1. Greg Phillips
Veterinary Microbiology

2. Next Generation DNA Sequencing
Review of DNA sequencing technology
3 Next Gen platforms-technology (and advertising)
New applications of Next Gen technology
Next Next Gen technology
$1,000 genome

3. The First Generation-
Sanger DNA Sequencing

4. Sanger DNA Sequencing by Capillary Electrophoresis & Fluorescent Detection
Long read length >1000 bp)

5. Traditional Applications of
DNA Sequencing
Gene sequencing
New gene discovery
Mutations (natural variants & synthetic)
Genome sequencing
New genomes (microbial to human)
Comparative genomics
Metagenomics
Environmental samples of mixed populations

6. Shotgun Sequencing Approach

7. Next-Generation DNA Sequencing:
3 Platforms
454 (Roche)
Pyrosequencing
Medium read length (~500 bp)
Illumina (Solexa)
Sequencing by synthesis
Short read sequencing (up to 100 bp)
SOLiD (Applied Biosystems)
Sequencing by ligations
Short read sequencing (~35 bp)

8. Next-Generation DNA Sequencing Advantages/Disadvantages
Depth of coverage of genome sequencing
Sanger: 4-5x
454: >20x
Illumina, SOLiD: up to 100x
Library preparation less laborious
Methods unique to each platform
Lower cost
VS.
Shorter read lengths
De novo sequence assembly difficult
Lower accuracy in homopolymer tracts
Large amount of data generated

9. 454 Sequencing Technology
Pyrosequencing
Library Preparation
Mardis Annu. Rev. Genomics Human Genet. 9:387.

10. 454 Sequencing Technology
Amplification
Mardis Annu. Rev. Genomics Human Genet. 9:387.

11. 454 Sequencing Technology
Sequencing Reaction
Mardis Annu. Rev. Genomics Human Genet. 9:387.

12. 454 Sequencing Technology
Detection

13. Roche/454 DNA Sequencing
Pyrosequencing

14. Illumina Sequencing Technology
Sequencing by Synthesis
Library Preparation
Mardis Annu. Rev. Genomics Human Genet. 9:387.

15. Illumina Sequencing Technology
Bridge Amplification
Mardis Annu. Rev. Genomics Human Genet. 9:387.

16. Illumina Sequencing Technology
Detection
Mardis Annu. Rev. Genomics Human Genet. 9:387.

17. Illumina Sequencing Technology

18. ABI/SOLiD DNA Sequencing Technology
Sequencing by ligation
Mardis Annu. Rev. Genomics Human Genet. 9:387.

19. ABI/SOLiD DNA Sequencing Technology
Sequencing by ligation
Mardis Annu. Rev. Genomics Human Genet. 9:387.

20. ABI/SOLiD DNA Sequencing Technology

21. Characteristics of New DNA Sequencing Technology
Rothberg &Leamon Nat. Biotechnol. 26:1117

22. New DNA Sequencing Technology is Well-Suited for Microbial Genomes

23. Next-Generation DNA Sequencing-
Applications
Genome sequencing
Short read de novo assembly requires new bioinformatic tools
Genome resequencing of mutants
Metagenomics
…the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species -Chen, K; Pachter L (2005). . PLoS Comp Biol 1: 24.

24. Next Gen Sequencing & Metagenomics
• Microbial communities in a natural environment
Seawater
Soil
• Host-associated microbial communities
Human microbiome
Gut
Oral cavity
• Microbial communities in a “managed-environment”
Sewage treatment plant
Aquaculture facility

25. Next-Generation DNA Sequencing-
More Applications
RNA-seq
Transcriptional profiling
Consensus sequencing
ChIP sequencing
Chromatin immunoprecipitation
Identification of protein binding sites
Small RNA-seq
Epigenetics

26. Next-Generation DNA Sequencing
RNA-seq
Marguerat, et al Biochem. Soc. Trans. 36: 1091–1096.

27. Next-Generation DNA Sequencing ChIP-seq
Chromatin immunoprecipitation

28. Next-Generation DNA Sequencing Small RNA-seq
Animals/Plants/Bacteria encode small,
non-coding RNAs that function in:
Gene regulation
Antiviral defense mechanisms
Development
Unknown functions

29. Next-Generation DNA Sequencing
Epigenetics

30. The Next-Next Generation- Helicos Single Molecule DNA Sequencing

31. The Next-Next Generation- Helicos Single Molecule DNA Sequencing

32. Single Molecule DNA Sequencing-
Pacific Biosciences
Eid et al Science

33. Nanopore DNA Sequencing

34. What do YOU think?