1. DNA Sequencing Technology and its Applications in Evolution Research Julie Urban, Ph.D. Assistant Director, Genomics & Microbiology Laboratory NC Museum of Natural Sciences

2. Planthopper Evolution

3. 1953

5. 15 Feb, 2001

6. Planthopper Evolution (2001)

7. Planthopper Evolution (2001) (2008) XX

8. Today Ion Proton ($149,000) human genome for < $5,000, ~1 day (?) MiSeq ($126,000) Ion Torrent ($90,000)

9. Planthopper Evolution

12. Capillary sequencing Sequencing by synthesis Single molecule methods

13. Planthopper Evolution Capillary (Sanger) Sequencing

15. Fluorescence Important in (most) Next Gen Sequencing

16. Planthopper Evolution Steps Common to all Next Generation Sequencing Systems 1. Make Library: -- fragment DNA into unique fragments -- add custom adaptors (“linkers”) 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface 3. Sequencing by Synthesis -- fragments are copied again (sequencing reaction) -- incorporation of each base “fires” a signal -- system detects signal coming from all clones “firing” at once for that unique fragment All sequence DNA in “massive parallel” fashion

17. Planthopper Evolution Roche/454 Sequencing 1. Make Library: -- fragment DNA into unique fragments -- add custom adaptors (“linkers”)

18. Planthopper Evolution Roche/454 Sequencing 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface

19. Planthopper Evolution Roche/454 Sequencing 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface

20. Planthopper Evolution Roche/454 Sequencing 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface

21. Planthopper Evolution Roche/454 Sequencing 3. Sequencing by Synthesis -- fragments are copied again (sequencing reaction) -- incorporation of each base “fires” a signal -- system detects signal coming from all clones “firing” at once for that unique fragment

22. Planthopper Evolution Illumina Sequencing 1. Make Library: -- fragment DNA into unique fragments -- add custom adaptors (“linkers”)

23. Planthopper Evolution Illumina Sequencing 1. Make Library: -- fragment DNA into unique fragments -- add custom adaptors (“linkers”)

24. Planthopper Evolution 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface Illumina Sequencing

25. Planthopper Evolution 3. Sequencing by Synthesis -- fragments are copied again (sequencing reaction) -- incorporation of each base “fires” a signal -- system detects signal coming from all clones “firing” at once for that unique fragment Illumina Sequencing

26. Planthopper Evolution 3. Sequencing by Synthesis -- fragments are copied again (sequencing reaction) -- incorporation of each base “fires” a signal -- system detects signal coming from all clones “firing” at once for that unique fragment Illumina Sequencing

27. Planthopper Evolution Life Tech/ABI Ion Torrent & Proton Sequencing 1. Make Library: -- fragment DNA into unique fragments -- add custom adaptors (“linkers”)

28. Planthopper Evolution 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface Life Tech/ABI Ion Torrent & Proton Sequencing

29. Planthopper Evolution 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface Life Tech/ABI Ion Torrent & Proton Sequencing

30. Planthopper Evolution Life Tech/ABI Ion Torrent & Proton Sequencing 3. Sequencing by Synthesis -- fragments are copied again (sequencing reaction) -- incorporation of each base “fires” a signal -- system detects signal coming from all clones “firing” at once for that unique fragment

31. Planthopper Evolution Life Tech/ABI Ion Torrent & Proton Sequencing 3. Sequencing by Synthesis -- fragments are copied again (sequencing reaction) -- incorporation of each base “fires” a signal -- system detects signal coming from all clones “firing” at once for that unique fragment

32. Planthopper Evolution Life Tech/ABI Ion Torrent & Proton Sequencing 3. Sequencing by Synthesis -- fragments are copied again (sequencing reaction) -- incorporation of each base “fires” a signal -- system detects signal coming from all clones “firing” at once for that unique fragment

33. Planthopper Evolution Life Tech/ABI Ion Torrent & Proton Sequencing 3. Sequencing by Synthesis -- fragments are copied again (sequencing reaction) -- incorporation of each base “fires” a signal -- system detects signal coming from all clones “firing” at once for that unique fragment

34. Planthopper Evolution Life Tech/ABI Ion Torrent & Proton Sequencing 3. Sequencing by Synthesis -- fragments are copied again (sequencing reaction) -- incorporation of each base “fires” a signal -- system detects signal coming from all clones “firing” at once for that unique fragment

35. Planthopper Evolution Single Molecule Systems: Pacific Biosciences

36. Planthopper Evolution Single Molecule Systems: Oxford Nanopore

37. Capillary sequencing Sequencing by synthesis Single molecule methods

40. Planthopper Evolution Applications of Next Generation Sequencing Metagenomics

41. Planthopper Evolution Applications of Next Generation Sequencing Microbiome http://commonfund.nih.gov/hmp/ Human Microbiome Project

42. Planthopper Evolution Applications of Next Generation Sequencing Microbiome

43. Planthopper Evolution Applications of Next Generation Sequencing Bacterial Genome Sequencing Bacteriome = insect organ made of bacteriocytes Planthoppers

44. Planthopper Evolution Applications of Next Generation Sequencing Ancient (=degraded) DNA Sequencing

46. Planthopper Evolution

47. Annual Review of Genomics and Human Genetics, 2008. 9: 387-402. Youtube: Current Topics in Genome Analysis 2012; NHGRI Lecture Series. -- Elaine Mardis -- Jonathan Eisen -- Julie Segre (Microbiome)

48. Collaborators and Funding Megan Ehlers NCSU undergrad Greg Pahel NCMNS Rob Dunn NCSU Holly Menninger NCSU Dan Fergus NCMNS Megan Thoemmes NCSU undergrad Jason Cryan NCMNS Julie Horvatn NCMNS