Identifying sequences with

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Identifying sequences with
Speaker S. Gaj
BioInformatics Lunch Meeting
Date 04-03-2005
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Annotation

• Annotation
• Best possible description available for a given
  sequence at the current time.
• How to annotate?
• Combining
• Alignment Tools
• Databases
• Datamining (scripts)

Background
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Microarrays
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Part ISequence Alignment
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Introduction

• Global alignment
• Optimal alignment between two sequences
  containing as many characters of the query as
  possible.
• Ex predicting evolutionary relationship
  between genes,
• Local alignment
• Optimal specific alignment between two sequences
  identifying identical area(s)
• Ex Identifying key molecular structures
  (S-bonds, a- helices, )

Background
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Global vs Local Alignment
Global Alignment
Score -42 at (seq1)1..90 (seq2)1..90 1
MA-----STVTSCLEPTEVFMDLWPEDHSNWQELSPLEPSDPLNPPTPPR
AAPSPVVPST
1 MSHGIQMSTIKKRRSTDEE
VFCLPIKGREIYEILVKIYQIENYNMECAPPAGASSVSVGA 56
EDYGGDFDFRVGFVEAGTAKSVTCTYSPVLNKVYC
61
TEAEPTEVFMDLWPEDHSNWQELSPLEPSD-----

• Includes total sequence

• The highest score

Local Alignment
Score 148 at (seq1)10..36 (seq2)64..90
10 EPTEVFMDLWPEDHSNWQELSPLEPSD
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EPTEVFMDLWPEDHSNWQELSPLEPSD

• The highest score

• Stop the alignment extension if it is not
  profitable

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BLAST

• Basic Local Alignment Search Tool
• Aligning an unknown sequence (query) against all
  sequences present in a chosen database based on a
  score-value.
• Aim
• Obtaining structural or functional information
  on the unknown sequence.

Introduction
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Programs

• Different BLAST programs available
• Parameters
• Maximum E-Value, Gap Opening Penalty (GOP), Gap
  Extension Penalty (GEP),
• Terms
• Query Sequence which will be aligned
• Subject Sequence present in database
• Hit Alignment result.

Nucleic Protein
Nucleic BlastN BlastX
Protein - BlastP
BLAST
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Substition Matrices What?

• Estimates the rate at which each possible residue
  in a sequence changes to each other residue over
  time.
• For example, hydrophobic residue is more likely
  to stay hydrophobic than not.
• Each matrix is tailored to look for certain types
  of sequences KNOW WHAT YOU ARE LOOKING FOR!

BLAST Matrices
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Substition Matrices Why?

1. Determine likelihood of homology between two
   sequences
2. Substitutions that are more likely should get a
   higher score
3. Substitutions that are less likely should get a
   lower score.

BLAST Matrices
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Matrices - PAM

• Point Accepted Mutation
• Mostly used in global amino acid alignments
• PAM1 represents 1 of change
• PAM250 (PAM1)250
• PAM1
• Applied for a time period over which we expect
  1 of the amino acids to undergo accepted point
  mutations within the species of interest.

BLAST Matrices
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BLOSUM

• Mostly used in local AA alignments
• Based on observed alignments, not predicted ones.
• BLOSUM 80, BLOSUM 62, BLOSUM 45
• Default BLOSUM 62
• Matrix calculated from comparisons of sequences
  with no less than 62 divergence.

BLAST Matrices
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PAM vs BLOSUM

• Closely related
• High PAM
• Low BLOSUM
• Distantly related
• Low PAM
• High BLOSUM

BLAST Matrices
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BlastN Example
BLAST
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BlastN Example
BLAST
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Common BLAST problems

• BlastN

Clone seq
mRNA
Sequencing Error
BLAST

• Solution
• Low penalty for GOP and GEP 1

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Translation Problems

• 6-Frame translation

gtemblJ03801HSLSZ Human lysozyme mRNA, complete
cds with an Alu repeat in the 3' flank.
BLAST
L A L P S S Q H
E G S H C S G A
1
ctagcactctgacctagcagtcaacatgaaggctctcattgttctggggc
t...
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Translation Problems

• 6-Frame translation

gtemblJ03801HSLSZ Human lysozyme mRNA, complete
cds with an Alu repeat in the 3' flank.
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S T L T Q S T
R L S L F W G
2
H S D L A V N M
K A L I V L G
BLAST
L A L P S S Q H
E G S H C S G A
1
0
ctagcactctgacctagcagtcaacatgaaggctctcattgttctggggc
t...
V L Q S L L E C
L Y F L H H A
-1
-2
C C K A F N N V
C I F Y I M H
-3
V A K P L I R M F
V F F T S C I
http//searchlauncher.bcm.tmc.edu/cgi-bin/seq-util
/sixframe.pl
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Common BLAST problems
intron
exon
Gene X
Translation
BLAST
full mRNA
Splicing
mRNA
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Common BLAST problems
mRNA
Clones derived from mRNA
BLAST
BlastX against protein sequence
3 possible hit-situations
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Common BLAST problems

• ? Yields no protein hit

? Aligns with protein in 1 of the 6 frames.
BLAST
? Part perfect alignment
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Questions?
End