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Psi-BLAST,
Prosite,
UCSC Genome
Browser
Lecture 3
Searching for remote homologs
 Sometimes
BLAST isn’t enough
 Large protein family, and BLAST only finds
close members. We want more distant
members
PSI-BLAST
 Position
Specific Iterative BLAST
Regular blast
Construct profile from
blast results
Blast profile search
Final results
Consensus, Pattern, PSSM
1
Seq1
Seq2
Seq3
2
3
4
5
6
A T C T T G
A A C T T G
A A C T T C
Consensus:
Pattern:
Profile = PSSM:
the most frequent
character in the
column is chosen
represents the
alignment as a
regular expression
Position Specific
Score Matrix
Pos
1
2
3
4
5
6
A
1
.67
0
0
0
0
C
0
0
1
0
0
.33
G
0
0
0
0
0
.67
T
0
.33
0
1
1
0
Nuc
AAC T T G
A-[TA]-C-T-T-[GC]
Pos
Nuc
A
C
G
T
1
1
0
0
0
2
.67
.33
0
0
3
0
1
0
0
4
0
1
0
0
5
.25
.25
.25
.25
6
.33
0
.33
.33
S(AACCAA)=1*0.67*1*1*.25*.33
S(GACCAA)=0
Sequences with higher scores ->
higher chance of being related
to the PSSM
PSI-BLAST
 Position
Specific Iterative BLAST
Regular blast
Construct profile from
blast results
Blast profile search
Final results
BLAST – PSI-Blast
PSI-Blast - results
PSI-BLAST
PSI-BLAST looks for seq’s
that are close to the query, and learns from
them to extend the circle of friends
 Disadvantage: if we obtained a WRONG
hit, we will get to unrelated sequences
(contamination). This gets worse and
worse each iteration
 Advantage:
PSI-BLAST
Which of the following is/are correct?
1. PSI-BLAST is expected to give more hits
than BLAST
2. PSI-BLAST is an iterative search method
3. PSI-BLAST is faster than BLAST
4. Each iteration of PSI-BLAST can only
improve the results of the previous
iteration
Turning information into knowledge
 The
outcome of a sequencing project are
masses of raw data
 The challenge is to turn these raw data
into biological knowledge
 A valuable tool for this challenge is an
automated diagnostic pipe through which
newly determined sequences can be
streamlined
From sequence to function


Nature tends to innovate rather than invent
Proteins are composed of functional
elements: domains and motifs


Domains are structural
units that carry out a
certain function. They are
shared between different
proteins
Motifs are shorter
and are usually critical
for the biological activity
http://www.expasy.ch/prosite
Prosite
 From
analyzing conserved regions in
protein sequences it is possible to derive
signatures of motifs and domains
 Prosite consists of annotated
sites/motifs/signatures/fingerprints
 Given an uncharacterized translated
protein sequence, prosite tries to predict
which motifs and domains make up the
protein and thus identify the family to
which it belongs
Prosite
Prosite represents entries with patterns or profiles
ATCTTG
AA C T T G
AA C T T C
profile
pattern
A-[TA]-C-T-T-[GC]

1
2
3
4
5
6
A
1
0.67
0
0
0
0
T
0
0.33
0
1
1
0
C
0
0
1
0
0
0.33
G
0
0
0
0
0
0.67
Profiles are used in prosite when the motif is relatively
divergent, and is difficult to represent as a pattern
 Profiles also characterize domains over their entire length, not
just the motif
Prosite sequence query
Patterns with a high probability of
occurrence

Entries describing commonly found posttranslational modifications or compositionally
biased regions
 Found in the majority of known protein
sequences
 High probability of occurrence
 Prosite filters them by default
Scanning Prosite
Query:
sequence
Result: all patterns
found in the sequence
Query:
pattern
Result: all sequences
which adhere to this
pattern
Prosite pattern query
UCSC Genome Browser
UCSC Genome Browser Gateway
Reset all
settings of
previous uses
UCSC Genome Browser Gateway
Results
Annotation tracks
Base position
UCSC Genes
UTR
RefSeq Genes
mRNAs (GenBank)
Intron
Mammal
conservation
Species
alignment
SNPs
Repeats
Coding
Gene
Direction
UCSC Gene
UCSC Genome Browser - movement
Zoom x3 +
Center
Controlling
annotation
tracks
Sickle-cell
anemia distr.
Malaria
distr.
BLAT

BLAT = Blast-Like Alignment Tool
 BLAT is designed to find similarity of >95% on
DNA, >80% for protein
 Rapid search by indexing entire genome
Good for:
1. Finding genomic coordinates of cDNA
2. Determining exons/introns
3. Finding human (or chimp, dog, cow…)
homologs of another vertebrate sequence
BLAT on UCSC Genome Browser
BLAT search
BLAT Results
BLAT Results
query
Match
hit
Non-Match
(mismatch/indel)
Indel
boundaries