PSI-BLAST

Introduction

BLAST stands for Basic Local Alignment Search Tool.The emphasis of this tool is to find regions of sequence similarity, which will yield functional and evolutionary clues about the structure and function of your novel sequence. Position specific iterative BLAST (PSI-BLAST) refers to a feature of BLAST 2.0 in which a profile is automatically constructed from the first set of BLAST alignments. PSI-BLAST is similar to NCBI BLAST2 except that it uses position-specific scoring matrices derived during the search, this tool is used to detect distant evolutionary relationships. PHI-BLAST functionality is available to use patterns to restrict search results.

Official Website
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How to use this tool

Running a tool from the web form is a simple multiple steps process, starting at the top of the page and following the steps to the bottom.

Each tool has at least 2 steps, but most of them have more:

  • The first steps are usually where the user sets the tool input (e.g. sequences, databases...)
  • In the following steps, the user has the possibility to change the default tool parameters
  • And finally, the last step is always the tool submission step, where the user can specify a title to be associated with the results and an email address for email notification. Using the submit button will effectively submit the information specified previously in the form to launch the tool on the server

Note that the parameters are validated prior to launching the tool on the server and in the event of a missing or wrong combination of parameters, the user will be notified directly in the form.

Step 1 - Database

Databases

The databases to run the sequence similarity search against. Multiple databases can be used at the same time

Database Name Description Abbreviation
UniProt Knowledgebase The UniProt Knowledgebase (UniProtKB) is the central access point for extensive curated protein information, including function, classification, and cross-references. Search UniProtKB to retrieve "everything that is known" about a particular sequence uniprotkb
UniProtKB/Swiss-Prot The manually curated subsection of the UniProt Knowledgebase uniprotkb_swissprot
UniProtKB/Swiss-Prot isoforms The isoform sequences for the manually curated subsection of the UniProt Knowledgebase uniprotkb_swissprotsv
UniProtKB/TrEMBL Subsection of the UniProt Knowledgebase derived from ENA Sequence (formerly EMBL-Bank) coding sequence translations with annotation produced by an automated process. uniprotkb_trembl
UniProtKB Taxonomic Subsets
UniProtKB Archaea Taxonomic subset of the UniProt Knowledgebase for archaea uniprotkb_archaea
UniProtKB Arthropoda Taxonomic subset of the UniProt Knowledgebase for arthropoda uniprotkb_arthropoda
UniProtKB Bacteria Taxonomic subset of the UniProt Knowledgebase for bacteria uniprotkb_bacteria
UniProtKB Complete Microbial Proteomes Taxonomic subset of the UniProt Knowledgebase for complete microbial proteomes uniprotkb_complete_microbial_proteomes
UniProtKB Eukaryota Taxonomic subset of the UniProt Knowledgebase for eukaryota uniprotkb_eukaryota
UniProtKB Fungi Taxonomic subset of the UniProt Knowledgebase for fungi uniprotkb_fungi
UniProtKB Human Taxonomic subset of the UniProt Knowledgebase for human uniprotkb_human
UniProtKB Mammals Taxonomic subset of the UniProt Knowledgebase for mammals uniprotkb_mammals
UniProtKB Nematoda Taxonomic subset of the UniProt Knowledgebase for nematoda uniprotkb_nematoda
UniProtKB PDB Taxonomic subset of the UniProt Knowledgebase for PDB uniprotkb_pdb
UniProtKB Rodents Taxonomic subset of the UniProt Knowledgebase for rodents uniprotkb_rodents
UniProtKB Vertebrates Taxonomic subset of the UniProt Knowledgebase for vertebrates uniprotkb_vertebrates
UniProtKB Viridiplantae Taxonomic subset of the UniProt Knowledgebase for viridiplantae uniprotkb_viridiplantae
UniProtKB Viruses Taxonomic subset of the UniProt Knowledgebase for viruses uniprotkb_viruses
UniProt Clusters The UniProt Reference Clusters (UniRef) databases combine closely related sequences into a single record to speed up searches.
UniProt Clusters 100% The UniProt Reference Clusters (UniRef) containing sequences which are 100% identical. uniref100
UniProt Clusters 100% (SEG filtered) UniProt Reference Clusters database (SEG filtered) with entries that have 100% mutual sequence identity. uniref100_seg
UniProt Clusters 90% The UniProt Reference Clusters (UniRef) containing sequences which are 90% identical. uniref90
UniProt Clusters 50% The UniProt Reference Clusters (UniRef) containing sequences which are 50% identical. uniref50
Patents
EPO Patent Protein Sequences Protein sequences appearing in patents from the European Patent Office (EPO) epop
JPO Patent Protein Sequences Protein sequences appearing in patents from the Japanese Patent Office (JPO) jpop
KIPO Patent Protein Sequences Protein sequences appearing in patents from the Korean Intelectual Property Office (KIP0). kpop
USPTO Patent Protein Sequences Protein sequences appearing in patents from the United States Patent and Trademark Office (USPTO) uspop
NR Patent Proteins Level-1 Non-redundant Patent Protein sequences Level 1 covering data from the EPO, JPO, KIPO and USPTO nrpl1
NR Patent Proteins Level-2 Non-redundant Patent Protein sequences Level 2 covering data from the EPO, JPO, KIPO and USPTO nrpl2
Structure
Protein Structure Sequences Protein sequences from structures described in the Brookhaven Protein Data Bank (PDB) pdb
Structural Genomics Targets Structural Genomic Targets (SGT) database sgt
Other Protein Databases
UniProt Archive The UniProt Archive (UniParc) contains available protein sequences collected from many different sources. The sequence data are archived to facilitate examination of changes to sequence data. Search UniParc if you want to examine the "history" of a particular sequence. uniparc
IntAct The IntAct sequence database is derived from UniProt entries and data from MassSpec experiments submitted to the IntAct protein-interaction database. intact
IMGT/HLA The human major histocompatibility complex (HLA) section of the the international immunogenetics (IMGT) database. imgthlap
IPD-KIR Human Killer-cell Immunoglobulin-like Receptors (KIR) sequence in the Immuno Polymorphism Database (IPD) ipdkirp
IPD-MHC Major Histocompatibility Complex (MHC) section of the Immuno Polymorphism Database (IPD) ipdmhcp

