Gapped%20BLAST%20and%20PSI-BLAST:%20a%20new%20generation%20of%20protein%20database%20search%20programs

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Gapped BLAST and PSI-BLAST a new generation of
protein database search programs

• By Stephen F. Altschul, Thomas L. Madden,
  Alejandro A. Schäffer, Jinghui Zhang, Zheng
  Zhang, Webb Miller and David J. Lipman

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Introduction to BLAST

• BLAST is a heuristic approximation to dynamic
  programming based local alignment.
• Finds locally maximal segment pairs with scores
  over a cutoff.
• Has a formal statistical theory to assess the
  significance of scores.

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Basic Algorithm

• Looks for words of length w with score greater
  than T.
• These hits are then extended to check for segment
  pairs with score greater than S (gtT.)
• Tradeoff Lowering T reduces probability of
  missing segment pairs (increases sensitivity) but
  increases number of hits to be extended.

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Scanning for hits

• Two Approaches
• Positions of length w words in query with score
  higher than T stored in a 20w sized array and
  hits detected by array lookup.
• A DFA for the appropriate words is generated and
  used to scan the sequences. A Mealy machine
  (acceptance on transitions) is used for
  efficiency.

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Other Issues

• Hit extension is simplified by stopping when
  score falls below a threshold compared to the
  best score found for shorter extensions.
• The various parameters are chosen based on
  experiments using random sequences.
• Combinations of MSPs can be used to get better
  scores for matching sequences.

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Two-Hit Method

• Original BLAST One-Hit
• Extend each hit to determine if it is in a
  high-scoring alignment
• Extension consumes gt90 of processing time
• hit short word pair whose aligned score T

• Two-Hit Method
• Extension invoked only if there are two
  non-overlapping word pairs on the same diagonal
• Lowering T yields more hits, but only a few are
  extended
• 3x faster

T threshold parameter as T ?, speed ?,
probability of missing weak similarities ?
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Two-Hit Method Algorithm

• Scan db for hits (word pair scoring T)
• Seek pairs of non-overlapping hits found with
  distance A of one another on same diagonal
• Invoke (ungapped) extension to determine if hits
  lie within a statistically significant alignment
  with query. Extend until alignment score has
  dropped X below max score yet attained.

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Gapped Alignments

• Original BLAST
• Implicitly treat gapped alignments
• Locate several distinct HSPs within same db
  sequence
• Calculate statistical significance on combined
  result

• Gapped BLAST
• Trigger gapped extension for any HSP exceeding
  moderate score Sg
• Gapped extension longer to execute, few undergo
  this extension

HSP high-scoring segment pair locally optimal
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Advantage of New Heuristic for Generating Gapped
Alignments

• Two or more HSPs may each have low scores
  independently, but can have a statistically
  significance together
• Only one of the constituent HSPs need to be found
  to generate a successful combined result can
  increase T

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Older Gapped Alignments

• Confine the dynamic programming to a banded
  section of the full path graph
• Optimal gapped alignment may be outside this band
• As width of band ?, speed ?

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New Heuristic for Generating Gapped Alignments

• Starting from a seed HSP, dynamic programming
  proceeds both bidirectionally through the path
  graph
• Consider only cells for which optimal local
  alignment score falls Xg below best score yet
  found
• Region of path graph explored adapts to alignment
  being constructed
• Seed central residue pair of segment with
  highest alignment along HSP

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New Gapped BLAST

• Ungapped extension of second hit invoked for two
  non-overlapping hits of score T within distance
  A of one another
• If HSP generated has normalized score Sg,
  gapped extension is triggered
• Resulting gapped alignment reported if
  statistically significant (low enough E-value)
• Runs on average 3x faster than original BLAST

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PSI-BLAST Overview

• Results of initial BLAST search used to construct
  position-specific scores.
• BLAST is repeated using the new scores till no
  more sequences are found.
• Position-specific scores improve the ability of
  successive BLAST iterations for detecting remote
  homologs.

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Position-specific score matrix

• Dimensions Lx20
• Multiple Alignment created using all segments
  with e-value above a threshold.
• Alignment based on pairwise alignments.
• Columns with gaps in query ignored.
• For each column C a reduced alignment MC is
  created.

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• MC includes all sequences with a residue in C and
  all columns which have the above sequences.
• Sequence weighting method used to generate
  observed residue frequencies.
• Score for residue i in column C given by log(Qi/
  Pi)
• Qi is the weighted sum of observed frequencies
  and a pseudocount.