Kun:LabNotes/ExonomeSeq/2009-10-16
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Test the 1-adaptor ligation protocol
- Billy sent me two tubes of sheared NA12878 samples captured by #6&7.
Batch 1
- I did size selection on ~80ul of the sheared DNA from tube 1 using the SizeSelect gel, and extract ~100bp(L100) and ~120bp(L120) fragments.
- I did End-repair on the extracted DNAs without further purification.
DNA 45ul 10X End-repair buffer 5.3ul Enzyme Mix 2.5ul 25C for 45 min -> 70C for 10min -> purified with Qiaquick columns
Ligation: Adaptor ligation: 20ul Solexa_1_up (200uM) + 20ul Solexa_1_lo_noP (200uM) + 5ul 10X Stoffel buffer + 55ul H2O -> 94C 3min -> -0.1C/sec to 20C DNA 30ul 40uM adaptors 3ul 2x QuickLigase Buffer 34ul QuickLigase 1ul RT 15min -> purified with Qiaquick columns -> extract 120bp+ and 150bp+ fragments with SizeSelect gel.
PCR F+R1 F+R2 F Template 5ul 5ul 5ul 2x iProof Mix 50ul 50ul 50ul Solexa_PCR_up(10uM) 2ul 2ul 2ul AmpR6.3Sol(100uM) 0.4ul - - AmpF6.3rSol(100uM) - 0.4ul - 50X SYBGI 0.4ul 0.4ul 0.4ul H2O 44ul 44ul 44ul 98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 10sec) x 8 -> (98C 10sec -> 72C 15sec) x 10 -> 72C 2min -> 15C hold.
Batch 2
- I also did End-repair on another 80ul sheared DNA from tube 2 (prior to size selection).
DNA 85ul 10X End-repair buffer 10ul Enzyme Mix 5ul RT 30min -> 70C 10min -> purified with a Qiaquick column.
- Perform size selection with SizeSelect gel, and extract ~120bp (EL120) and ~140bp(EL140) bands.
After extracting the DNA, I refilled each well with 20ul EB, ran for additional 3 minutes. File:2009-10-16 CloneWell-1.png
Ligation: DNA 30ul 40uM adaptors 2ul 2x QuickLigase Buffer 33ul QuickLigase 1ul RT 15min -> purified with Qiaquick columns, eluted with 30ul EB.
- Perform PCR on a small fraction of the ligation products without size selection.
PCR F+R1 F+R2 F Template 2ul 2ul 2ul 2x iProof Mix 50ul 50ul 50ul Solexa_PCR_up(10uM) 2ul 2ul 2ul AmpR6.3Sol(100uM) 0.4ul - - AmpF6.3rSol(100uM) - 0.4ul - 50X SYBGI 0.4ul 0.4ul 0.4ul H2O 45ul 45ul 45ul 98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 10sec) x 8 -> (98C 10sec -> 72C 15sec) x 4 -> 72C 2min -> 15C hold. File:2009-10-17 ligation PCR.jpg A: Batch 1, 100bp B: Batch 1, 120bp C: Batch 2, 140bp D: Batch 2, 120bp
- Perform PAGE size selection on ~24ul of the ligation products in 2 wells of the 5-well TBE gel.
File:2009-10-17-PAGE-sizeSelect.png
- Perform PCR on the PAGE size selected DNA:
PCR F+R1 F+R1+R2 Template 5ul 5ul 2x iProof Mix 50ul 100ul Solexa_PCR_up(10uM) 2ul 4ul AmpR6.3Sol(100uM) 0.2ul 0.4ul AmpF6.3rSol(100uM) - 0.4ul 50X SYBGI 0.2ul 0.4ul H2O 45ul 90ul 98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 20sec) x 10 -> 72C 2min -> 15C hold. File:2009-10-19-ligation-PCR.png
Prepare amplicons for sequencing
- Pooled the Batch 2 amplicons that were amplified from non-size-select ligation products with two primer sets. Labeled them EL-120.2 & EL140.2, purified with Qiaquick columns.
- Labeled the amplicons that were amplified from size-selected ligation products using the three primers with EL-120.3, EL140.3. Purified with Qiaquick columns.
EL-120.2: 70ng/ul x 30ul EL-120.3: 68ng/ul x 30ul EL-140.2: 80ng/ul x 30ul EL-140.3: 66ng/ul x 30ul
Conclusion
- The one-adaptor protocol worked.
- After end-repair, size selection can be performed with SizeSelect gel, which is fast and easy. The fragments to select should center around 120bp.
- No size selection is necessary on the ligation products prior to PCR.
- The ligation PCR amplicons seem to be clean enough to be sequenced directly without further size selection.