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Database Searches. BLAST. BLAST. Basic Local Alignment Search Tool Altschul, Gish, Miller, Myers, Lipman, J. Mol. Biol. 215 (1990) Altschul, Madden, Schaffer, Zhang, Zhang, Miller, Lipman, Nucleic Acids Res . 25 (1997) Main ideas:

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blast
BLAST
  • Basic Local Alignment Search Tool
    • Altschul, Gish, Miller, Myers, Lipman, J. Mol. Biol. 215 (1990)
    • Altschul, Madden, Schaffer, Zhang, Zhang, Miller, Lipman, Nucleic Acids Res. 25 (1997)
  • Main ideas:
    • Increase search speed by finding fewer, but better, hot spots during initial screening phase
    • Uses longer word sizes
    • Integrate scoring matrix into first phase
      • Compare with FASTA, which requires exact matches
blast terminology
BLAST Terminology
  • Segment pair: equal-length substrings of sequences S1 and S2
  • Locally maximal segment pair: segment pair whose alignment score cannot be improved by extending or shortening it
  • Maximum segment pair(MSP) = segment pair with maximum score over all segment pairs in the sequences S1 and S2
  • High-scoring segment pair (HSP): A segment pair with score higher than some cutoff score, s.
  • wis the length parameter; t is the threshold parameter
blast hits
BLAST: Hits
  • A hit is a w-length word in the database that aligns with a word from the query sequence with score > t
  • BLAST looks for hits instead of exact matches
    • Allows word size to be kept high for speed, without sacrificing sensitivity
  • Typically, w= 3-5 for amino acids, ~11-12 for DNA
  • t is the most critical parameter:
    • ↑t ↓ “background” hits (faster)
    • ↓t ↑ ability to detect more distant relationships (at cost of increased noise
slide5
Hits
  • For each word, evaluate score of match (exact or not) according to BLOSUM62
    • E.g., for PQG, score is 7+5+6 = 18
  • There are 20wpossible w-length words, but considering only those with score >t, greatly reduces number of matches
    • E.g., there are 203 = 8000 possible matches to PQG, but only 50 achieve score > t = 13
extending a hit
Extending a hit
  • After locating a hit, BLAST attempts to extend hit in both directions, until score has drops more than Xbelow the maximum score yet attained.
  • Extension step typically accounts for > 90% of execution time.
improvement 2 hit method
Improvement: 2-hit method
  • Do extensions only when there are two hits on the same diagonal within some distance Aof each other (e.g., A =40)
  • Reduces sensitivity (ability to detect distantly related sequences)
    • To compensate, use lower tvalue (e.g., 11 rather than 13)
  • Since we only extend when there are two nearby hits, many fewer regions are extended
gapped blast
Gapped BLAST
  • Allows local alignments with indels (similar to FASTA)
  • Local alignments from different diagonal are merged into a different local alignment followed by some indels followed by a second local alignment, etc.
    • equivalent to a path through the dynamic programming matrix composed of alternating diagonal sections and paths connecting them
gapped blast1
Gapped BLAST
  • Original BLAST implicitly handled gaps by finding several distinct HSPs and calculating a statistical assessment of the combined result
    • Two or more HSPs each below the cutoff value might in combination rise to statistical significance
  • Gapped BLAST, extend hits by allowing gaps when hits are promising (exceed sg):
    • Advantage: We can afford to miss some HSPs as long as at least one is found
  • Use dynamic programming, starting from center of each high-scoring region if s > sg
    • sgis chosen such that gapped alignment is triggered in about 1/50 of the sequences compared
psi blast
PSI-BLAST
  • Position-Specific Iterated BLAST
  • Generates a multiple alignment from statistically significant alignments produced by BLAST
  • Produces a position-specific score matrix (PSSM)
    • Can search the database using the PSSM
    • Match sequences to profile
    • Generate new profiles
    • Repeat (iteration)
    • Search gradually extends to increasingly divergent sequences
flavors of blast
Flavors of BLAST
  • BLASTP - protein query against protein DB
  • BLASTN - DNA/RNA query against GenBank (DNA)
  • BLASTX - 6 frame trans. DNA query against proteinDB
  • TBLASTN - protein query against 6 frame GB transl.
  • TBLASTX - 6 frame DNA query to 6 frame GB transl.
  • PSI-BLAST - protein ‘profile’ query against protein DB
  • PHI-BLAST - protein pattern against protein DB
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