Revision as of 19:13, 13 September 2014 by >Noi
Preparation of WGBS libraries of small fragment DNA in cancer patient plasma samples (no DNA fragmentation step)[edit]
Calendar
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
|
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
|
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
|
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
- - 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
- - 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