Step 2 - Sequence

Sequence Input Window

The query sequence can be entered directly into this form. The sequence can be be in GCG, FASTA, EMBL, GenBank, PIR, NBRF, PHYLIP or UniProtKB/Swiss-Prot format. A partially formatted sequence is not accepted. Adding a return to the end of the sequence may help certain applications understand the input. Note that directly using data from word processors may yield unpredictable results as hidden/control characters may be present.

Sequence File Upload

A file containing a valid sequence in any format (GCG, FASTA, EMBL, GenBank, PIR, NBRF, PHYLIP or UniProtKB/Swiss-Prot) can be used as input for the sequence similarity search. Word processors files may yield unpredictable results as hidden/control characters may be present in the files. It is best to save files with the Unix format option to avoid hidden Windows characters.

Step 3 - Parameters

Matrix

The comparison matrix to be used to score alignments when searching the database

Matrix Name Abbreviation
BLOSUM45 BLOSUM45
BLOSUM62 BLOSUM62
BLOSUM80 BLOSUM80
PAM30 PAM30
PAM70 PAM70

Default value is: BLOSUM62

Additional information

Gap Open Penalty

Penalty taken away from the score when a gap is created in sequence. Increasing the gap openning penalty will decrease the number of gaps in the final alignment.

Default value is: 11

Additional information

Gap Extend Penalty

Penalty taken away from the score for each base or residue in the gap. Increasing the gap extension penalty favors short gaps in the final alignment, conversly decreasing the gap extension penalty favors long gaps in the final alignment.

Default value is: 1

Additional information

Results E() Limit

Limits the number of scores and alignments reported based on the expectation value. This is the maximum number of times the match is expected to occur by chance.

Default value is: 10.0

PSSM E-Value cut-off

Expectation value threshold for automatic selection of matched sequences for inclusion in PSSM at each iteration.

Default value is: 1.0e-3

Scores

Maximum number of match score summaries reported in the result output.

