BLAST

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BLAST+ user manual

Introduction
Enter a sequence
Choose a database
- Protein databases
- Nucleotide databases
Select options
Programmatic access

Introduction

The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families [more...].

Programs available for the BLAST search

Programs available on ExPASy

blastp: compares an amino acid query sequence against a protein sequence database
blastn: compares a nucleotide query sequence against a nucleotide sequence database
tblastn: compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames
blastx: compares a nucleotide query sequence translated in all reading frames against a protein sequence database

Other programs

blastp with different algorithms: PSI-BLAST, PHI-BLAST and DELTA-BLAST: compare an amino acid query sequence against a protein sequence database. Available at the NCBI.
blastn with different algorithms: megablast and discontiguous megablast: compare a nucleotide query sequence against a nucleotide sequence database. Available at the NCBI.
tblastx: compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. Available at the NCBI.

Enter a sequence

Enter a query protein or nucleotide sequence into the text area. Accepted input:

UniProt Knowledgebase (UniProtKB) accession or identifier
bare sequence
sequence in FASTA format

e.g. P00750, P05067-5, A4_HUMAN or acccgtggtcgctgctg...

The format and the nature of the input (protein or nucleotide) are determined automatically.

Choose a database

Protein databases

UniProt Knowledgebase

The UniProt Knowledgebase (UniProtKB) is the central hub for the collection of functional information on proteins, with accurate, consistent and rich annotation. UniProtKB is composed of two sections:

Reviewed (Swiss-Prot) - Manually annotated: Records with information extracted from literature and curator-evaluated computational analysis.
Unreviewed (TrEMBL) - Computationally analyzed: Records that await full manual annotation.

In addition to capturing the core data mandatory for each UniProtKB entry (mainly, the amino acid sequence, protein name or description, taxonomic data and citation information), as much annotation information as possible is added [more...].

N.B. Since UniProtKB contains a huge number of sequences, if you are interested in a particular taxon that is listed under 'UniProtKB taxonomic subsets', you are strongly advised to select from that menu. Moreover, you can also greatly reduce the search space by choosing to perform the BLAST against UniProtKB/Swiss-Prot only instead of the whole UniProtKB (UniProtKB/Swiss-Prot + UniProtKB/TrEMBL) and this whether you choose UniProtKB 'Complete database', 'Proteomes', 'Reference proteomes' or any of the 'UniProtKB taxonomic subsets'.

Complete database: UniProtKB/Swiss-Prot + UniProtKB/TrEMBL
Proteomes: Proteomes are sets of proteins thought to be expressed by organisms whose genomes have been completely sequenced [more...].
Reference proteomes: Reference proteomes are 'Complete proteomes' which have been selected as reference proteomes. Reference proteomes are both manually defined and algorithmically selected according to a number of criteria [more...].

Notes:

For database option 'Complete database', you may filter the displayed results to a particular taxon by entering a species name, a TaxID or the latin name of a taxonomic group (elements of the UniProtKB OC, OS and OX lines). If you wish to enter more than one term, separate them with an exclamation mark e.g. 'Fungi! Homo sapiens' or '4751! 9606'.
This filter is applied only if the selected output format is 'html' (default).
WARNING: This is post-processing of the results: the BLAST is performed on 'Complete database', and only results fulfilling the taxonomic criteria you have entered are shown. This will decrease your hits and statistically bias your results.
ADVICE: If the taxon you're interested in is in the 'UniProtKB taxonomic subsets' select menu, we strongly suggest you use that list instead.
For any of the options 'Complete database', 'Proteomes' or 'Reference proteomes', if the checkbox 'UniProtKB/Swiss-Prot (manually annotated) only' is checked, only UniProtKB/Swiss-Prot sequences will be considered.

UniProtKB taxonomic subsets

Each option constitutes a taxonomic subset of UniProtKB. If the checkbox 'UniProtKB/Swiss-Prot (manually annotated) only' is checked, only UniProtKB/Swiss-Prot sequences will be considered.

