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