Pyromania

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Pyromania
USAFSAM EPI Lab
MSgt Stephen Christian, USAF, MSgt, MT
(ASCP) Lucinda Sinclair, GS-11
Distribution Statement A Approved for Public
release distribution is unlimited. 311 ABG/PA
No. 10-080, 12 Mar 2010
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Overview

• Introduction to Influenza
• Why\What to Sequence
• Sequencing (Pyro versus Sanger)
• Pyrosequencing for influenza antiviral resistance
  markers (Tamiflu resistance in 2009 A (H1/N1)

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Influenza
http//www.ncbi.nlm.nih.gov/genomes/FLU/
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Types of influenza
1918 H1N1 50,000,000 deaths 1957 H2N2
1,000,000 deaths 1968 H3N2 500,000
deaths Annual flu 250,000 deaths H5N1
annual 25 - 75
A,B, and C (Epidemics A B) Pandemics A only
2009 A/H1N1
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Influenza Genome
PB2 PB1 PA HA NP NA M NS
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Why/What to Sequence
HA1 region of HA gives the most bang for the buck.
PB2 PB1 PA HA NP NA M NS
13,588 bps
Tamiflu
Amantadine
Presence/Absence of Antiviral resistance markers
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Sequencing (Pyro versus Sanger)

• Nucleic Acid Extraction
• Traditional RT PCR
• Sequencing

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Sanger Sequencing

• Sequencing by Chain Termination
• Extract Nucleic Acid
• Produce amplicon
• Clean Amplicon
• Big Dye Rxn (Chain Termination)
• Clean Big Dye Rxn
• Electrophoresis of Big Dye (Sequencer)
• Analyze Sequence Data

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Produce Amplicon (HA1)
Template (Amplicon)
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Clean Amplicon (HA1)
(dATPs) (dGTPs) (dCTPs) (dTTPs)
Polymerase
Salts Buffers
Clean PCR Product Amplicon
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Big Dye Reaction
Template (Amplicon)
Primer
dNTPs A (dATPs) G(dGTPs) C (dCTPs) T (dTTPs)
ddNTPs A G C T
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Big Dye Reaction
Template (Amplicon)
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Clean Big Dye Reaction
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Electrophoresis
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Results from Electrophoresis
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Sequence Analysis

• Build Single Contig
• Analyze constructed nucleotide sequence (Protein)
• Relate to current vaccine strain on phylogenetic
  tree

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Pyrosequencing

• Sequencing by Addition
• Extract Nucleic Acid
• Produce amplicon
• Pyrosequencer

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Pyrosequencing
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Simple Sample Preparation
1. Amplify relevant region by PCR (100 - 300 bp),
using 1 biotinylated primer
2. Immobilize biotinylated PCR products onto
streptavidin-coated beads
3. Separate strands by denaturation in NaOH
4. Wash /neutralize the immobilized strand
5. Anneal sequencing primer
96 samples are processed in parallel
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Pyrosequencing
1.) Hybridize sequence primer along with DNA
polymerase, ATP sulfurylase, luciferase, apyrase,
adenosine 5 phosphosuflate (APS) and luciferin.
luciferin
luciferase
Apyrase
APS
sulfurylase
Polymerase
Polymerase
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Pyrosequencing
2.) Add one of four dNTPs, DNA polymerase
incorporates dNTPs (if it is complementary)
releasing PPi.
luciferin
luciferase
Apyrase
APS
sulfurylase
dNTPs added one at a time
Polymerase
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Pyrosequencing
2.) Add one of four dNTPs, DNA polymerase
incorporates dNTPs (if it is complementary)
releasing PPi.
luciferin
luciferase
Apyrase
APS
sulfurylase
Polymerase
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Pyrosequencing
2.) Add one of four dNTPs, DNA polymerase
incorporates dNTPs (if it is complementary)
releasing PPi.
luciferin
luciferase
APS
sulfurylase
Apyrase
PPi
Polymerase
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Pyrosequencing
ATP sulfurylase converts PPi and APS to ATP.
Luciferase converts luciferin and ATP to
oxyluciferin which generates light read by a
camera.
sulfurylase
luciferin
luciferase
ATP
APS
PPi
Apyrase
Polymerase
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Pyrosequencing
ATP sulfurylase converts PPi and APS to ATP.
Luciferase converts luciferin and ATP to
oxyluciferin which generates light read by a
camera.
sulfurylase
luciferase
ATP
APS
oxyluciferin
PPi
luciferin
Apyrase
Polymerase
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Pyrosequencing
Hybridize sequence primer, DNA polymerase, ATP
sulfurylase, luciferase, apyrase, adenosine 5
phosphosuflate (APS) and luciferin. Add one of
four dNTPs, DNA polymerase incorporates dNTPs
(if it is complementary) releasing PPi. ATP
sulfurylase converts PPi and APS to ATP.
Luciferase converts luciferin and ATP to
oxyluciferin which generates light read by a
camera. Apyrase degrades dNTPs and ATP, when
complete next dNTP is added. As process
continues, the correct DNA sequence is built up
and displayed.
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(No Transcript)
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• Sanger sequencing steps
• VS.

2 Pyrosequencing steps
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Neuraminidase Pyrosequencing
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Questions