Noi/NOTES/2014-8-3
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Preparation of WGBS libraries of small fragment DNA in cancer patient plasma samples (no DNA fragmentation step)[edit]
Select plasma DNA samples for WGBS[edit]
Pancreas (UCSD-004)[edit]
Diagnosis | Conc. (ng/uL) | Volume (ul) | Yields (ng) | Selected for WGBS | Zhang lab ID |
Adenocarcinoma, moderately to poorly differentiated, c/w pT3N0MX | 0.473 | 30.00 | 14.19 | PC-P-1 | |
Adenocarcinoma, ductal type, moderately to poorly differentiated, pT3N1MX | 0.852 | 30.00 | 25.56 | YES | PC-P-2 |
Poorly differentiated adenocarcinoma, ductal type, pT3N1MX | 1.360 | 30.00 | 40.80 | YES | PC-P-3 |
Adenocarcinoma, ductal type, moderately to poorly differentiated, involving head and body of pancreas and invading into duodenal submucosa and peripancreatic soft tissue, c/w pT3N1MX | 0.340 | 30.00 | 10.20 | YES | PC-P-4 |
Poorly differentiated pancreatic ductal adenocarcinoma, invading into duodenum, consistent with pT3N0MX | 0.813 | 30.00 | 24.39 | YES | PC-P-5 |
Invasive pancreatic duct adenocarcinoma, moderately to poorly differentiated, Stage: ypT3N1MX, Stage IIB | 0.767 | 30.00 | 23.01 | YES | PC-P-6 |
Invasice ductal adenocarcinoma of the pancreas, moderately to poorly differentiated, T3N1MX, stage IIB | 0.644 | 30.00 | 19.32 | YES | PC-P-7 |
Metastatic pancreatic adenocarcinoma | 0.524 | 30.00 | 15.72 | YES | PC-P-8 |
Metastatic adenocarcinoma | 0.238 | 30.00 | 7.14 | YES | PC-P-9 |
Adenocarcinoma, moderately to poorly differentiated, c/w pT3N0MX | 0.370 | 30.00 | 11.10 | PC-P-10 |
Colon (UCSD-006)[edit]
Diagnosis | Conc. (ng/uL) | Volume (ul) | Yields (ng) | Selected for WGBS | Zhang lab ID |
Poorly differentiated adenocarcinoma, consistent with recurrent colonic adenocarcinoma | 0.200 | 30.00 | 6.00 | YES | 6-P-1 |
Metastatic malignant hemangiopericytoma/ malignant solitary fibrous tumor | 1.100 | 30.00 | 33.00 | YES | 6-P-2 |
Sigmoid colon, rectum, and anus, resection. Recurrent invasive moderately- to poorly- differentiated | 1.570 | 30.00 | 47.10 | YES | 6-P-3 |
Metastatic adenocarcinoma. | 0.889 | 30.00 | 26.67 | YES | 6-P-4 |
Metastatic mucinous adenocarcinoma. | 0.487 | 30.00 | 14.61 | YES | 6-P-5 |
Metastatic moderately-differentiated adenocarcinoma with mucinous features. | 0.249 | 30.00 | 7.47 | YES | 6-P-6 |
Adenocarcinoma | 0.247 | 30.00 | 7.41 | 6-P-7 | |
Metastatic moderately-differentiated adenocarcinoma with municous features | 0.226 | 30.00 | 6.78 | YES | 6-P-8 |
Adenocarcinoma with municous features, moderately differentiated, pT4aN2b | 0.408 | 30.00 | 12.24 | YES | 6-P-9 |
Mucinous adenocarcinoma, moderately differentiated | 0.333 | 30.00 | 9.99 | 6-P-10 |
- Note: In 2014-08-18 exp, sample 6-P-6 was replace with sample 6-P-10
Lung (UCSD-007)[edit]
Diagnosis | Conc. (ng/uL) | Volume (ul) | Yields (ng) | Selected for WGBS | Zhang lab ID |
