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README for standalone MEGABLAST
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(last updated 7/2/2004)
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Mega BLAST uses the greedy algorithm of Zhang et al. [1] for nucleotide
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sequence alignment search and concatenates many queries to save time spent
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scanning the database. This program is optimized for aligning sequences that
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differ slightly as a result of sequencing or other similar "errors". It is up to
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10 times faster than more common sequence similarity programs and therefore can
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be used to swiftly compare two large sets of sequences against each other.
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Most of the options are similar to those in the blastall binary (see README.bls
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file for their descriptions). Note that megablast binary does not require the
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program option. Below are the more detailed explanations of some of the options
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either specific to Mega BLAST or having different meaning:
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-----------------------------
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When W is divisible by 4, it guarantees that all perfect matches of length
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W + 3 will be found by Mega BLAST search, however perfect matches of length
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as low as W might also be found, although the latter is not guaranteed. Any
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value of W not divisible by 4 is equivalent to the nearest value divisible by
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4 (with 4*i+2 equivalent to 4*i).
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-----------------------------
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-G, -E Affine gapping penalties.
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If these options are not set (both are 0), then non-affine gapping is assumed
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with gap opening penalty 0 and gap extension penalty E, that can be computed
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from match reward r and mismatch penalty q by the formula: E = r/2 - q. The
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affine version of Mega BLAST requires significantly more memory, so it should
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be avoided if possible, especially when some of the query or database
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sequences are very long.
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-----------------------------
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-D Type of the Mega BLAST output.
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0: Produce one-line output for each alignment, in the form
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'subject-id'=='[+-]query-id' (s_off q_off s_end q_end) score
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Here subject(query)-id is a gi number, an accession or some other type of
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identifier found in the FASTA definition line of the respective sequence.
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+ or - corresponds to same or different strand alignment.
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Score for non-affine gapping parameters means the total number of
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differences (mismatches + gaps). For affine case it is the actual (raw)
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score of the alignment.
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1: Show the same output as level 0, plus the endpoints and percentage
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of identical nucleotides for each ungapped segment in the alignment.
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2: Show the traditional BLAST (blastn) output.
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3: Show one-line output for each alignment, with the following fields
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Query id, Subject id, percent of identity, alignment length, number of
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mismatches (not including gaps), number of gap openings, start of
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alignment in query, end of alignment in query, start of alignment in
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subject, end of alignment in subject, expected value, bit score.
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If the alignment is from a reverse strand, the subject start and end are
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printed in the reverse order, reflecting the actual direction of the
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-----------------------------
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This option is described in the README.bls file and in general works
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identically to other BLAST programs. It actually contains two different
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options: the type of filtering and what stages of the search should mask the
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filtered regions. The option is specified by a string that contains all types
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of filters the user wants to apply, separated by semicolons or spaces. The
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available filters for nucleotide BLAST or Mega BLAST searches are:
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L - low complexity (equivalent to D)
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Finally, if letter 'm' is included in the filter string, all types of
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filters are used to mask the query sequence regions only on the word finding
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stage and do not affect the extension stage.
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E.g. if the option -F "m D;R" is specified, then both dust and human repeats
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filtering will be applied, but the alignments will be extended through the
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filtered areas. With option -F "L;V" the dust and vector screen filters will
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be applied, and the filtered areas will be masked for all stages of the
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The -F m option affects the lower case filtering (specified by the -U option)
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as well. Therefore if one wants to use lower case filtering, but allow the
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extension through lower case regions of the query sequence, the -F m -U T
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combination of options must be used.
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-----------------------------
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As in BLAST, this values provides a cutoff threshold for the extension
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algorithm tree exploration. When the score of a given branch drops below the
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current best score minus the X-dropoff, the exploration of this branch
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-y X-dropoff for ungapped extensions.
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-Z X-dropoff for the final gapped extension.
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Both -y and -Z are used only in conjunction with -n T option, i.e. when
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non-greedy gapped extension is performed, like in blastn.
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Note that all of these are the raw values, as opposed to bit values for other
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-----------------------------
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-e The cutoff expectation value.
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By default this value is set to a very large number, i.e. effectively there
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is no expectation value cutoff.
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-----------------------------
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-v Maximal number of database sequences to report alignments from.
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-b Maximal number of reported alignments for a given database sequence.
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These options are meaningful only in conjunction with -D 2.
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-----------------------------
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-J Believe the query defline.
