Noi/NOTES/2014-8-18

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Preparation of WGBS libraries of small fragment DNA in cancer patient plasma samples (no DNA fragmentation step, REPEAT with dAdGdCdT for end repair)[edit]

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1) End-repair/dA-tailing[edit]

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

Pancreas[edit]

Zhang lab ID Conc. (ng/ul) Volume for 1ng Exp ID H2O Klenow,exo-:dAdCdG Mix Total Exp ID
PC-P-2 0.852 1.17 wPC-P-2 8.83 10.00 20.00 wPC-P-2
PC-P-3 1.360 0.74 wPC-P-3 9.26 10.00 20.00 wPC-P-3
PC-P-4 0.340 2.94 wPC-P-4 7.06 10.00 20.00 wPC-P-4
PC-P-5 0.813 1.23 wPC-P-5 8.77 10.00 20.00 wPC-P-5
PC-P-6 0.767 1.30 wPC-P-6 8.70 10.00 20.00 wPC-P-6
PC-P-7 0.644 1.55 wPC-P-7 8.45 10.00 20.00 wPC-P-7
PC-P-8 0.524 1.91 wPC-P-8 8.09 10.00 20.00 wPC-P-8
PC-P-9 0.238 4.20 wPC-P-9 5.80 10.00 20.00 wPC-P-9

Colon[edit]

Zhang lab ID Conc. (ng/ul) Volume for 1ng Exp ID H2O Klenow,exo-:dAdCdG Mix Total Exp ID
6-P-1 0.200 5.00 w6-P-1 5.00 10.00 20.00 w6-P-1
6-P-2 1.100 0.91 w6-P-2 9.09 10.00 20.00 w6-P-2
6-P-3 1.570 0.64 w6-P-3 9.36 10.00 20.00 w6-P-3
6-P-4 0.889 1.12 w6-P-4 8.88 10.00 20.00 w6-P-4
6-P-5 0.487 2.05 w6-P-5 7.95 10.00 20.00 w6-P-5
6-P-10 0.333 3.00 6-P-10 7.00 10.00 20.00 6-P-10
6-P-8 0.226 4.42 w6-P-8 5.58 10.00 20.00 w6-P-8
6-P-9 0.408 2.45 w6-P-9 7.55 10.00 20.00 w6-P-9

Lung[edit]

Zhang lab ID Conc. (ng/ul) Volume for 1ng Exp ID H2O Klenow,exo-:dAdCdG Mix Total Exp ID
7-P-1 0.692 1.45 w7-P-1 8.55 10.00 20.00 w7-P-1
7-P-2 1.140 0.88 w7-P-2 9.12 10.00 20.00 w7-P-2
7-P-3 0.208 4.81 w7-P-3 5.19 10.00 20.00 w7-P-3
7-P-5 1.110 0.90 w7-P-5 9.10 10.00 20.00 w7-P-5
7-P-6 0.233 4.29 w7-P-6 5.71 10.00 20.00 w7-P-6
7-P-7 0.229 4.37 w7-P-7 5.63 10.00 20.00 w7-P-7
7-P-8 0.167 5.99 w7-P-8 4.01 10.00 20.00 w7-P-8
7-P-10 0.203 4.93 w7-P-10 5.07 10.00 20.00 w7-P-10
NTC_1 0.000 0.00 ntc1 10.00 10.00 20.00 ntc1
NTC_2 0.000 0.00 ntc2 10.00 10.00 20.00 ntc2
PC-S_5 1.580 0.63 wPC-S-5 9.37 10.00 20.00 wPC-S-5
PC-S_8 0.953 1.05 wPC-S-8 8.95 10.00 20.00 wPC-S8
  • I also included 2 matched paired serum samples for pancreatic cancer patients (PC-S-5 and PC-S-8) to compare the level of small fragment DNA. I expected to get lower amount of DNA in the library from serum sample than plasma as the total amount of DNA in serum is derived from lysed WBC. This can also confirm that the library is generated from small fragment DNA.