Invasive moderately differentiated adenocarcinoma, pT26N0 | 0.692 | 30.00 | 20.76 | YES | 7-P-1 |
Poorly differentiated squamous cell carcinoma. Consistent with pT2N0MX, stage IB | 1.140 | 30.00 | 34.20 | YES | 7-P-2 |
Metastatic melanoma | 0.208 | 30.00 | 6.24 | YES | 7-P-3 |
Squamous cell carcinoma, clear cell variant, moderately differentiated | 0.202 | 30.00 | 6.06 | 7-P-4 | |
Squamous cell carcinoma, pT1bN0 | 1.110 | 30.00 | 33.30 | YES | 7-P-5 |
Adenocarcinoma, mix subtype, including 60% bronchioalveolar carcinoma, mucinous subtype with 40% invasive carcinoma, acinar and papillary subtypes | 0.233 | 30.00 | 6.99 | YES | 7-P-6 |
Invasive squamous carcinoma, moderately differentiated, consistent with pT1N0MX | 0.229 | 30.00 | 6.87 | YES | 7-P-7 |
Invasive adenocarcinoma, poorly differentiated | 0.167 | 30.00 | 5.01 | YES | 7-P-8 |
Non-small-cell carcinoma | 0.231 | 30.00 | 6.93 | 7-P-9 | |
Poorly differentiated carcinoma, possibly adenoSCC | 0.203 | 30.00 | 6.09 | YES | 7-P-10 |
1) End-repair/dA-tailing[edit]
- Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG (final conc. 1:0.1:0.1mM) in total volume 20ul
Pancreas[edit]
Zhang lab ID | Conc. (ng/ul) | Volume for 1ng | H2O | Klenow,exo-:dA:dC:dG Mix | Total | # in strip | Exp ID |
PC-P-2 | 0.852 | 1.17 | 8.83 | 10.00 | 20.00 | #4_1 | wPC-P-2 |
PC-P-3 | 1.360 | 0.74 | 9.26 | 10.00 | 20.00 | #4_2 | wPC-P-3 |
PC-P-4 | 0.340 | 2.94 | 7.06 | 10.00 | 20.00 | #4_3 | wPC-P-4 |
PC-P-5 | 0.813 | 1.23 | 8.77 | 10.00 | 20.00 | #4_4 | wPC-P-5 |
PC-P-6 | 0.767 | 1.30 | 8.70 | 10.00 | 20.00 | #4_5 | wPC-P-6 |
PC-P-7 | 0.644 | 1.55 | 8.45 | 10.00 | 20.00 | #4_6 | wPC-P-7 |
PC-P-8 | 0.524 | 1.91 | 8.09 | 10.00 | 20.00 | #4_7 | wPC-P-8 |
PC-P-9 | 0.238 | 4.20 | 5.80 | 10.00 | 20.00 | #4_8 | wPC-P-9 |
Colon[edit]
Zhang lab ID | Conc. (ng/ul) | Volume for 1ng | H2O | Klenow,exo-:dA:dC:dG Mix | Total | # in strip | Exp ID |
6-P-1 | 0.200 | 5.00 | 5.00 | 10.00 | 20.00 | #6_1 | w6-P-1 |
6-P-2 | 1.100 | 0.91 | 9.09 | 10.00 | 20.00 | #6_2 | w6-P-2 |
6-P-3 | 1.570 | 0.64 | 9.36 | 10.00 | 20.00 | #6_3 | w6-P-3 |
6-P-4 | 0.889 | 1.12 | 8.88 | 10.00 | 20.00 | #6_4 | w6-P-4 |
6-P-5 | 0.487 | 2.05 | 7.95 | 10.00 | 20.00 | #6_5 | w6-P-5 |
6-P-6 | 0.249 | 4.02 | 5.98 | 10.00 | 20.00 | #6_6 | w6-P-6 |
6-P-8 | 0.226 | 4.42 | 5.58 | 10.00 | 20.00 | #6_7 | w6-P-8 |
6-P-9 | 0.408 | 2.45 | 7.55 | 10.00 | 20.00 | #6_8 | w6-P-9 |
Lung[edit]
Zhang lab ID | Conc. (ng/ul) | Volume for 1ng | H2O | Klenow,exo-:dAdCdG Mix | Total | # in strip | Exp ID |
7-P-1 | 0.692 | 1.45 | 8.55 | 10.00 | 20.00 | #7_1 | w7-P-1 |
7-P-2 | 1.140 | 0.88 | 9.12 | 10.00 | 20.00 | #7_2 | w7-P-2 |
7-P-3 | 0.208 | 4.81 | 5.19 | 10.00 | 20.00 | #7_3 | w7-P-3 |
7-P-5 | 1.110 | 0.90 | 9.10 | 10.00 | 20.00 | #7_4 | w7-P-5 |
7-P-6 | 0.233 | 4.29 | 5.71 | 10.00 | 20.00 | #7_5 | w7-P-6 |
7-P-7 | 0.229 | 4.37 | 5.63 | 10.00 | 20.00 | #7_6 | w7-P-7 |
7-P-8 | 0.167 | 5.99 | 4.01 | 10.00 | 20.00 | #7_7 | w7-P-8 |