Prokaryotic proteomes

The HAMAP proteomes are non-redundant sets of all the proteins from selected complete genome sequencing projects, compiled from UniProtKB.

Other databases

UniRef100, UniRef90 and UniRef50: The UniProt Reference Clusters (UniRef) provide clustered sets of sequences from the UniProt Knowledgebase (including isoforms) and selected UniParc records in order to obtain complete coverage of the sequence space at several resolutions while hiding redundant sequences (but not their descriptions) from view. UniRef100 contains all UniProt Knowledgebase records plus selected UniParc records. In UniRef100, all identical sequences and subfragments with 11 or more residues are placed into a single record. UniRef50 and UniRef90 are built based on UniRef100 [more...].
PDB: The Protein Data Bank (PDB) is a database of protein 3D structures [more...]. Sequences extracted from the PDB SEQRES lines are processed into a non-redundant set where identical sequences are merged into single records.

Nucleotide databases

Sequence databases

The European Nucleotid Archive (ENA, formerly EMBL-Bank) constitutes the world's primary nucleotide sequence resource. Main sources for DNA and RNA sequences are direct submissions from individual researchers, genome sequencing projects and patent applications [more...].
ENA records are broken down into different 'Data classes' e.g. 'Annotation', 'Reads' and each of theses data classes can be further broken down into 'Data domain' such as 'HTC' and 'HTG' for 'Annotation' [more...].

ENA: Standard annotated assembled sequences
HTG: High throughput assembled gemomic sequences with optional annotation
EST: Raw expressed sequence tag sequence data (no qualities) and sample/library information
Unigene - EST: Database of EST clusters (list of ESTs known to match the same cDNA) from the NCBI (updated occasionally). This database contains also useful information like Sequence Tagged Site (STS) matches, tissue distribution, or transcript map [more...].
GSS: Genome survey sequences; single pass, single direction sequences
STS: Sequence tagged sites
Patents: Sequence associated with a patent process

Taxonomic groups

Each option represents a subset of the ENA Standard annotated assembled sequences (STD) database. Each substet is taxonomy-based except for the last one 'Synthetic'.

Eukaryota: Sequences belonging to superkingdom 'Eukaryota'
Vertebrata: Sequences belonging to 'Vertebrata'
Mammalia: Sequences belonging to class 'Mammalia'
Homo sapiens: Sequences belonging to species 'Homo sapiens'
Rodentia: Sequences belonging to order 'Rodentia'
Other mammalia: Sequences belonging to class 'Mammalia' but not species 'Homo sapiens', nor order 'Rodentia'
Other vertebrata: Sequences belonging to 'Vertebrata' but not to the 'Mammalia' class
Invertebrata: Sequences belonging to superkingdom 'Eukaryota' but not to 'Vertebrata'
Fungi: Sequences belonging to kingdom 'Fungi'
Viridiplantae: Sequences belonging to kingdom 'Viridiplanta'
Prokaryota: Sequences belonging to superkingdom 'Bacteria' or superkingdom 'Archaea'
Bacteriophages: Sequences belonging to diffent groups of superkingdom 'Viruses'
Viruses: Sequences belonging to supekingdom 'Viruses'
Unclassified: Sequences not belonging to superkingdom 'Eukaryota', 'Bacteria', 'Archaea' or 'Viruses'
Synthetic: Sequences corresponding to synthetic molecules and constructs [more...]

Microbial genomes

One of the selected genomes released in the form of a complete, assembled sequence.

