1. Molecular Biology Dr. Chaim Wachtel April 4, 2013

2. Topics Microarray Deep Sequencing PCR –Real-Time –Digital –Nanostring GENE EXPRESSION

3. Microarray Affymetrix Agilent Same idea different implementation Pros and cons to each method

4. The Chips AgilentAffymetrix

5. Printing of Affymetrix Chips

6. Printing of Agilent Chips

7. Probes are 60 nucleotides in length vs 25 for Affymetrix

8. Affymetrix workflow

9. Agilent workflow

10. Feature Extraction Bioinformatics Analysis “home-made”=low-density Agilent array

11. Pros and Cons Cost Dual color vs single Specificity

12. Deep Sequencing Next Generation Sequencing (NGS)

14. Sanger Sequencing

15. Multiple Platforms Illumina 454 Solid Nanopore/Oxford Ion Torrent

16. Different companies different methods

17. Workflow for Sequencing

18. Depends on project- RNA or DNA Protocols are ~90% the same for all systems

19. Illumina

21. Workflow for Sequencing Bridge PCR Emulsion PCR

26. Sequencing with Illumina

27. Sequencing with Solid

28. The Ion Torrent

30. Scalability

31. The Chip

52. Nanopore Technology

53. * all images are of an alpha prototype of the MinION device, final device expected to be at least half this size

54. How does it work?

56. What’s the big deal?? GridION, consists of an array of sequencers, each containing 2,000 nanopores. That is expected to increase to 8,000 nanopores in the next- generation device. Twenty of these, running simultaneously, could sequence a full human genome in 15 minutes, the company said, describing both devices as "plug-and-play." MinION, with 512 nanopores will be capable of sequencing 150,000 DNA bases per hour the 48,490-bp genome of lambda DNA was sequenced as a complete fragment—in one read. This read length is three orders of magnitude larger than the typical length of DNA fragments that are read in current sequencing approaches using fluorescent nucleotide labeling 3. 3 $$$$- MinION will cost $900