1. Pyromania USAFSAM “EPI Lab” MSgt Stephen Christian, USAF, MSgt, MT (ASCP) Lucinda Sinclair, GS-11 1 Distribution Statement A: Approved for Public release; distribution is unlimited. 311 ABG/PA No. 10-080, 12 Mar 2010

2. Team Aerospace Begins Here! 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) 2

3. Team Aerospace Begins Here! Influenza http://www.ncbi.nlm.nih.gov/genomes/FLU/ 3

4. Team Aerospace Begins Here! 4 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

5. Team Aerospace Begins Here! Influenza Genome PB2 PB1 PA HA NP NA M NS 5

6. Team Aerospace Begins Here! 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 6 Amantadine Presence/Absence of Antiviral resistance markers Tamiflu

7. Team Aerospace Begins Here! Nucleic Acid Extraction Traditional RT PCR Sequencing 7 Sequencing (Pyro versus Sanger)

8. Team Aerospace Begins Here! 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 8

9. Team Aerospace Begins Here! Produce Amplicon (HA1) Template (Amplicon) 9

10. Team Aerospace Begins Here! Clean Amplicon (HA1) 10 Template (Amplicon) Clean PCR Product Amplicon (dATP’s) (dGTP’s) (dCTP’s) (dTTP’s) Polymerase Salts Buffers

11. Team Aerospace Begins Here! Template (Amplicon) Primer dNTP’s A (dATP’s) G(dGTP’s) C (dCTP’s) T (dTTP’s) ddNTP’s A G C T Big Dye Reaction

12. Team Aerospace Begins Here! Big Dye Reaction 12 Template (Amplicon)

13. Team Aerospace Begins Here! Clean Big Dye Reaction 13

14. Team Aerospace Begins Here! Electrophoresis 14

15. Team Aerospace Begins Here! Results from Electrophoresis 15

16. Team Aerospace Begins Here! Sequence Analysis Build Single Contig Analyze constructed nucleotide sequence (Protein) Relate to current vaccine strain on phylogenetic tree 16

17. Team Aerospace Begins Here!Pyrosequencing 17 Sequencing by Addition Extract Nucleic Acid Produce amplicon Pyrosequencer

18. Team Aerospace Begins Here!Pyrosequencing

19. 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

20. Team Aerospace Begins Here! 1.) Hybridize sequence primer along with DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosphosuflate (APS) and luciferin. Polymerase Apyrase sulfurylase luciferase APS luciferin PolymerasePyrosequencing

21. Team Aerospace Begins Here! dNTP’s added one at a time Apyrase sulfurylase luciferase APS luciferin Polymerase 2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi.Pyrosequencing

22. Team Aerospace Begins Here! Apyrase sulfurylase luciferase APS luciferin Polymerase 2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi.Pyrosequencing

23. Team Aerospace Begins Here! Apyrase sulfurylase luciferase APS luciferin Polymerase 2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi. PPiPyrosequencing

24. Team Aerospace Begins Here! Apyrase sulfurylase luciferase 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 = ATPPyrosequencing

25. Team Aerospace Begins Here! Apyrase sulfurylase luciferase 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 oxyluciferinPyrosequencing

26. Team Aerospace Begins Here! 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.Pyrosequencing

27. Team Aerospace Begins Here! 27

28. Team Aerospace Begins Here! 5Sanger sequencing steps VS. 2 Pyrosequencing steps

29. Team Aerospace Begins Here! Neuraminidase Pyrosequencing 29

30. Team Aerospace Begins Here! 30 Questions