Dr' Wishart

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Dr. Wishart

• Office hours
• Generally available right after class
• Best time to catch me is after 400 pm in my
  office in Athabasca Hall
• Usually around til 600 pm
• Arranging an appointment is always best (email)
• Email responses take 1-2 days

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Where to get your notes

• http//redpoll.pharmacy.ualberta.ca/
• Look under Courses

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General Course Outline

• Bioinformatics introduction
• Bionformatics Databases
• Sequence Alignment
• Protein Feature ID
• Computational microbiology
• Peptide/Protein Analysis
• Protein Structure (Xray)

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General Course Outline

• Protein Structure (NMR)
• Mass Spectrometry 1
• Mass Spectrometry 2
• Proteomics
• Systems Biology
• Enzymology/Systems Biology
• Protein Structure (Xray)

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Introduction to Bioinformatics

• Microbiology 343
• David Wishart Rm. 3-41 Ath
• david.wishart_at_ualberta.ca

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Objectives Outline

• Definitions and roles of bioinformatics
• DNA sequencing (foundation to bioinformatics)
• Genomes and Genomics
• Gene finding in prokaryotes
• From genes to proteins

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Bioinformatics
Definition - A field of information
technology which endeavours
to improve the storage,
management and analysis of
biological, medical and
pharmaceutical data. A blend information
technology and biotechnology
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Bioinformatics - Converting Data to Knowledge
Data
Knowledge
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Bioinformatics Software

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Whats the Appeal?

• No spills or smells
• No need for a lab coat
• No need to get hands or clothes messy
• Provides a faster or alternative route to create
  hypotheses, perform difficult experiments, avoid
  unnecessary experiments, compare, visualize and
  analyze data, make predictions, see what is
  unseeable and handle a growing tidal wave of
  both data and knowledge

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Bioinformatics
Genomics
Proteomics
Bioinformatics
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High Throughput DNA Sequencing
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Shotgun Sequencing
Isolate Chromosome
ShearDNA into Fragments
Clone into Seq. Vectors
Sequence
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Principles of DNA Sequencing
Primer
DNA fragment
Amp
PBR322
Tet
Ori
Denature with heat to produce ssDNA
Klenow ddNTP dNTP primers
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The Secret to Sanger Sequencing
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Principles of DNA Sequencing
3 Template
G C A T G C
5
5 Primer
GddC
GCddA
GCAddT
ddG
GCATGddC
GCATddG
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Principles of DNA Sequencing
G
T
_
_
short
C
A
G C A T G C


long
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Capillary Electrophoresis
Separation by Electro-osmotic Flow
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Multiplexed CE with Fluorescent detection
ABI 3700
96x700 bases
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Shotgun Sequencing
Assembled Sequence
Sequence Chromatogram
Send to Computer
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Shotgun Sequencing

• Very efficient process for small-scale (10 kb)
  sequencing (preferred method)
• First applied to whole genome sequencing in 1995
  (H. influenzae)
• Now standard for all prokaryotic genome
  sequencing projects
• Successfully applied to D. melanogaster
• Moderately successful for H. sapiens

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The Finished Product
GATTACAGATTACAGATTACAGATTACAGATTACAG ATTACAGATTACA
GATTACAGATTACAGATTACAGA TTACAGATTACAGATTACAGATTACA
GATTACAGAT TACAGATTAGAGATTACAGATTACAGATTACAGATT AC
AGATTACAGATTACAGATTACAGATTACAGATTA CAGATTACAGATTAC
AGATTACAGATTACAGATTAC AGATTACAGATTACAGATTACAGATTAC
AGATTACA GATTACAGATTACAGATTACAGATTACAGATTACAG ATTA
CAGATTACAGATTACAGATTACAGATTACAGA TTACAGATTACAGATTA
CAGATTACAGATTACAGAT
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Sequenced Genomes
http//www.genomenewsnetwork.org/
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Genomes to Date

• 8 vertebrates (human, mouse, rat, fugu, dog,
  chimp)
• 3 plants (arabadopsis, rice, poplar)
• 2 insects (fruit fly, mosquito)
• 2 nematodes (C. elegans, C. briggsae)
• 1 sea squirt
• 4 parasites (plasmodium, guillardia)
• 4 fungi (S. cerevisae, S. pombe)
• 200 bacteria and archebacteria
• 2000 viruses

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Prokaryotes

• Simple gene structure
• Small genomes (0.5 to 10 million bp)
• No introns (uninterrupted)
• Genes are called Open Reading Frames of ORFs
  (include start stop codon)
• High coding density (gt90)
• Some genes overlap (nested)
• Some genes are quite short (lt60 bp)

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Prokaryotic Gene Structure
ORF (open reading frame)
TATA box
Stop codon
Start codon
ATGACAGATTACAGATTACAGATTACAGGATAG
Frame 1
Frame 2
Frame 3
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Simple Gene Finding

• Scan forward strand until a start codon is found
• Staying in same frame scan in groups of three
  until a stop codon is found
• If of codons between start and end is greater
  than 50, identify as gene and go to last start
  codon and proceed with step 1
• If codons between start and end is less than
  50, go back to last start codon and go to step 1
• At end of chromosome, repeat process for reverse
  complement

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Advanced Gene Finding

• Identify all ORFs (open reading frames) gt 200
  bases on both strands using normal and alternate
  start/stop codons
• Find high scoring -10,-35 and RBS sites at 5
  ends of putative ORFs
• Find high scoring rho terminators at 3 ends of
  putative ORFs
• Exclude ORFs without identified signals at 5 or
  3 ends

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Key Prokaryotic Gene Signals

• Alternate start codons
• RNA polymerase promoter site (-10, -35 site or
  TATA box)
• Shine-Dalgarno sequence (Ribosome binding
  site-RBS)
• Stem-loop (rho-independent) terminators
• High GC content (CpG islands)

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Alternate Start Codons (E. coli)
ATG Met GTG Val TTG Leu
Class I Class IIa
CTG Met ATT Val ATA Leu ACG Thr
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-10, -35 Site (RNA pol Promoter)
-36 -35 -34 -33 -32 . -13 -12 -11 -10 -9 -8 T
T G A C T A t A A T
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RBS (Shine Dalgarno Seq)
-13 -12 -11 -10 -9 -8 .. -1 0 1 2 3 4 G G
G G G G n A T G n C
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Terminator Stem-loops
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More Sophisticated Methods
RBS site
promoter site
HMM
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Really Sophisticated Methods

• GLIMMER
• http//www.tigr.org/software/glimmer/
• Uses interpolated markov models (IMM)
• Requires training of sample genes
• Takes about 1 minute/genome
• GeneMark.hmm
• http//opal.biology.gatech.edu/GeneMark/gmhmm2_pro
  k.cgi
• Available as a web server
• Uses hidden markov models (HMM)

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Glimmer Performance
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What Next?

• Raw DNA sequence ? Gene sequences
• Gene seqs ? Protein sequences
• Gene Protein seqs ? Databases
• Gene Protein info ? Databases
• Most protein and DNA sequence data is entered
  into GenBank through XXX
• Next Lecture Databases

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Sample Exam Question

• Describe an algorithm or sketch a flowchart for
  gene finding in prokaryotes
• What are the key features of a prokaryotic ORF?
• Following is a gene sequence identify and label
  all major features