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.