Select options

Comparison matrix

The matrix assigns a probability score for each position in an alignment. The BLOSUM matrix assigns a probability score for each position in an alignment that is based on the frequency with which that substitution is known to occur among consensus blocks within related proteins.
BLOSUM62 is among the best of the available matrices for detecting weak protein similarities. The PAM set of matrices is also available.
If the "Auto-select" option is selected (default), the matrix will be selected depending on the query sequence length, based on the following (empirically constructed) table:

Default values

Query length	Substitution matrix
<35	PAM-30
35-50	PAM-70
50-85	BLOSUM-80
>85	BLOSUM-62

Note: If the input sequence is shorter than 30 amino acids and the blast program is blastp, the matrix is automatically set to be PAM-30 [more...].

E-value threshold

Default value: 10
The expectation value (E) threshold is a statistical measure of the number of expected matches in a random database. The lower the e-value, the more likely the match is to be significant. E-values between 0.1 and 10 are generally dubious, and over 10 are unlikely to have biological significance. In all cases, those matches need to be verified manually. You may need to increase the E threshold in the following cases :

if you have a very short query sequence
to detect very weak similarities, or similarities in a short region
if your sequence has a low complexity region and you use the masking option ('Filter low complexity regions').

Filter low complexity regions

Default value: yes
'yes' translates to '-seg yes', 'no' to ''

Low-complexity regions, e.g. stretches of cysteine in CSP_DROME (Q03751), hydrophobic regions in membrane proteins, tend to produce spurious, insignificant matches with sequences in the database which have the same kind of low-complexity regions, but are unrelated biologically.
If this option is checked, the query sequence will be run through the program SEG, and all amino acids in low-complexity regions will be replaced by X's which will appear in the alignment. The masked regions will also be visible as slashed regions in the PaintBlast image.

Note: If the input sequence is shorter than 30 amino acids and the blast program is blastp, this parameter is automatically set to 'no' [more...].

Allow introduction of gaps in alignments

Default value: yes
'yes' translates to '', 'no' to '-comp_based_stats F -ungapped'

This will allow gaps to be introduced in the sequences when the comparison is done.

Note: If the input sequence is shorter than 30 amino acids and the blast program is blastp, this parameter is automatically transformed to '-comp_based_stats 0' [more...].

blastp and short sequences

For comparison of amino acid sequences shorter than 30 amino acids against protein sequence databases, some input parameters are automatically overridden to optimize the search following The BLAST^® Command Line Applications User Manual, (table 2, "blastp-short" task):

The chosen matrix is set to PAM30 (-matrix PAM30).
The filter low complexity regions is set to 'no'.
The 'Gapped alignments' parameter is set to '-comp_based_stats 0'.

Additional parameters, that not may not be tuned using the BLAST form on ExPASy are added to end up with the following parameters:

-word_size 2 -gapopen 9 -gapextend 1 -matrix PAM30 -threshold 16 -comp_based_stats 0 -window_size 15

Programmatic access

Examples

https://web.expasy.org/cgi-bin/blast/blast.pl?seq=P00750&prot_db1=UniProtKB&curated=on&ethr=10&Gap=T&matrix=auto&Filter=T&showal=100&showsc=100&format=html
blastp
https://web.expasy.org/cgi-bin/blast/blast.pl?seq=P00750&curated=on
blastp - generates the same blast command as the one in the previous example.
https://web.expasy.org/cgi-bin/blast/blast.pl?seq=P00750&curated=on&Tax=9989! 9605
blastp - generates the same blast command as the one in the previous example, the results are then filtered to keep only matches belonging taxa 'Rodentia' or 'Homo sapiens'.
Note that this filter is applied only if prot_db1=UniProtKB ('UniProtKB - Complete database' in the form, default) and 'format=html' (default).
https://web.expasy.org/cgi-bin/blast/blast.pl?seq=ACACG...&nt_db1=embl&format=txt
blastn
https://web.expasy.org/cgi-bin/blast/blast.pl?seq=A4_HUMAN&nt_db2=rod&format=xml
tblastn
https://web.expasy.org/cgi-bin/blast/blast.pl?seq=ACACG...&prot_db4=pdbaa&matrix=PAM30&ethr=1&Filter=F&Gap=F&showsc=50&showal=50
tblastx

