PubMed Entrez BLAST OMIM Taxonomy Structure

  Set Subsequence

A region of the query sequences can be used to be used for BLAST searching. You can enter the range in nucleotides or protein residues in the "Form" and "To" boxes provided under "Set Subsequence". For example to limit matches to the region from nucleotide 24 to nucleotide 200 of a query sequence, you would enter From= 24 To= 200. If one of the limits you enter is out of range, the intersection of the [From,To] and [1,length] intervals will be searched, where length is the length of the whole query sequence.
  Databases available for BLAST search
Learn more

The BLAST  pages offer several different databases for searching. some of these, like SwissProt, PDB and Kabat are complied outside of NCBI. Other like ecoli, dbEST and month, are subsets of the NCBI databases. Other "virtual Databases" can be created using the Limit by Entrez Query option. 

Peptide Sequence Databases

All non-redundant GenBank CDS translations+RefSeq Proteins+PDB+SwissProt+PIR+PRF
Last major release of the SWISS-PROT protein sequence database (no updates)
Proteins from the Patent division of GenPept.
yeast (Saccharomyces cerevisiae) genomic CDS translations
Escherichia coli genomic CDS translations
Sequences derived from the 3-dimensional structure from Brookhaven Protein Data Bank
Drosophila genome
Drosophila genome proteins provided by Celera and Berkeley Drosophila Genome Project (BDGP).
All new or revised GenBank CDS translation+PDB+SwissProt+PIR+PRF released in the last 30 days.
Nucleotide Sequence Databases
All GenBank+RefSeq Nucleotides+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS sequences). No longer "non-redundant". 
Database of GenBank+EMBL+DDBJ sequences from EST Divisions
Human subset of GenBank+EMBL+DDBJ sequences from EST Divisions
Mouse subset of GenBank+EMBL+DDBJ sequences from EST Divisions
Non-Mouse, non-Human sequences of GenBank+EMBL+DDBJ sequences from EST Divisions
Genome Survey Sequence, includes single-pass genomic data, exon-trapped sequences, and Alu PCR sequences.
Unfinished High Throughput Genomic Sequences: phases 0, 1 and 2 (finished, phase 3 HTG sequences are in nr)
Nucleotides from the Patent division of GenBank.
Yeast (Saccharomyces cerevisiae) genomic nucleotide sequences
Database of mitochondrial sequences
Vector subset of GenBank(R), NCBI, in
E. coli
Escherichia coli genomic nucleotide sequences
Sequences derived from the 3-dimensional structure from Brookhaven Protein Data Bank
Drosophila genome 
Drosophila genome provided by Celera and Berkeley Drosophila Genome Project (BDGP).
All new or revised GenBank+EMBL+DDBJ+PDB sequences released in the last 30 days.
Select Alu repeats from REPBASE, suitable for masking Alu repeats from query sequences. It is available by anonymous FTP from (under the /pub/jmc/alu directory). See "Alu alert" by Claverie and Makalowski, Nature vol. 371, page 752 (1994).
Database of GenBank+EMBL+DDBJ sequences from STS Divisions .
Searches Complete Genomes, Complete Chromosome, or contigs form the NCBI Reference Sequence project..

Human Genome Blast DataBases

human genomic contig sequences with NT_#### accessions.
human RefSeq mrna with NM_#### or XM_#### accessions
human RefSeq proteins with NP_#### or XP_#### accessions
gscan mrna 
predicted mRNA sequences generated by running GenomeScan program on human genomic contigs
gscan protein 
CDS translations from gscan mrna set.

CDD Search 

 Compares protein sequences to the Conserved Domain Database. The CDD is a database containing a collection of functional and/or structural domains derived from two popular collections, Smart and Pfam, plus contributions from colleagues at NCBI. For more information please see the CDD homepage

  BLAST Search main parameters

Limit by Entrez Query

BLAST searches can be limited to the results of an Entrez query against the database chosen. This can be used to limit searches to subsets of the BLAST databases. Any terms can be entered  that would normally be allowed in an Entrez search session. For example: 
protease NOT hiv1[Organism] 
This will limit a BLAST search to all proteases, except those in HIV 1. This can also be used to limit searches to a particular molecule type: 
biomol_mrna[PROP] AND brain
To limit to a specific organism you can either select using the pulldown menu, form a  list of the most common organism in the databases. Or enter the name of the organism in the Entrez Query field with the [Organism] qualifier. For example: 
Mus musculus[Organism]
Or  For help in constructing Entrez queries please see the "Writing Advanced Search Statements" section of the Entrez Help document. 