7-P-10 | 0.203 | 4.93 | 5.07 | 10.00 | 20.00 | #7_8 | w7-P-10 |
NTC_1 | 0.000 | 0.00 | 10.00 | 10.00 | 20.00 | NA | ntc1 |
NTC_2 | 0.000 | 0.00 | 10.00 | 10.00 | 20.00 | NA | ntc2 |
JURKAT_1 | 1.000 | 1 | 9.00 | 10.00 | 20.00 | NA | JK_1 |
JURKAT_1 | 1.000 | 1.00 | 9.00 | 10.00 | 20.00 | NA | JK_2 |
Klenow exo- and dA:dG:dC reaction mix[edit]
Components | 1x rxn | 30x rxn |
10X Tango buffer | 2.00 | 60.00 |
dA:dC:dG (20:2:2mM) | 1.00 | 30.00 |
Klenow fragment, exo- (5U/ul) | 1.00 | 30.00 |
H2O | 6.00 | 180.00 |
Total | 10.00 |
-Aliquot 32.50 of Klwnow, exo- and dA:dC:dG reaction mix to 8-tube strip
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 10ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA with multichannel pipette. Pipette few times
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- - 75C, 10min --> hold at 4C (heat lid on)
- No AMPure bead purification
2) Methylated adaptor ligation[edit]
- Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul
Exp ID | TruSeq Index | Exp ID | TruSeq Index | Exp ID | TruSeq Index |
wPC-P-2 | 1 | w6-P-1 | 13 | w7-P-1 | 11 |
wPC-P-3 | 2 | w6-P-2 | 14 | w7-P-2 | 12 |
wPC-P-4 | 4 | w6-P-3 | 15 | w7-P-3 | 21 |
wPC-P-5 | 5 | w6-P-4 | 16 | w7-P-5 | 22 |
wPC-P-6 | 6 | w6-P-5 | 27 | w7-P-6 | 3 |
wPC-P-7 | 7 | w6-P-6 | 18 | w7-P-7 | 9 |
wPC-P-8 | 8 | w6-P-8 | 19 | w7-P-8 | 23 |
wPC-P-9 | 10 | w6-P-9 | 20 | w7-P-10 | 25 |
Ligation reaction mix[edit]
Components | 1x rxn | 30x rxn |
dA-tailed DNA | 20.00 | 0.00 |
10X Tango buffer | 0.50 | 15.00 |
HC T4 DNA ligase (30units/ul) | 1.00 | 30.00 |
10mM ATP | 1.25 | 37.50 |
H2O | 1.25 | 37.50 |
Total | 24.00 |
- - Aliquot 13ul of ligation reaction mix to 8-tube strip
- - Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- - Add 4ul of ligation reaction mix with multichannel pipette
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 20h (no heat lid)
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- - Continue to bisulfite conversion
Bisulfite conversion[edit]
- Performed bisulfite conversion using EZ DNA Methylation-Gold™ Kit and did the same procedure following manufacturer's instruction and elute with 42ul Elution buffer. This kit is the same kit as LifeTech MethylCode™ Bisulfite Conversion Kit.
Prep
- - Prepare 3 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- - Mix well and spin down the tube
- - Put on thermomixer at 37C for 15min, 1,000rpm
- - Solution should be clear
- - Spin down the tube
- - Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul
- - Mix by pipetting 10X with multi-channel pipette
- - Spin down the tube
- - Incubate following below program
- - 98°C for 10 minutes (DNA denaturation)
- - 64°C for 2.5 hours (Bisulfite conversion)
- - 4°C storage for up to 20 hours or continue to desulfonation
- - Mix 600ul of Binding Buffer with 1ul of 10ng/ul tRNA. For many reaction, make multiple mix.
