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)

Calendar

Select plasma DNA samples for WGBS

Pancreas (UCSD-004)

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)

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)

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

  • Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG (final conc. 1:0.1:0.1mM) in total volume 20ul

Pancreas

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

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

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

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

  • 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

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

  • 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

1st round PCR (fix 12 cycles)

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

  • 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

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.