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The default is T (TRUE) for all types of output except -D 2. In the latter
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case, the default is F (FALSE), unless a SeqAlign ASN.1 output is required,
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specified by the -O option.
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Note: this option must be set to F (FALSE) if the sequence IDs in the FASTA
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-----------------------------
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-M Maximal total length of queries to be concatenated for a single megablast
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Setting this value to smaller than default (20,000,000) can reduce the memory
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image of the program for large searches.
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-----------------------------
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-P Maximal number of positions for a hash value.
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This option provides for a very simple type of filtering if it is set to a
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non-zero value. Namely, any pattern of length 12 when word size is greater
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than or equal to 16 (8 for smaller word sizes), that appears in all of the
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query sequences together more than P times, is masked and not included in the
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search look-up table. If such masking occurs, megablast shows a warning
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message on the standard output. This can be useful when running megablast for
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very long unmasked sequences, in which case when -P option is not set, the
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search might take a very long time.
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-----------------------------
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-O ASN.1 Seqalign file.
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This option specifies a file name for writing ASN.1 output. It is only
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meaningful in conjunction with -D 2. The ASN.1 will consist of separate
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ASN.1 codes for each query sequence:
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All hits for first query
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All hits for second query
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-----------------------------
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-s Minimal hit score to report.
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By default this value is set to W, where W is the wordsize (-W option),
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i.e. is ignored (since all found alignments are extended from an exact match
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of length at least W).
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-----------------------------
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-Q Masked query output.
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All regions of the query sequences, that were hit by any found alignment, are
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masked by N's. The output is written to a file specified by the -Q option. It
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can be used only in conjunction with -D 2.
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-----------------------------
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-f Show full IDs in the output.
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By default, for -D 0 and -D 1 outputs, the sequence IDs are reported as GIs
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or accession numbers (if GIs are not available). If -f is set to T, full IDs
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will be shown, unless -J option is set to F. In the latter case full deflines
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will be shown for the query sequences.
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-----------------------------
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-U Use lower case filtering of FASTA sequences.
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Like in blastall binary, this option allows to treat lower case in the query
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sequences as masked residues. The deafult for this option is set to FALSE,
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in which case the lower case is treated identically to upper case.
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-----------------------------
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-p Cutoff by percentage of identity
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The alignments with identity percentage below the value of this option are
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not reported in all output formats except -D 0 (with the latter the traceback
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is not performed, so it is impossible to calculate the percentage of identical
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-----------------------------
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-t Discontiguous word template length.
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If this is not zero, the discontiguous word approach is used. The supported
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template lengths are 16, 18, and 21. The word size (-W parameter) must be 11 or
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-N Discontiguous template type: coding (0), non-coding (1), or both (2).
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For each of the three template lengths, two discontiguous templates are
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supported. One of them, called coding, is based on the '110' pattern, the other
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is optimal, or close to optimal, based on the hit probability simulations for
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The exact templates are:
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W = 11, t = 16, coding: 1101101101101101
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W = 11, t = 16, non-coding: 1110010110110111
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W = 12, t = 16, coding: 1111101101101101
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W = 12, t = 16, non-coding: 1110110110110111
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W = 11, t = 18, coding: 101101100101101101
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W = 11, t = 18, non-coding: 111010010110010111
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W = 12, t = 18, coding: 101101101101101101
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W = 12, t = 18, non-coding: 111010110010110111
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W = 11, t = 21, coding: 100101100101100101101
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W = 11, t = 21, non-coding: 111010010100010010111
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W = 12, t = 21, coding: 100101101101100101101
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W = 12, t = 21, non-coding: 111010010110010010111
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If 'both' option is chosen, then all initial matches satisfying either one of the
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two types of templates are extended.
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-----------------------------
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-g Generate words for every base of the database.
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Both in blastn and traditional megablast, the database sequences are compressed
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4:1, and words are looked up only at the beginning of each byte, i.e. at every 4th
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base. This option prescribes to lookup words starting at any arbitrary base
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of the database sequence.
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-----------------------------
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-H Maximal number of HSPs to save per database sequence.
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-----------------------------
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<p>[1] Zhang Z., Schwartz S., Wagner L., & Miller W. (2000),
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"A greedy algorithm for aligning DNA sequences", J Comput Biol 2000; 7(1-2):203-14.
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<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&list_uids=10890397&dopt=Citation">
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