Pyromania
USAFSAM “EPI Lab”
MSgt Stephen Christian, USAF, MSgt, MT (ASCP)Lucinda Sinclair, GS-11
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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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Flu Mortality
1918
1957
1968
annual
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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
Types of influenza
2009 A/H1N1
A,B, and C (Epidemics = A &B)
Pandemics = A only
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Influenza Genome
PB2
PB1
PA
HA
NP
NA
M
NS
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HA1 region of HA gives the most bang for the buck.
Why/What to Sequence
PB2
PB1
PA
HA
NP
NA
M
NS
13,588 bp’s
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AmantadinePresence/Absence of Antiviral resistance markers
Tamiflu
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Nucleic Acid Extraction
Traditional RT PCR
Sequencing
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Sequencing (Pyro versus Sequencing (Pyro versus Sanger) Sanger)
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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)
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Template (Amplicon)
Template (Amplicon)
Clean PCR Product
Amplicon
(dATP’s)
(dGTP’s)
(dCTP’s)
(dTTP’s)
Polymerase Salts
Buffers
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Template (Amplicon)
Primer Primer
Primer
Primer
Primer
Primer
Primer
dNTP’s
A (dATP’s)
G(dGTP’s)
C (dCTP’s)
T (dTTP’s)
ddNTP’s
A
G
C
T
Big Dye Reaction
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Big Dye Reaction
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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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PyrosequencingPyrosequencing
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Sequencing by Addition
Extract Nucleic Acid
Produce amplicon
Pyrosequencer
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PyrosequencingPyrosequencing
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2. Immobilize biotinylated PCR products onto streptavidin-coated beads
3. Separate strands by denaturation in NaOH
5. Anneal sequencing primer
96 samples are processed in parallel
4. Wash /neutralize the immobilized strand
1. Amplify relevant region by PCR (100 - 300 bp), using 1 biotinylated primer
Simple Sample Preparation
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1.) Hybridize sequence primer along with DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosphosuflate (APS) and luciferin.
Polymerase
Apyrase
sulfurylase
luciferaseAPS
luciferin
Polymerase
PyrosequencingPyrosequencing
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dNTP’s added one at atime
Apyrase
sulfurylase
luciferaseAPS
luciferin
Polymerase
2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi.
PyrosequencingPyrosequencing
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Apyrase
sulfurylase
luciferaseAPS
luciferin
Polymerase
2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi.
PyrosequencingPyrosequencing
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Apyrase
sulfurylase
luciferaseAPS
luciferin
Polymerase
2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi.
PPi
PyrosequencingPyrosequencing
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Apyrase
sulfurylase
luciferaseAPS
luciferin
Polymerase
ATP sulfurylase converts PPi and APS to ATP. Luciferase converts luciferin and ATP to oxyluciferin which generates light read by a camera.
+ PPi = ATP
PyrosequencingPyrosequencing
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Apyrase
sulfurylaseluciferase
APS
luciferin
Polymerase
ATP sulfurylase converts PPi and APS to ATP. Luciferase converts luciferin and ATP to oxyluciferin which generates light read by a camera.
+ PPi = ATP
oxyluciferin
PyrosequencingPyrosequencing
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Hybridize sequence primer, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosphosuflate (APS) and luciferin.
Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (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 dNTP’s and ATP, when complete next dNTP is added.
As process continues, the correct DNA sequence is built up and displayed.
PyrosequencingPyrosequencing
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5 Sanger sequencing steps
VS.
2 Pyrosequencing steps
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Neuraminidase Pyrosequencing
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Questions
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