2. Comparison between old generation and new generation of sequencing machines

3. Review over the sequencing process

4. Genome sequencing project Human genome

5. Genome sequencing project Shot gun method DNA library Sequencing Assembly

7. Efficiency of old generation of DNA sequencer How many bases it can sequence and how long it takes?

8. Now with new generation You can sequence bacterial genome in less than a day Arabidopsis genome in days Human genome in less than 3 weeks

9. Prepare the library

10. Nebulizing the DNA

11. DNA is fragmented into small sizes 50 bp-1Kbp Is it a wide range??????

12. How to select a narrower DNA size?

13. DNA size selection Select from 500-800 bp

14. Repair purified nebulized DNA by end polishing Three enzymes are used : Extend by ……………….. Remove by ……………… Capping by ……………….

15. Adaptor ligation A : Green B: Red and biotinylated

16. After Adaptor ligation 4 products are produced

17. DNA immobilization Biotin::striptavidin interaction Which product will be washed away? Which product Will be sequenced or non sequenced

18. ss DNA library formation Predict what will happen if we add NaOH soln. ?

19. What you will have in the supernatant ?

20. The final ssDNA library

21. PCR of DNA

22. Mixing the beads and DNA

23. Beads contains on their surface ………… The beads and ssDNA are mixed in ………………… PCR reaction results in …………….. Microreactors contain ………………………. Mix.

24. PCR preparation 4 Of 96 wells micro titter plate

25. DNA amplification

26. DNA orientation on the beads The beads contain DNA coplementary to …………………. Side. The B adaptor will be ……….., the A adaptor will be ……………

27. Isolation of the positive amplified DNA

28. Separation of amplified DNA Magnetic particle concentrator

30. Sequencing reaction Another PCR

31. After this sequencing the beads will be added onto the slide

32. Titanium slide

34. One bead goes in one well on the titanium slide and then this slide will be placed for sequencing