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Blast Introducción A Blast
Blast Introducción A Blast
David Fristrom
Bibliographer/Librarian
Science and Engineering Library
fristrom@bu.edu
617 358-4124
What is BLAST?
Free, online service from National Center for
Biotechnology Information (NCBI)
http://blast.ncbi.nlm.nih.gov/Blast.cgi
What is BLAST?
BLAST :
Nucleotide/Protein
Sequence Databases
as
Google : Internet
What is BLAST?
Alignment
AACGTTTCCAGTCCAAATAGCTAGGC
===--===
=-===-==-======
AACCGTTC
TACAATTACCTAGGC
Hits(+1): 18
Misses (-2): 5
Gaps (existence -2, extension -1): 1 Length: 3
Score = 18 * 1 + 5 * (-2) 2 2 = 6
Global Alignment
Compares total length of two
sequences
Needleman, S.B. and Wunsch, C.D. A
general method applicable to the search
for similarities in the amino acid sequence
of two proteins. J Mol Biol. 48(3):44353(1970).
Local Alignment
Compares segments of sequences
Finds cases when one sequence is a part
of another sequence, or they only match in
parts.
Smith, T.F. and Waterman, M.S. Identification of
common molecular subsequences. J Mol Biol.
147(1):195-7 (1981)
Search Tool
By aligning query sequence against all
sequences in a database, alignment can
be used to search database for similar
sequences
But alignment algorithms are slow
What is BLAST?
Quick, heuristic alignment algorithm
Divides query sequence into short words,
and initially only looks for (exact) matches
of these words, then tries extending
alignment.
Much faster, but can miss some
alignments
Altschul, S.F. et al. Basic local alignment search
tool. J Mol Biol. 215(3):403-10(1990).
What is BLAST?
BLAST is not Google
BLAST is like doing an experiment: to get
good, meaningful results, you need to
optimize the experimental conditions
Sample Search
Human beta globin (HBB)
Subunit of hemoglobin
Interpreting Results
Score: Normalized score of alignment
(substitution matrix and gap penalty). Can
be compared across searches
Max score: Score of single best aligned
sequence
Total score: Sum of scores of all aligned
sequences
Interpreting Results
Query coverage: What percent of query
sequence is aligned
E Value: Number of matches with same
score expected by chance. For low
values, equal to p, the probability of a
random alignment
Typically, E < .05 is required to be
considered significant
Step 0:
Do you need to use BLAST?
Step 1:
Nucleotide BLAST vs. protein BLAST
Largely determined by your query sequence
BUT
If your nucleotide sequence can be translated to a
peptide sequence, you probably want to do it (use tool
such as ExPASy Translate Tool)
Protein blasts are more sensitive and biologically
significant
Protein Databases
Non-redundant protein sequences (nr)
Kitchen-sink:
Protein Databases
Patented protein sequences (pat)
Patented sequences
Nucleotide Databases
Human genomic + transcript
http://www.ncbi.nlm.nih.gov/genome/guide/human/
GenBank (NCBI)
EMBL (European Nucleotide Sequence Database)
DDBJ (DNA Databank of Japan)
PDB (RCSB Protein Data Bank - 3d-structure)
Nucleotide Databases
Reference mRNA sequences (refseq_rna)
Reference genomic sequences
(refseq_genomic)
NCBI Reference Sequences: Comprehensive,
integrated, non-redundant, well-annotated set
of sequences
Nucleotide Databases
Expressed sequence tags (est)
Non-human, non-mouse ESTs (est_others)
http://www.ncbi.nlm.nih.gov/About/primer/est.html
http://www.ncbi.nlm.nih.gov/dbEST/index.html
http://www.ncbi.nlm.nih.gov/dbGSS/index.html
Nucleotide Databases
High throughput genomic sequences (HTGS)
Unfinished sequences (phase 1-2). Finished are
already in nr/nt
http://www.ncbi.nlm.nih.gov/HTGS/
Nucleotide Databases
Human ALU repeat elements
(alu_repeats)
Database of repetitive elements
Nucleotide Databases
Environmental samples (env_nt)
Nucleotide sequences from environmental
samples (not associated with known
organism)
Database Options
Limit to (or exclude) an organism
Exclude Models (XM/XP)
Model reference sequences produced by
NCBI's Genome Annotation project. These
records represent the transcripts and proteins
that are annotated on the NCBI Contigs
which may have been generated from
incomplete data.
Entrez Query
Use Entrez query syntax to limit search
Step 3:
Choose an Algorithm
How close a match are you looking for?
Determines how similarities are scored
Affects speed of search and chance of
missing match
Again, what is the goal of the search?
blastp
Protein-protein BLAST
Standard protein BLAST
PSI-BLAST
Protein-protein BLAST
Position-Specific Iterated BLAST
Finds more distantly related matches
Iterates: Initial search results provide
information on allowed mutations;
subsequent searches use these to create
custom substitution matrix
PHI-BLAST
Protein-protein BLAST
Pattern Hit Initiated BLAST
Variation of PSI-BLAST
Specify a pattern that hits must match
Use when you know protein family has a
signature pattern: active site, structural domain,
etc.
Better chance of eliminating false positives
Example: VKAHGKKV
megablast
Nucleotide BLAST
Finds highly similar sequences
Very fast
Use to identify a nucleotide sequence
blastn
Nucleotide BLAST
Use to find less similar sequences
discontiguous megablast
Nucleotide BLAST
Bioinformatics. 2002 Mar;18(3):440-5.
PatternHunter: faster and more sensitive
homology search. Ma B, Tromp J, Li M.
Step: 4
Algorithm Parameters
Fine-tune the algorithm
Short Queries
Expect threshold: The lower it is, the fewer
false positives (but you might miss real
hits)
Algorithm Parameters
Scoring Matrix:
PAM: Accepted Point Mutation
Empirically derived chance a substitution will be accepted,
based on closely related proteins
Higher PAM numbers correspond to greater evolutionary
distance
Algorithm Parameters
Filters and Masking
Can ignore low complexity regions in
searching
Additional Sources
Pevsner, Jonathan Bioinformatics and Functional
Genomics, 2nd ed. (Wiley-Blackwell, 2009)
BLAST help pages:
http://blast.ncbi.nlm.nih.gov/Blast.cgi?
CMD=Web&PAGE_TYPE=BlastDocs
Slides from class on similarity searching; lots of
technical details on algorithms and similarity
matrices:
http://www.ncbi.nlm.nih.gov/Class/NAWBIS/Mod
ules/Similarity/simsrchlast.html