pyromania usafsam “epi lab” msgt stephen christian, usaf, msgt, mt (ascp) lucinda sinclair,...

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