Kun:LabNotes/ExonomeSeq/2009-10-16: Difference between revisions
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>Sam Chiang |
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EL-140.3: 66ng/ul x 30ul | EL-140.3: 66ng/ul x 30ul | ||
==Sequencing results== | |||
*17 millions reads were obtained from both the 140.2 and 140.3 libraries, and over 16 millions are mappable. | |||
===Variants called (140.2)=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Depth''' | |||
| align="center" style="background:#f0f0f0;"|'''2''' | |||
| align="center" style="background:#f0f0f0;"|'''8''' | |||
| align="center" style="background:#f0f0f0;"|'''10''' | |||
| align="center" style="background:#f0f0f0;"|'''12''' | |||
| align="center" style="background:#f0f0f0;"|'''20''' | |||
| align="center" style="background:#f0f0f0;"|'''30''' | |||
| align="center" style="background:#f0f0f0;"|'''40''' | |||
|- | |||
| Q=20||3124(88.3%)||2501(92.4%)||2343(92.7%)||2215(92.9%)||1768(93.9%)||1463(95.1%)||1260(95.4%) | |||
|- | |||
| Q=25||2961(90.2%)||2404(93.2%)||2252(93.4%)||2133(93.5%)||1708(94.4%)||1415(95.2%)||1217(95.5%) | |||
|- | |||
| Q=30||2831(91.2%)||2316(93.4%)||2178(93.6%)||2062(93.7%)||1649(94.5%)||1364(95.2%)||1172(95.6%) | |||
|- | |||
| Q=35||2391(92.8%)||2178(93.9%)||2047(94.0%)||1939(94.1%)||1555(94.9%)||1287(95.5%)||1100(95.9%) | |||
|- | |||
| Q=40||1961(94.1%)||1944(94.4%)||1915(94.7%)||1817(94.6%)||1471(95.2%)||1208(95.9%)||1029(96.3%) | |||
|- | |||
| | |||
|} | |||
===Variants called (140.3)=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Depth''' | |||
| align="center" style="background:#f0f0f0;"|'''2''' | |||
| align="center" style="background:#f0f0f0;"|'''8''' | |||
| align="center" style="background:#f0f0f0;"|'''10''' | |||
| align="center" style="background:#f0f0f0;"|'''12''' | |||
| align="center" style="background:#f0f0f0;"|'''20''' | |||
| align="center" style="background:#f0f0f0;"|'''30''' | |||
| align="center" style="background:#f0f0f0;"|'''40''' | |||
|- | |||
| Q=20||2747(83.5%)||2129(88.7%)||1987(89.4%)||1856(90.4%)||1521(91.5%)||1255(92.3%)||1077(92.9%) | |||
|- | |||
| Q=25||2580(85.9%)||2044(89.3%)||1910(90.1%)||1791(91.1%)||1465(92.2%)||1206(92.7%)||1034(93.2%) | |||
|- | |||
| Q=30||2442(87.7%)||1951(90.1%)||1825(91.0%)||1707(92.0%)||1399(92.9%)||1153(93.5%)||984(94.1%) | |||
|- | |||
| Q=35||2073(89.4%)||1853(90.6%)||1733(91.4%)||1624(92.3%)||1336(93.2%)||1100(93.7%)||937(94.3%) | |||
|- | |||
| Q=40||1702(90.2%)||1675(91.0%)||1637(91.9%)||1542(92.7%)||1273(93.5%)||1045(94.1%)||893(94.7%) | |||
|- | |||
| | |||
|} | |||
==Conclusion== | ==Conclusion== | ||
*The one-adaptor protocol worked. | *The one-adaptor protocol worked. |
Revision as of 18:44, 26 October 2009
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
Sequencing results
- 17 millions reads were obtained from both the 140.2 and 140.3 libraries, and over 16 millions are mappable.
Variants called (140.2)
Depth | 2 | 8 | 10 | 12 | 20 | 30 | 40 |
Q=20 | 3124(88.3%) | 2501(92.4%) | 2343(92.7%) | 2215(92.9%) | 1768(93.9%) | 1463(95.1%) | 1260(95.4%) |
Q=25 | 2961(90.2%) | 2404(93.2%) | 2252(93.4%) | 2133(93.5%) | 1708(94.4%) | 1415(95.2%) | 1217(95.5%) |
Q=30 | 2831(91.2%) | 2316(93.4%) | 2178(93.6%) | 2062(93.7%) | 1649(94.5%) | 1364(95.2%) | 1172(95.6%) |
Q=35 | 2391(92.8%) | 2178(93.9%) | 2047(94.0%) | 1939(94.1%) | 1555(94.9%) | 1287(95.5%) | 1100(95.9%) |
Q=40 | 1961(94.1%) | 1944(94.4%) | 1915(94.7%) | 1817(94.6%) | 1471(95.2%) | 1208(95.9%) | 1029(96.3%) |
Variants called (140.3)
Depth | 2 | 8 | 10 | 12 | 20 | 30 | 40 |
Q=20 | 2747(83.5%) | 2129(88.7%) | 1987(89.4%) | 1856(90.4%) | 1521(91.5%) | 1255(92.3%) | 1077(92.9%) |
Q=25 | 2580(85.9%) | 2044(89.3%) | 1910(90.1%) | 1791(91.1%) | 1465(92.2%) | 1206(92.7%) | 1034(93.2%) |
Q=30 | 2442(87.7%) | 1951(90.1%) | 1825(91.0%) | 1707(92.0%) | 1399(92.9%) | 1153(93.5%) | 984(94.1%) |
Q=35 | 2073(89.4%) | 1853(90.6%) | 1733(91.4%) | 1624(92.3%) | 1336(93.2%) | 1100(93.7%) | 937(94.3%) |
Q=40 | 1702(90.2%) | 1675(91.0%) | 1637(91.9%) | 1542(92.7%) | 1273(93.5%) | 1045(94.1%) | 893(94.7%) |
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.