1. Sequencing Transcriptomes Do Me a SOLiD

2. Overview – Library Construction RNA ◦Isolate & Bioanalyze ◦rRNA Depletion ◦Fragment ◦Bioanalyze Amplified Library Construction ◦Hybridize & Ligate RNA ◦Reverse Transcription ◦cDNA Size Selection/Amplification RNA ◦Isolate & Bioanalyze ◦rRNA Depletion ◦Fragment ◦Bioanalyze Amplified Library Construction ◦Hybridize & Ligate RNA ◦Reverse Transcription ◦cDNA Size Selection/Amplification

3. RNA ◦Isolate & Bioanalyze ◦rRNA Depletion ◦Fragment ◦Bioanalyze Amplified Library Construction ◦Hybridize & Ligate RNA ◦Reverse Transcription ◦cDNA Size Selection/Amplification RNA ◦Isolate & Bioanalyze ◦rRNA Depletion ◦Fragment ◦Bioanalyze Amplified Library Construction ◦Hybridize & Ligate RNA ◦Reverse Transcription ◦cDNA Size Selection/Amplification Overview – Library Construction

4. Emulsion PCR (ePCR) Emulsion break/bead wash Enrich templated beads Modify 3’ ends Emulsion PCR (ePCR) Emulsion break/bead wash Enrich templated beads Modify 3’ ends Overview – Templated Bead Prep

5. Library Construction RNA – Isolate -> Bioanalyzer

6. Library Construction RNA – Bioanalyzer

7. Library Construction RNA – Total, mRNA, Fragmented

8. Library Construction RNA – Total, mRNA, Fragmented

9. Library Construction Fragmented RNA – Ligate, Reverse Transcribe Adaptor ligated to fragmented RNA Primer binding site during cDNA amplification Adaptor ligated to fragmented RNA Primer binding site during cDNA amplification

10. Library Construction cDNA – Size Selection 6% TBE-Urea gel 180V for ~17 mins 6% TBE-Urea gel 180V for ~17 mins 100-200bp range excised Cut vertically 4 equal pieces (~1x6mm) In-gel PCR Only 15-18 cycles Primers 1 & 2 bind to adaptors Barcode? 100-200bp range excised Cut vertically 4 equal pieces (~1x6mm) In-gel PCR Only 15-18 cycles Primers 1 & 2 bind to adaptors Barcode?

11. Library Construction Amplified cDNA – Yield, Size Distribution

12. Library Construction Amplified cDNA – Yield, Size Distribution

13. Templated Bead Prep Amplified cDNA – ePCR 500-1500 pM Template (~60-180pg/uL) Empirically determined  500-1500 pM Template (~60-180pg/uL) Empirically determined 

14. Templated Bead Prep Amplified cDNA – ePCR Before PCR

15. Templated Bead Prep Amplified cDNA – ePCR

16. Templated Bead Prep Amplified cDNA – ePCR

17. Templated Bead Prep Amplified cDNA – ePCR After PCR

18. Templated Bead Prep Amplified cDNA – ePCR Centrifuge

19. Templated Bead Prep ePCR – Emulsion Break

20. Templated Bead Prep ePCR – Emulsion Break/Bead Counts

21. Templated Bead Prep Bead Enrichment Templated Beads + P2 Enrichment Beads on Glycerol Cushion

22. Templated Bead Prep Bead Enrichment Low templated bead recovery at interphase.

23. Templated Bead Prep Bead Enrichment Ideal templated bead recovery at interphase.

24. Templated Bead Prep Bead Enrichment 3’ Modification Bead Counts # Templated Beads (75-100 million = Good) % Templated Beads (<20% = Good) 3’ Modification Bead Counts # Templated Beads (75-100 million = Good) % Templated Beads (<20% = Good)

25. SOLiD Run Sequencing

26. SOLiD Run Sequencing Bead Depostion Chambers

27. SOLiD Run Sequencing

28. SOLiD Run Sequencing - Reagents

29. SOLiD Run Sequencing – Focus Beads WFA Run (~5500 beads/panel) WFA Run (~5500 beads/panel) Sequencing Run (~220,000 beads/panel) Sequencing Run (~220,000 beads/panel)

30. SOLiD Run Sequencing - Cycling

31. SOLiD Run Sequencing - Interrogation

32. SOLiD Run Sequencing – Satay Plots Quality Check Spectral Purity Signal Intensity Quality Check Spectral Purity Signal Intensity

33. SOLiD Run Sequencing – Satay Plots Cycle #1 Cycle #10

34. SOLiD Run Sequencing – Cycle Scans

35. SOLiD Run Sequencing – Exposure Time Quality Check Focus Reagents Quality Check Focus Reagents

36. SOLiD Run Sequencing – Color Balance Quality Check Library Prep (Bias?) Reagents Quality Check Library Prep (Bias?) Reagents

37. SOLiD Run Sequencing – Heat Maps Quality Check Bead Count Deposition Bead Signal Image Signal Background Quality Check Bead Count Deposition Bead Signal Image Signal Background

38. SOLiD Time cDNA Library (RNA to cDNA) = 3 days ePCR (ePCR to WFA) = 2 days WFA = 1 day SOLiD Run = 7 days Total Time = 13 days Nucleotides/Day* = 1.54x10^8 bp/day * - 40M reads of 50bp each

39. SOLiD Money mRNA Library Construction = $225 = $20 Bead Prep = $350 SOLiD Reagents = $500 (?) Total Cost = ~$1200** Cost/Nucleotide* = 6x10^-7 dollars/bp * - 40M reads of 50bp each ** - Includes miscellaneous/incidentals

40. That’s SOLiD!