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Greg Phillips Veterinary Microbiology gregory@iastate

Greg Phillips Veterinary Microbiology gregory@iastate.edu. 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. The First Generation-

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Greg Phillips Veterinary Microbiology gregory@iastate

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  1. Greg Phillips Veterinary Microbiology gregory@iastate.edu

  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. TheFirst 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. 2008. Annu. Rev. Genomics Human Genet. 9:387.

  10. 454 Sequencing Technology Amplification Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

  11. 454 Sequencing Technology Sequencing Reaction Mardis. 2008. 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. 2008. Annu. Rev. Genomics Human Genet. 9:387.

  15. Illumina Sequencing Technology Bridge Amplification Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

  16. Illumina Sequencing Technology Detection Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

  17. Illumina Sequencing Technology

  18. ABI/SOLiD DNA Sequencing Technology Sequencing by ligation Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

  19. ABI/SOLiD DNA Sequencing Technology Sequencing by ligation Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

  20. ABI/SOLiD DNA Sequencing Technology

  21. Characteristics of New DNA Sequencing Technology Rothberg &Leamon. 2008. 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. 2008. 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 http://www.pacificbiosciences.com/video_lg.html Eidet al. 2009 Science

  33. Nanopore DNA Sequencing http://www.nanoporetech.com/sequences/index/34

  34. What do YOU think?

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