- - Add 601ul Binding Buffer and tRNA mix to each column
- - Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
- - Mix by pipetting 5x
- - Spin down column at 14,000 rpm for 30s
- - Discard spnt
- - Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- - Spin down column at 14,000 rpm for 30s (do not discard spnt)
- - Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
- - Spin down column at 14,000 rpm for 30s (do not discard spnt)
- - Add 200ul Wash Buffer
- - Spin down column at 14,000 rpm for 30s
- - Discard spnt
- - Add 200ul Wash Buffer
- - Spin down column at 14,000 rpm for 3min (do not discard spnt)
- - Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
- - Add 32ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- - Wait for 2min
- - Spin down column at 12,000 rpm for 1min
- - Continue to amplification (During elution step, PCR mix should be prepared)
Amplification[edit]
1st round PCR (fix 12 cycles)[edit]
Components | Conc | unit | Final conc./amount | unit | Volume (ul) | 30 rxn mix |
Bis-cvt DNA | 30.00 | 0.00 | ||||
10X Reaction buffer | 10 | X | 1 | X | 5.00 | 150.00 |
dNTP mix | 10 | mM | 0.25 | mM | 1.25 | 37.50 |
TruS_F/R | 10 | uM | 0.3 | uM | 1.50 | 45.00 |
50X SYBG | 50 | X | 0.4 | X | 0.40 | 12.00 |
PfuTurbo Cx | 2.5 | Unit/ul | 1 | unit | 1.00 | 30.00 |
H2O | 10.85 | 325.50 | ||||
Total | 50.00 |
- - Aliquot 20 + 30ul BIS-DNA template
- - Mix well
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 or 14X cycles --> 72C for 2min
- - Purify with 1.25X AMPure beads
- - Elute with 42ul EB Buffer
- - Continue to 2nd round PCR. No PAGE verification.
2nd round PCR[edit]
- Even I have done optimization for PCR cycle number for 2nd round PCR in previous experiment, I still need to do in this experiment since I want to make sure that PCR cycle number I am going to use works well for all sample. I
Quick test[edit]
Components | Conc | unit | Final conc. | unit | Volume (ul) | 60 rxn mix |
Purified 1st round DNA | 3.00 | 0.00 | ||||
5X Phusion HF buffer | 5 | X | 1 | X | 2.00 | 120.00 |
dNTP mix | 10 | mM | 0.25 | mM | 0.25 | 15.00 |
TruS_F/R | 10 | uM | 0.3 | uM | 0.30 | 18.00 |
50X SYBR | 50 | X | 0.4 | X | 0.08 | 4.80 |
Phusion HF | 2 | unit/ul | 0.10 | 6.00 | ||
H2O | 4.27 | 256.20 | ||||
Total | 10.00 |
- - Aliquot 14 + 6ul DNA template
- - Mix well.
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 7 and 9 cycles --> 72C for 2min
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 7 and 9 cycles --> 72C for 2min
- - I originally want to do 8 and 10 cycles, but qPCR curve getting saturated ~7 cycles. I then stopped at 7 and 9 cycles.
- - Added 2ul 6X loading dye to each strip tube & mix well
- - Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min.
- - To save TBE gel, I loaded only 7 cycles. I planned to load 9 cycles when 7 cycles give very faint signal.
File:ZhangLab 2 2014-08-05 13hr 58min wgbs PCP-7X.jpg File:ZhangLab 2 2014-08-05 13hr 53min wgbs 6P-7X.jpg File:ZhangLab 2 2014-08-05 14hr 02min wgbs 7P-7x.jpg
- From gel image I decided to do 2nd round PCR in larger volume at 8 cycles as Dr. Zhang suggested (1st: 12x, 2nd: 8x)
Components | Conc | unit | Final conc. | unit | Volume (ul) | 24.5 rxn mix |
Purified 1st round DNA | 30.00 | 0.00 | ||||
5X Phusion HF buffer | 5 | X | 1 | X | 20.00 | 490.00 |
dNTP mix | 10 | mM | 0.25 | mM | 2.50 | 61.25 |
TruS_F/R | 10 | uM | 0.3 | uM | 3.00 | 73.50 |
50X SYBR | 50 | X | 0.4 | X | 0.80 | 19.60 |
Phusion HF | 2 | unit/ul | 1.00 | 24.50 | ||
H2O | 42.70 | 1046.15 | ||||
Total | 100.00 |
- - Aliquot 70 + 30ul DNA template
- - Mix well.
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 8 cycles --> 72C for 2min
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 8 cycles --> 72C for 2min
- - I pool ~95ul PCR product from the two replicates and elute with 65ul EB buffer
- - I did PAGE quantification by loading 4ul of bead-purified sequencing libraries
File:ZhangLab 2 2014-08-05 17hr 52min PQ PC-P 4ul.jpg File:ZhangLab 2 2014-08-05 17hr 54min PQ 6P-4ul.jpg File:ZhangLab 2 2014-08-05 17hr 50min PQ 7P-4ul.jpg
- Note that for this WGBS library prep from patient plasma DNA and previous experiment of RRBS, I only used dA:dC:dG for end repair. I forgot that there could be the end of fragmented DNA that is not digested by MspI, so I should include dT in end repair mix. EspecialIy for WGBS of small fragment DNA in plasma, I must include all 4 bases. I then have generated the new WGBS of above samples using all 4 dAdCdGdT(10:1:1:1 ratio) with minor change of sample list.
- Continued on 2014-08-18