Filter (Low-complexity) 

 Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (Computers and Chemistry, 1993) or, for BLASTN, by the DUST program of Tatusov and Lipman (in preparation). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences. 
Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs. 
It is not unusual for nothing at all to be masked by SEG, when applied to sequences in  SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect.

Filter (Human repeats) 

 This option masks Human repeats (LINE's and SINE's) and is especially useful for human sequences that may contain these repeats. Filtering for repeats can increase the speed of a search especially with very long sequences (>100 kb) and against databases which contain large number of repeats (htgs). For more information please see "Why does my search timeout on the BLAST servers?" in the BLAST Frequently Asked Questions. Human Repeat Filtering is still experimental and under development, so it may change in the near future.

Filter (Mask for lookup table only) 

This option masks only for purposes of constructing the lookup table used by BLAST. BLAST searches consist of two phases, finding hits based upon a lookup table and then extending them. The option to "Mask for lookup table only" masks only for the lookup table so that no hits are found based upon low-complexity sequence. The BLAST extensions are performed without masking and so they can be extended through low-complexity sequence. This option is still experimental and may change in the near future. 

Mask Lower Case 

With this option selected you can cut and paste a FASTA sequence in upper case characters and denote areas you would like filtered with lower case. This allows you to customize what is filtered from the sequence during the comparison to the BLAST databases  


The statistical significance threshold for reporting matches against database sequences; the default value is 10, meaning that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Increasing the threshold shows less stringent matches. Fractional values are acceptable. 

Learn more

Inclusion Threshold

The statistical significance threshold for including a sequence in the model used by PSI-BLAST to create the PSSM on the next iteration. 

Query Genetic Code

Genetic code to be used in blastx translation of the query. (See List of Genetic Codes)

Number of hits

It is possible to speed up search by specifying maximum number of hits to be computed.


If AutoFormat is disabled (unchecked) the "Status = Ready" and a change of background color to blue indicates the search is complete. However, it will not perform actual formatting. Formatting can be performed by pressing the 'Format' button on a previous page.
When the AutoFormat option is enabled (checked) clicking the Format button will show the status and time stamps and then automatically format BLAST results when they are ready.

Send Results by E-mail

By entering a e-mail address in the "Send Results by E-mail" field the BLAST server will send a copy of your BLAST results to the address provided. The default format of these results is in HTML however, you can have plain text results send in an e-mail by setting the "Format" pull-down menu from "HTML" to "Plain text" on the main BLAST search page. The BLAST graphic is not available through the e-mail service.

  Format Options

Graphical Overview

An overview of the database sequences aligned to the query sequence is shown. The score of each alignment is indicated by one of five different colors, which divides the range of scores into five groups. Multiple alignments on the same  database sequence are connected by a striped line. Mousing over a hit sequence causes the definition and score to be shown in the window at the top, clicking on a hit sequence takes the user to the associated alignments. 


Causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name. 


Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. See also EXPECT. 


Restricts database sequences to the number specified for which high-scoring segment pairs (HSPs) are reported; the default limit is 100. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT below), only the matches ascribed the greatest statistical significance are reported.

Database LinkOuts

Enabling this option provides cross reference links from the BLAST results to other NCBI specialized databases. If a database sequence matches your query and it also found in LocusLink or UniGene (more databases to be included in the future) there will be links () from the BLAST search results to these resources.

Alignments Views 


Standard BLAST alignment in pairs of query sequence and database match.

Query-anchored with identities

The databases alignments are anchored (shown in relation to) to the query sequence. Identities are displayed as dashes, with mismatches displayed as single letter  nucleotide abbreviations.

Query-anchored without identities

Identities are shown as single letter nucleotide abbreviations. 

Flat Query-anchored with identities

The 'flat' display shows inserts as deletions on the query. 

Identities are displayed as dashes, with mismatches displayed as single letter nucleotide abbreviations. 

Flat Query-anchored without identities

The 'flat' display shows inserts as deletions on the query. Identities are shown as single letter nucleotide abbreviations. 

Get ASN.1 for SeqAnnot

SeqAnnot format for importation into NCBI Toolkit programs. 

Get ASN.1 for the BLAST Object

Object format for NCBI toolkit programs.

The translations include: 

compares a nucleotide query sequence translated in all reading frames against a protein sequence database

compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames.

compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. Please note that tblastx program cannot be used with the nr database on the BLAST Web page.
  Matrix and Gap Costs
Learn more


A key element in evaluating the quality of a pairwise sequence alignment is the "substitution matrix", which assigns a score for aligning any possible pair of residues.  The matrix used in a BLAST search can be changed depending on the type of sequences you are searching with (see the BLAST Frequently Asked Questions). 

