BCB 444/544

1
BCB 444/544

• Lab 3
• BLAST
• Scoring Matrices Alignment Statistics
• Sept6

2
Exhaustive vs Heuristic Methods

• Exhaustive - tests every possible solution
• guaranteed to give best answer
• (identifies optimal solution)
• can be very time/space intensive!
• e.g., Dynamic Programming
• as in Smith-Waterman algorithm
• Heuristic - does NOT test every possibility
• no guarantee that answer is best
• (but, often can identify optimal
  solution)
• sacrifices accuracy (potentially) for speed
• uses "rules of thumb" or "shortcuts"
• e.g., BLAST FASTA

3
Today's Lab focus on BLAST Basic Local
Alignment Search Tool

• STEPS
• Create list of very possible "word" (e.g., 3-11
  letters) from query sequence
• Search database to identify sequences that
  contain matching words
• Score match of word with sequence, using a
  substitution matrix
• Extend match (seed) in both directions, while
  calculating alignment score at each step
• Continue extension until score drops below a
  threshold (due to mismatches)
• Contiguous aligned segment pair (no gaps) is
  called
• High Scoring Segment Pair (HSP)

4
Today's Lab focus on BLAST Basic Local
Alignment Search Tool

• Results?
• Original version of BLAST?
• List of HSPs Maximum Scoring Pairs
• More recent, improved versionof BLAST?
• Allows gaps Gapped Alignment
• How? Allows score to drop below threshold,
• (but only temporarily)

5
BLAST - a few details

• Developed by Stephen Aultschul at NCBI in 1990
• Word length?
• Typically 3 aa for protein sequence
• 11 nt for DNA sequence
• Substitution matrix?
• Default is BLOSUM62
• Can change under Algorithm Parameters
• Choose other BLOSUM or PAM matrices
• Stop Extension Threshold?
• Typically 22 for proteins
• 20 for DNA

6
BLAST - a few more details

• BLAST is family of programs with several
  "variants"
• BLASTN -
• BLASTP -
• BLASTX -
• TBLASTM -
• TBLASTX -
• Statistical Significance?
• E-value E m x n x P
• m total number of residues in database
• n number of residues in query sequence
• P probability that an HSP is result of random
  chance
• lower E-value, less likely to result from random
  change, thus higher significance
• Bit Score S' is normalized, to account for
  sequence length differences size of database
• Low Complexity Masking - remove repeats that
  confound scoring

7
"Scoring" or "Substitution" Matrices

• 2 Major types for Amino Acids PAM BLOSUM
• PAM Point Accepted Mutation
• relies on "evolutionary model" based on
  observed differences in alignments of closely
  related proteins
• BLOSUM BLOck SUbstitution Matrix
• based on aa substitutions observed in blocks
  of conserved sequences within evolutionarily
  divergent proteins

8
PAM Matrix

• PAM Point Accepted Mutation
• relies on "evolutionary model" based on observed
  differences in closely related proteins
• Model includes defined rate for each type of
  sequence change
• Suffix number (n) reflects amount of "time"
  passed rate of expected mutation if n of amino
  acids had changed
• PAM1 - for less divergent sequences (shorter
  time)
• PAM250 - for more divergent sequences (longer
  time)

9
BLOSUM Matrix

• BLOSUM BLOck SUbstitution Matrix
• based on aa substitutions observed in blocks
  of conserved sequences within evolutionarily
  divergent proteins
• Doesn't rely on a specific evolutionary model
• Suffix number (n) reflects expected similarity
  average aa identity in the MSA from which the
  matrix was generated
• BLOSUM45 - for more divergent sequences
• BLOSUM62 - for less divergent sequences

10
BLOSUM62 Substitution Matrix

• s(a,b) corresponds to score of aligning character
  a with character b
• Match scores are often calculated
• based on frequency of mutations in very similar
  sequences
• (more details later)

11
(No Transcript)
12
Affine Gap Penalty Functions

• Affine Gap Penalties Differential Gap
  Penalties used to reflect cost differences
  between opening a gap and extending an existing
  gap
• Total Gap Penalty is linear function of gap
  length
• W ? ? X (k - 1)
• where ? gap opening penalty
• ? gap extension penalty
• k length of gap
• Sometimes, a Constant Gap Penalty is used, but it
  is usually least realistic than the Affine Gap
  Penalty

Can also be solved in O(nm) time using DP