Technology developement for high – throughput sequencing of microRNAs

1. Francois Vigneault Church Lab CEGS meeting Oct 16th 2008 Technologydevelopement for high –throughputsequencing of microRNAs

3. How to clone miRNAs? 5’ 3’ X PO4 5’ 3’-OH PO4 T4 RNA ligase + 5’ + PO4 3’ X ATP

4. How to capture miRNAs? 5’ 3’ X PO4 5’ 3’-OH PO4 T4 RNA ligase + 5’ + PO4 5’ 3’ 3’ App X X ATP

5. How does one make app-Oligo • Lau et al. Science 2001 (Bartel lab) • Yield 10-20% • IDT (5 nmole $ 595)

6. Enzymatic adenylation ATP 5’ 3’ App T4 DNA Ligase X PO4 3’ 5’ .

7. Pre-adenylation

8. 3’adapter ligation

9. 5’ adapter ligation

10. High-throughput sequencing • Illumina ~5 000 000 reads/track for 1200$ • Less than ~1000 miRNAs in human cells • Multiplexing up to 50 samples per track (100 fold dynamic range) bring cost per samples to 24$

11. Bar-coded adenylation 5’ 3’ T4 DNA Ligase App X PO4 ATAT NNNN NNNN NNNN NNNN 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 3’ T4 DNA Ligase App X PO4 GCGC 5’ 3’ T4 DNA Ligase App X PO4 TAGC 5’ 3’ T4 DNA Ligase App X PO4 CCAA .

12. Bar-coded adenylation 5’ 3’ App X ATAT 5’ 3’ App X GCGC 5’ 3’ App X TAGC 5’ 3’ App X CCAA

13. miRNAs ligation 5’ 3’ T4 DNA Ligase App X ATAT 5’ 5’ 5’ 5’ 3’-OH 3’-OH 3’-OH 3’-OH PO4 PO4 PO4 PO4 5’ 3’ T4 DNA Ligase App X GCGC 5’ 3’ T4 DNA Ligase App X TAGC 5’ 3’ T4 DNA Ligase App X CCAA

14. miRNAs ligation 3’ T4 DNA Ligase X ATAT 5’ 5’ 5’ 5’ PO4 PO4 PO4 PO4 3’ T4 DNA Ligase X GCGC 3’ T4 DNA Ligase X TAGC 3’ T4 DNA Ligase X CCAA

15. 5’adapter ligation 5’ PO4 3’ 5’ T4 DNA Ligase 3’-OH X NNNN

16. 5’adapter ligation 3’ 5’ T4 DNA Ligase X NNNN .

17. RT-PCR TAQ pol 3’ 5’ X NNNN RT pol

18. Sequencing miRNA barcode

19. Sequencing

20. Sequencing ATC TCA AAT CCA GCC TGA AAT GCC ATAT TTG… TGA AAT ATC ACG ATC ATC ATC GGG GCGC TTG… GCC ATC GGG TTC TGA GTG ATC TGA TAGC TTG… GTG GGG TCA GTC GTG GCC TCA GTG CCAA TTG… .

21. Analysis ATC TCA AAT CCA GCC TGA AAT GCC ATAT TTG… TGA AAT ATC ACG ATC ATC ATC GGG GCGC TTG… GCC ATC GGG TTC TGA GTG ATC TGA TAGC TTG… GTG GGG TCA GTC GTG GCC TCA GTG CCAA TTG…

22. Analysis miRNA profiling Sample1 Sample2 Sample3 Sample4 ATC ATC ATC TCA TCA TCA AAT AAT AAT AAT CCA CCA CCA CCA GCC GCC GCC TGA TGA TGA AAT AAT AAT GCC GCC GCC GCC ATAT ATAT ATAT ATAT TTG… TTG… TTG… TTG… ATC TCA GCC TGA AAT TGA TGA TGA TGA AAT AAT AAT AAT ATC ATC ATC ATC ACG ACG ACG ACG ATC ATC ATC ATC ATC ATC ATC ATC ATC ATC ATC ATC GGG GGG GGG GGG GCGC GCGC GCGC GCGC TTG… TTG… TTG… TTG… Expression levels GCC GCC GCC ATC ATC ATC GGG GGG GGG TTC TTC TTC TGA TGA TGA GTG GTG GTG ATC ATC ATC TGA TGA TGA TAGC TAGC TAGC TTG… TTG… TTG… GTG GTG GTG GGG GGG GGG TCA TCA TCA GTC GTC GTC GTG GTG GTG GGG GGG TCA TCA GTC GTC GTG GTG GTG GTG GCC GCC GCC GCC GCC TCA TCA TCA TCA TCA GTG GTG GTG GTG GTG CCAA CCAA CCAA CCAA CCAA TTG… TTG… TTG… TTG… TTG… miRNA sequences .

23. Projections • Personal Genome Project (PGP) • Induced pluripotent stem cells (iPS) • VDJ-ome

24. VDJ-ome

25. Acknowledgment • George M Church • Michael Sismour • Greg Porreca • Andrew Z Fire and lab • All Church lab members…