Enter a sequence

seq=	Input sequence
P00750	UniProtKB accession
A4_HUMAN	UniProtKB identifier
ACACGGTCATCGCGCGCCTGCGCAAGGAG...	bare protein or nucleotide sequence, remove FASTA header if any for programmatic access

Choose a database

prot_db1=	UniProt Knowledgebase (UniProtKB)
UniProtKB	Complete database (default)
Complete_proteomes	Proteomes
Reference_proteomes	Reference proteomes

Tax=	Taxonomic filter only applied if protdb1=UniProtKB (default) and format=html (default)
Fungi! Homo sapiens	Fungi or Homo sapiens
4751! 9606	Fungi or Homo sapiens

prot_db2=	UniProtKB taxonomic subsets
Archaea	Archaea
Bacteria	Bacteria
Eukaryota	Eukaryota
Viruses	Viruses
Arthropoda	Arthropoda
Fungi	Fungi
Mammalia	Mammalia
Metazoa	Metazoa
Primates	Primates
Rodentia	Rodentia
Vertebrata	Vertebrata
Viridiplantae	Viridiplantae
ARATH	Arabidopsis thaliana
CAEEL	Caenorhabditis elegans
DICDI	Dictyostelium discoideum
DROME	Drosophila melanogaster
ECOLI	Escherichia coli
HUMAN	Homo sapiens
MOUSE	Mus musculus
PLAFA	Plasmodium falciparum
RAT	Rattus norvegicus
YEAST	Saccharomyces cerevisiae
SCHPO	Schizosaccharomyces pombe

curated=	UniProtKB/Swiss-Prot (manually annotated) only
on	UniProtKB/Swiss-Prot (manually annotated) only Only relevant if the target database is UniProtKB (prot_db1 or prot_db2)

prot_db3=	Prokaryotic proteomes
OS_code	OS_code OS_name
ACEP3	ACEP3 Acetobacter pasteurianus (strain NBRC 3283 / LMG 1513 / CCTM 1153)
prot_db4=	Other database
UniRef100	UniRef100
UniRef90	UniRef90
UniRef50	UniRef50
pdbaa	PDB

nt_db1=	Sequence databases
embl	ENA
htg	HTG
est	EST
unigene_est	Unigene EST
gss	GSS
sts	STS
pat	Patents
nt_db2=	ENA taxonomic subsets
euk	Eukaryota
all_vrt	Vertebrata
all_mam	Mammalia
hum	Homo sapiens
rod	Rodentia
mam	Other mammalia
vrt	Other vertebrata
inv	Invertebrata
fun	Fungi
pln	Viridiplantae
pro	Prokaryota
vrl	Viruses
phg	Bacteriophages
unc	Unclassified
syn	Synthetic
nt_db3=	Prokaryotic genomes
OS_code	OS_code OS_name
NANEQ	NANEQ Nanoarchaeume equitans (Strain Kin4-M)

Select options

matrix=	Comparison matrix
auto	Auto-select (default)
BLOSUM62	BLOSUM62
BLOSUM45	BLOSUM45
BLOSUM80	BLOSUM80
PAM30	PAM30
PAM70	PAM70
ethr=	E-value
0.0001	0.0001
0.001	0.001
0.01	0.01
0.1	0.1
1	1
10	10 (default)
100	100
1000	1000
10000	10000
Filter=	Filter low complexity regions
T	yes (translates to '-seg yes', default)
F	no (translates to '')
Gap=	Gapped alignments
T	yes (translates to '', default)
F	no (tranlates to -comp_based_stats F -ungapped)
showsc=	Number of best scoring sequences to show
50	50
100	100 (default)
250	250
1000	1000
3000	3000
showal=	Number of best alignments to show
50	50
100	100 (default)
250	250
1000	1000
format=	Output format
html	HTML (default)
txt	Plain text
xml	XML