More information on BLAST substitution matrices

Gap Cost and Lambda Ratio 

The pull down menu shows the Gap Costs (Penalty to open Gap and penalty to extend Gap) and the Lambda ratio settings for the matrix chosen. There can only be a limited number of options for these parameters. Increasing the Gap Costs and Lambda ratio will result in alignments which decrease the number of Gaps introduced.

Learn More


PSI-BLAST can save the Position Specific Score Matrix to be used in other protein searches. The PSSM can be stored in a text file and cut and pasted into the PSSM field.
To save a PSSM file: 
  • Run a protein BLAST search. 
  • Check the PSI-BLAST box on formatting page. 
  • Click the "Format" Button. 
  • On the PSI-BLAST results page, click the "Run PSI-BLAST Iteration 2" button. 
  • Now, on the Format page, "PSSM" from the "Show" pull down menu.
  • Click "Format".
  • This will display  text output with the ASCII-encoded PSSM. The "Save as..." option of the browser can be used to save this to a plain text file on your hard drive. 
From the protein BLAST page, chose any database, and paste the contents of the PSSM text file into the "PSSM" field. If the database is the same as when the PSSM was stored, you'll reproduce the iteration on which you've saved the PSSM; A different database will yield a different hit list. 

Composition-based statistics

BLAST and PSI-BLAST now permit calculated E-values to take into account the amino acid composition of the individual database sequences involved in reported alignments. This improves E-value accuracy, thereby reducing the number of false positive results.

The improved statistics are achieved with a scaling procedure [1,2] which in effect employs a slightly different scoring system for each database sequence. As a result, raw BLAST alignment scores in general will not correspond precisely to those implied by any standard substitution matrix. Furthermore, identical alignments can receive different scores, based upon the compositions of the sequences they involve. The improved statistics are now used by default for all rounds of searching on the PSI-BLAST page, but not on the BLAST page. Therefore, if one uses default settings, the results of the first round of searching will be different on the BLAST and PSI-BLAST pages. In addition adjustments have been made to two PSI-BLAST parameters: the pseudocount constant default has been changed from 10 to 7, and the E-value threshold for including matches in the PSI-BLAST model has been changed from 0.001 to 0.002.

[1] Altschul, S.F. et al. (1997) Nucl. Acids Res. 25:3389-3402.
[2] Schäffer, A.A. et al. (1999) Bioinformatics 15:1000-1011. 
  NCBI BLAST Advanced Options

Program Advanced Options

-G  Cost to open gap [Integer]
default = 5  for nucleotides 11 proteins
-E  Cost to extend gap [Integer]
default = 2 nucleotides 1 proteins
-q Penalty for nucleotide mismatch [Integer]
default = -3
-r reward for nucleotide match [Integer]
default = 1
-e expect value [Real]
default = 10
-W wordsize [Integer]
default = 11 nucleotides 3 proteins
-y  Dropoff (X) for blast extensions in bits (default if zero) 
default = 20 for blastn 7 for other programs
-X  X dropoff value for gapped alignment (in bits) 
default = 15 for al programs except for blastn for which it does not apply
-Z  final X dropoff value for gapped alignment (in bits) 
50 for blastn 25 for other programs 

Limited values for gap existence and extension are supported for these three programs.  Some supported and suggested values are: 

  Existence     Extension
     10             1
     10             2
     11             1
      8             2
      9             2
  PHI-BLAST Pattern
Learn more

PHI-BLAST (Pattern-Hit Initiated BLAST) is a search program that combines matching of regular expressions
with local alignments surrounding the match. Given a protein sequence S and a regular expression pattern P
occurring in S, PHI-BLAST helps answer the question: What other protein sequences both contain an occurrence of P
and are homologous to S in the vicinity of the pattern occurrences? PHI-BLAST may be preferable to just searching for pattern occurrences because it filters out those cases where the pattern occurrence is probably random and not indicative of homology.  Please see the Rules for Pattern Syntax
Learn more

The Position-Specific Iterated BLAST, or PSI-BLAST program performs an iterative search in which sequences found in one round of searching are used to build a score model for the next round of searching. In PSI-BLAST the algorithm is not tied to a specific score matrix. Traditionally, it has been implemented using an AxA substitution matrix where A is the alphabet size. PSI-BLAST instead uses a QxA matrix, where Q is the length of the query sequence; at each position the cost of a letter depends on the position w.r.t. the query and the letter in the subject sequence.
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Revised January 21, 2000