BLAST

• BLAST (Basic Local Alignment Search Tool): family of sequence alignment algorithms developed by Altschul et. al. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-402.  These programs are used for sequence similarity identification.  They identify regions of local alignment  to assist in detecting relationships among sequences
• Allows the user to identify similarities between the query nucleotide or protein sequence with sequences in public databases
• Identifies clusters of nearby or locally dense "similar" k-tuples (number of string of letters), see an example of a BLAST alignment
• Used to identify whether a given sequence is novel, homologous to a known sequence, or if the sequence contains motifs which may provide clues to a possible roles of the sequence being queried
• Most commonly used tools at the NCBI (National Center for Biotechnology Information)

• BLAST programs: each BLAST program performs a different task:

  blastp compares an amino acid query sequence against a protein sequence database
  blastn compares a nucleotide query sequence against a nucleotide sequence database
  blastx compares a nucleotide query sequence translated in all reading frames against a protein   sequence database
  tblastn compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames
  tblastx compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database
  BLAST databases:
   

  Nucleotide Sequence Databases
  nr	All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS sequences). No longer "non-redundant"
  month	All new or revised GenBank+EMBL+DDBJ+PDB sequences released in the last 30 days
  Drosophila genome	Drosophila genome provided by Celera and Berkeley Drosophila Genome Project (BDGP)
  dbest	Database of GenBank+EMBL+DDBJ sequences from  EST Divisions
  dbsts	Database of GenBank+EMBL+DDBJ sequences from  STS Divisions
  htgs	Unfinished High Throughput Genomic Sequences: phases 0, 1 and 2 (finished, phase 3 HTG sequences are in nr)
  gss	Genome Survey Sequence, includes single-pass genomic data, exon-trapped sequences, and Alu PCR sequences
  yeast	Yeast (Saccharomyces cerevisiae) genomic nucleotide sequences
  pdb	Sequences derived from the 3-dimensional structure from Brookhaven Protein Data Bank
  kabat  [kabatnuc]	Kabat's database of sequences of immunological interest
  vector	Vector subset of GenBank (R), NCBI, in ftp://ncbi.nlm.nih.gov/blast/db/
  mito	Database of mitochondrial sequences
  alu	Select Alu repeats from REPBASE, suitable for masking Alu repeats from query sequences
  epd	Eukaryotic Promotor Database

   

  Peptide Sequence Databases
  nr	All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF
  month	All new or revised GenBank CDS translation +PDB+SwissProt+PIR+PRF released in the last 30 days
  swissprot	Last major release of the SWISS-PROT protein sequence database (no updates)
  Drosophila    genome	Drosophila genome proteins provided by Celera and Berkeley, Drosophila Genome Project (BDGP)
  yeast	Yeast (Saccharomyces cerevisiae) genomic CDS translations
  ecoli	Escherichia coli genomic CDS translations
  pdb	Sequences derived from the 3-dimensional structure from Brookhaven Protein Data Bank
  kabat [kabatpro]	Kabat's database of sequences of immunological interest
  alu	Translations of select Alu repeats from REPBASE, suitable for masking Alu repeats from query sequences

  Sequence Input

  The preferred query sequence format for the BLAST program is the FASTA format. Advanced BLAST tolerates both spaces and numbers and is case insensitive.  

  Parameter Settings

  Parameter settings can be modified to optimize your BLAST search:

  sequence filtering: enables the program to mask regions of a query sequence in order to exclude regions of low compositional complexity such as repetitive elements (turned ON as the default setting) it inserts "x"s in regions of low complexity

  introduction of gaps: (regions of insertions and deletions); blastn and blastp search tools offer fully gapped alignments, while blastx and tblastn have "in-frame" gapped alignments; the tblastx search tool provides only ungapped alignments (turned ON as the default setting)

  Statistical matrices: are used both to identify sequences in a database, and to predict the biological significance of the match.  There are two main types of matrices, you can select the prefered matrix for your Advanced BLAST search

  PAM (Percent Accepted Mutation) matrices: predicted matrices, most sensitive for alignments of sequences with evolutionary related homologs. The greater the number in the matrix name, the greater the expected evolutionary (mutational) distance, i.e. PAM30 would be used for alignments expected to be more closely related in evolution than an alignment performed using the PAM250 matrix

  BLOSUM (Blocks Substitution Matrix): calculated matrices, most sensitive for local alignment of related sequences, ideal when trying to identify an unknown nucleotide sequence. BLOSUM62 is the default matrix set be the BLAST search tool