Default value is: 500

Alignments

Maximum number of match alignments reported in the result output.

Default value is: 500

Sequence Range

Specify a range or section of the input sequence to use in the search. Example: Specifying '34-89' in an input sequence of total length 100, will tell BLAST to only use residues 34 to 89, inclusive.

Default value is: START-END

Dropoff

The amount a score can drop before extension of word hits is halted

Default value is: 15 (default) [15]

Final Dropoff

Dropoff value for final gapped alignment

Default value is: 25 (default) [25]

Alignment Views

Formating for the alignments

Name Description Value
pairwise The query and match are output as a pairwsie alignment with a consensus line between the two sequences. In the consensus the match states are represented as: identical match as the base/residue, similarity as a '+' and missmatch as a space. 0
M/S identities The matches found are shown relative to the ungapped query sequence as a differences to the query. Identities appear as dots (.), similarites in upper case, missmatches in lower case and gaps as dash (-). Insertions are indictated with a line pointing to the insertion site with the inserted sequence on another line. 1
M/S non-identities The matches found are shown relative to the ungapped query sequence as a differences to the query. Identities and similarites appear in upper case, missmatches in lower case and gaps as dash (-). Insertions are indictated with a line pointing to the insertion site with the inserted sequence on another line. 2
Flat identities The matches found are shown relative to the gapped query sequence as a differences to the query. Identities appear as dots (.), similarites in upper case, missmatches in lower case and gaps as dash (-). 3
Flat non-identities The matches found are shown relative to the gapped query sequence as a differences to the query. Identities and similarites appear in upper case, missmatches in lower case and gaps as dash (-). 4

Default value is: pairwise [0]

Checkpoint File Upload

Checkpoint file from the previous iteration. Must be in ASN.1 Binary Format.

Usage Mode For PHI-BLAST

Usage mode for PHI-BLAST functionality

Usage Mode Name Description Value
blastpgp This is the default PHIBLAST/BLASTPGP program mode, without patseedp or seedp options. blastpgp
patseedp This function takes three inputs, an input pattern, a query protein sequence with the pattern, and a protein sequence database. It identifies the pattern in the query and aligns the query against the database entries that contains the same pattern. It reports the pattern position in the query, the total number of pattern occurrences in the database, and the actual database entries with pattern and alignment to the input query. Specifically, it reports the seqid of the database entry, its alignment (with the query) E-value, scores, and pattern position. patseedp
seedp Restrict the search for local alignments to a subset of the pattern occurrences in the query. This program option requires the user to specify the location(s) of the interesting pattern occurrence(s) in the pattern file (for the syntax see below). When there are multiple pattern occurrences in the query it may be important to decide how many are of interest because the E-value for matches is effectively multiplied by the number of interesting pattern occurrences. seedp

Default value is: blastpgp

Pattern File Upload

Pattern file for PHI-BLAST functionality. This file needs to be in the style of a prosite entry file, with at least an ID line, PA line and optional HI line.

Step 4 - Submission

Job title

It's possible to identify the tool result by giving it a name. This name will be associated to the results and might appear in some of the graphical representations of the results.

Email Notification

Running a tool is usually an interactive process, the results are delivered directly to the browser when they become available. Depending on the tool and its input parameters, this may take quite a long time. It's possible to be notified by email when the job is finished by simply ticking the box "Be notified by email". An email with a link to the results will be sent to the email address specified in the corresponding text box. Email notifications require valid email addresses.

Email Address

If email notification is requested, then a valid Internet email address in the form joe@example.org must be provided. This is not required when running the tool interactively (The results will be delivered to the browser window when they are ready).

References

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
(1997 Sep 01) Nucleic acids research 25 (17) :3389-402
Basic local alignment search tool.
(1990 Oct 05) Journal of molecular biology 215 (3) :403-10
A new bioinformatics analysis tools framework at EMBL-EBI.
(2010 Jul) Nucleic acids research 38 (Web Server issue) :W695-9
Analysis Tool Web Services from the EMBL-EBI.
(2013 Jul) Nucleic acids research 41 (Web Server issue) :W597-600