provide additional sequence context.
-X extended options. Less used options. Other options include
  -XB, -XM4G, -Xo, -Xx, and -Xy; see fasta_guide.pdf.
-z 1, 2, 3, 4, 5, 6
  Specify the statistical calculation. Default is -z 1 for local similarity searches, which uses regression against the length of the library sequence. -z -1 disables statistics. -z 0 estimates significance without normalizing for sequence length. -z 2 provides maximum likelihood estimates for lambda and K, censoring the 250 lowest and 250 highest scores. -z 3 uses Altschul and Gish’s statistical estimates for specific protein BLOSUM scoring matrices and gap penalties. -z 4,5: an alternate regression method. -z 6 uses a composition based maximum likelihood estimate based on the method of Mott (1992) Bull. Math. Biol. 54:59-75.
-z 11,12,14,15,16
  compute the regression against scores of randomly shuffled copies of the library sequences. Twice as many comparisons are performed, but accurate estimates can be generated from databases of related sequences. -z 11 uses the -z 1 regression strategy, etc.
-z 21, 22, 24, 25, 26
  compute two E()-values. The standard (library-based) E()-value is calculated in the standard way (-z 1, 2, etc), but a second E2() value is calculated by shuffling the high-scoring sequences (those with E()-values less than the threshold). For "average" composition proteins, these two estimates will be similar (though the best-shuffle estimates are always more conservative). For biased composition proteins, the two estimates may differ by 100-fold or more. A second -z option, e.g. -z "21 2", specifies the estimation method for the best-shuffle E2()-values. Best-shuffle E2()-values approximate the estimates given by PRSS (or in a pairwise SSEARCH).
-Z db_size
  Set the apparent database size used for expectation value calculations (used for protein/protein FASTA and SSEARCH, and for [T]FASTX/Y).

Reading sequences from STDIN

The FASTA programs can accept a query sequence from the unix "stdin" data stream. This makes it much easier to use fasta36 and its relatives as part of a WWW page. To indicate that stdin is to be used, use "@" as the query sequence file name. "@" can also be used to specify a subset of the query sequence to be used, e.g:

   cat query.aa | fasta36 @:50-150 s

would search the ’s’ database with residues 50-150 of query.aa. FASTA cannot automatically detect the sequence type (protein vs DNA) when "stdin" is used and assumes protein comparisons by default; the ’-n’ option is required for DNA for STDIN queries.

Environment variables:

  location of library choice file (-l FASTLIBS)
  format strings used to define options to re-search the database.
  the format string used to define the option to lookup the library sequence in entrez, or some other database.


Bill Pearson

Version: $ Id: $ Revision: $Revision: 210 $

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