  
  Results Format  

  Results returned in either text format (default) or HTML format (must supply an e-mail address and select the HTML results format option)

  A Request ID number is given such that the results be obtained at a later time, if you want the results immediately, click on the "Format Results" button

  Formatting items such as the results format option and the number of descriptions and alignments in the results output are needed only for formatting, these items may be specified from the BLAST query form or at the time you request your results

  Most results will be held for up to 24 hours; very large result files will be deleted after 30 minutes

  BLAST Output

  All BLAST programs produce a similar output consisting of:

  program introduction

  a schematic distribution of the ordered alignments of the query sequence to those in the databases

  coloured bars are distributed in a way to reflect the region of alignment onto the query sequence. The colour legend represents the significance of the alignment scores

  holding the mouse over a given bar will display a description of that specific alignment sequence in the above window; clicking on a specific bar will cause the browser to jump down to that particular alignment

  an ordered set of biological definition line of the database sequences which have been significantly aligned to the query sequence

  • sequence alignments and their corresponding line descriptions are listed  in order of lowest to highest E value

    E value:  the expect value is the probability that the associated match is due to randomness; the lower the E value, the more specific/significant the match

  • identifiers for the database sequences appear in the first column and are hyperlinkedto the associated GenBank entry
  • the Score (bits) is a sum value calculated for alignments using the scoring matrix; the higher the score value, the better the alignment

   

  • the percent identity (called "Identities" is given as a percent) is the percent of exact matches between your query sequence and the database sequence, this value also gives the number of nucleotide bases or amino acid residues that are matched in the database sequence versus the query sequence
  • alignments are listed in order of lowest to highest E value (as are the descriptions)
  • alignments are gapped unless specified by the user at the BLAST search submission page
    • gap value is the percent of the alignment sequence that has been gapped in the particular alignment

  and a list of statistics specific to the particular BLAST search are displayed at the bottom of the output, they include the BLAST version number, the database and matrices used for the search

  For more information on how to use BLAST see the following BLAST tutorials:

  • NCBI BLAST Tutorial
  • BISC BLAST Tutorial

  
  BLAST now comes in several flavors including (these programs can also be accessed from the BLAST home page at NCBI):
   

  MEGABLAST search: like a Basic BLAST search, but allows you to change certain parameters in order to perform a more specified BLAST search

• specify an organism or taxonomic class for your search
• set the E value
• filter for low complexity or human repeats
• query Genetic Codes (blastx and tblastx only)
• change you scoring matrix (BLOSUM62 is the default scoring matrix)
• there are other advanced BLAST options
• PSI-BLAST: Position Specific Iterated-BLAST

• this tool can be used when your BLAST search results give you very few matches, the PSI-BLAST will re-iterated the BLAST searches creating a defined profile, upon re-iteration (you just click on the button to re-iterate) you may reveal alignment matches that are significant that you would not have found using BLAST alone.  PSI-BLAST generates "on-the-fly" a scoring matrix specific to your BLAST search, and continues to specify this matrix upon each re-iteration
• 
  PHI-BLAST: Pattern Hit Initiated-BLAST

• this tool can be used to search for a specific pattern or motif in you sequence and in the databases
• the pattern designates the amino acid sequence you are searching for                                                 e.g.  [RG]-[M]-[X]-[YWF]-5[X]-[A]; this submission pattern would yeild a search for sequence patterns having "R" (Arginine) or "G" (Glycine) at position 1 (not necessarily position 1 or the N-terminus of the amino acid sequences in the databases), followed by a "M" (Methionine), followed by any amino acid "X", followed by any one of three AA : "Y" (Tyrosine) or "W" (Tryptophan) or "F" (Phenylalanine); followed by any 5 amino acids "X", followed by an "A" (Alanine)
• BLAST 2 Sequences:   this tool produces the alignment of two
                                    given sequences using BLAST engine for
                                    local alignment.

GENERAL GUIDELINES for analysis CUT-OFFS of BLAST OUTPUT

• E-value < 0.05
• % Identity >25% over 100 base pairs or 35 amino acid residues

2. Global Alignment: FASTA

• identifies clusters of nearby or locally dense "identical" k-tuples, takes this local alignment and extends it globally
• decreases the sensitivity, but gains speed
• Z-score: number of S.D. above the random
• default matrix: BLOSUM50
• output: local alignment score, global score, maximum score, E-values as well as alignments and descriptions