In order to get desirable search results and avoid those unwanted, you can specify some parameters and/or choose search options you need (if so) before the start of a search.
Common parameters nearly for all searches:
Subsequence from/to - start and stop position in the query sequence to specify a shorter section of the sequence to be searched.
Low-complexity filter - the function conceals segments of the query sequence that have low compositional complexity. Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output, leaving the more biologically interesting regions of the query sequence available for specific matching against database.
By pressing the “Automatically adjust parameters for short input sequences” button, word size and other parameters will be automatically adjusted for the improvement of short queries result.
Further algorithm parameters of dissimilar searches differ. Symbolically, the algorithm parameters of different searches could be split up into groups as introduced below, since some of them are pretty much alike.
Limit on Query Length
There is a limit on query length for Smith-Waterman, MSS and BestSeq algorithms. Alerts and notifications appear on exceeding the query limitation of 10.000 symbols.
Blast, MOTIF and Keyword algorithms have no limit on query length.
BlastN Search
General Parameters:
- Max target sequences — the maximum number of aligned sequences to display.
- Expectation value (E) — a parameter that describes the number of hits that can be "expected" to be seen by chance when searching a database of a particular size. The Expectation value can be used as a convenient way to create a significance threshold for reporting results. When the Expectation value is increased from the default value, a larger list with more low-scoring hits would be reported.
- Word size — another parameter, important for a short query search. The BLAST algorithm uses words to unite regions of similarity. The query length must be at least twice as long as the word size (e.g. if your query is a protein sequence of 4 residues, then the word size should be reduced to 2). Note that the smaller the word size is, the longer it will take you to get the search results.
- Nucleotide query strand — select plus, minus or both strands for searching.
- Alignment identities percent threshold % — show sequence hits with an alignment percent identity only above a chosen value.
-
Query coverage threshold % — show sequence hits with query coverage only above a chosen value. It's the percentage of query length over the length of the alignment, and can be a useful metric in assessing similarity. For example, a 100% identity alignment with query coverage of 20% means only 20% of the query length matches perfectly to the answer.
Scoring Parameters:
- Match/Mismatch Scores — a scoring parameter that associates particular numbers for a match and for a mismatch.
- Gap costs — (open / extend) the pull down menu shows the Gap Costs for the chosen Matrix. There is a limited number of options for these parameters. Increasing the Gap Costs will result in alignments which decrease the number of Gaps introduced and vice versa.
-
Perform ungapped alignment — decide whether to perform ungapped alignment.
Filters and Masking:
- Lower case filtering — the filter displays masked region in lower case letters.
- As in BLAST, “X dropoff” values provides a cutoff threshold for the extension algorithm tree exploration. The exploration of the branch stops, when its score drops below the current best score minus the X-dropoff:
- X dropoff value for ungapped extensions;
- X dropoff value for gapped extensions;
-
X dropoff value for final gapped extensions.
BlastP/ TBlastN/ BlastX/ TBlastX searches, MSS Protein
General Parameters and Filters and Masking have the same meaning and characteristics as for BlastN search, though the Scoring Parameters are different.
Scoring Parameters:
- Matrix — a substitution matrix is a key element in evaluating the quality of a pairwise sequence alignment. It assigns a score for aligning any possible pair of residues. There are different substitution matrices to detect similarities among sequences that are diverged by differing degrees. For proteins, a provisional table of recommended substitution matrices and gap costs for various query lengths is:
| Query Length | Substitution Matrix | Gap Costs |
|---|---|---|
| <35 | PAM30 | (9, 1) |
| 35-50 | PAM70 | (10, 1) |
| 50-85 | BLOSUM80 | (10, 1) |
| >85 | BLOSUM62 | (11, 1) |
*See more information on BLAST substitution matrices
- Gap costs — (open / extend) the pull down menu shows the gap costs for the chosen matrix. There is a limited number of options for these parameters. Increasing the Gap Costs will result in alignments which decrease the number of Gaps introduced and vice versa.
- Multiple hits window size — this specific search parameter of BLAST specifies how close the two hits have to be to trigger extension. When BLAST is set to two-hit mode, this option requires two word hits on the same diagonal to be within letters of each other in order to extend from either one. The larger the value, the more sensitive BLAST will be.
- Perform ungapped alignment — decide whether to perform ungapped alignment.
Also, pay attention to the fact that MSS Protein has another suite of substitution matrices to choose from:
The values of substitution matrices can be observed here.
Smith-Waterman Nucleotide/ Smith-Waterman Protein searches / MSS Nucleotide
General Parameters:
- Max target sequences — select maximum number of aligned sequences to display.
- Reverse (true/ false) — select “True” to calculate scores and perform alignments also for reverse complement of query sequences. (Firstly, nucleotides are substituted for their complementary ones, like: A<-->T, C<-->G; then the sequence is reversed).
- Alignment identities percent threshold % — show sequence hits with an alignment percent identity only above a chosen value.
- Query coverage threshold % — show sequence hits with query coverage only above a chosen value.
Scoring Parameters:
- Matrix — select the substitution matrix (DNA/ NUC.4.4). Matrices for the Smith-Waterman Protein search are the same as for MSS Protein:
- Minimum score — specify the minimum score.
- Cost to open a gap — specify the gap open penalty (0...255).
- Cost to extend a gap — specify the gap extension penalty (0...255).