Klenow exo- and dA:dG:dC reaction mix[edit]

Components 1x rxn 28x rxn
10X Tango buffer 2.00 56.00
dA:dC:dG:dT (20:2:2:2mM) 1.00 28.00
Klenow fragment, exo- (5U/ul) 1.00 28.00
H2O 6.00 168.00
Total 10.00

- Aliquot 32 of Klwnow, exo- and dA:dC:dG:dT 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 Exp ID TruSeq Index
wPC-P-2 1 w6-P-1 13 w7-P-1 11 ntc1 3
wPC-P-3 2 w6-P-2 14 w7-P-2 12 ntc2 9
wPC-P-4 4 w6-P-3 15 w7-P-3 21 wPC-S_5 23
wPC-P-5 5 w6-P-4 16 w7-P-5 22 wPC-S_8 25
wPC-P-6 6 w6-P-5 27 w7-P-6 3
wPC-P-7 7 w6-P-10 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]

Bisulfite conversion procedures

Amplification[edit]

  • Repeat all procedure exactly the same as previous experiment on 2014-08-03

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 --> 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]

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 8 cycles --> 72C for 2min
- 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. I loaded only PCR at 7 cycles first.

- Gel1: LM PC-Pv(8) PC-S-5 PC-S-8
- Gel2: LM 6-P (8) NTC1 NTC2
- Gel3: LM 7-P (8) NTCPCR

File:ZhangLab 2 2014-08-20 12hr 20min test-2ndPCR-pat-7x gel1.jpg File:ZhangLab 2 2014-08-20 12hr 20min test-2ndPCR-pat-7x gel2.jpg

File:ZhangLab 2 2014-08-20 12hr 20min test-2ndPCR-pat-7x gel3.jpg
  • From quick test qPCR, I continue to do qPCR of the remaining 1st round PCR in a large volume ( 120 ul total) and reduce PCR cycle number to 6 cycles
Components Conc unit Final conc. unit Volume (ul) 27 rxn mix
Purified 1st round DNA 36.00 0.00
5X Phusion HF buffer 5 X 1 X 24.00 648.00
dNTP mix 10 mM 0.25 mM 3.00 81.00
TruS_F/R 10 uM 0.3 uM 3.60 97.20
50X SYBR 50 X 0.4 X 0.96 25.92
Phusion HF 2 unit/ul 1.20 32.40
H2O 51.24 1383.48
Total 120.00
- Aliquot 84 + 36ul DNA template
- Mix well. Split 38ul X3
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 6 cycles --> 72C for 2min
- I pool ~110ul PCR product from the two replicates, purify with 2 rounds of 1X AMPure beads (fist round elute with EB buffer 60ul. 2nd round elute with 65ul EB buffer.
  • Note that the band of PCR after reducing PCR cycle to 6 cycles was different from the gel images above as the band around 300bp was stronger and less smear from over-amplification. Some examples was shown in the gel image below.
File:ZhangLab 2 2014-09-11 01hr 14min PAGE-verify cf1-4 6P-2-5.jpg
  • I will use Qbit dsDNA HS assay for library concentration quantification.

Qubit dsDNA HS quantification of WGBS libraries from cancer patient ctDNA[edit]

Sample Conc. in the Qubit Unit uL used Dilution Conc. (ng/ul) Volume (ul) Total amount (ng) Used for prev. exp (ng) Amount left (ng) > 400ng
PC-P-2 88.1 ng/mL 2 100 8.81 60.00 528.60 0.00 528.60 YES
PC-P-3 138 ng/mL 2 100 13.80 60.00 828.00 0.00 828.00 YES
PC-P-4 90.3 ng/mL 2 100 9.03 60.00 541.80 0.00 541.80 YES
PC-P-5 85.9 ng/mL 2 100 8.59 60.00 515.40 0.00 515.40 YES
PC-P-6 114 ng/mL 2 100 11.40 60.00 684.00 0.00 684.00 YES
PC-P-7 122 ng/mL 2 100 12.20 60.00 732.00 0.00 732.00 YES
PC-P-8 103 ng/mL 2 100 10.30 60.00 618.00 0.00 618.00 YES
PC-P-9 115 ng/mL 2 100 11.50 60.00 690.00 0.00 690.00 YES
6-P-1 95.8 ng/mL 2 100 9.58 60.00 574.80 0.00 574.80 YES
6-P-2 126 ng/mL 2 100 12.60 60.00 756.00 100.00 656.00 YES
6-P-3 61.6 ng/mL 2 100 6.16 60.00 369.60 100.00 269.60 NO
6-P-4 104 ng/mL 2 100 10.40 60.00 624.00 100.00 524.00 NO
6-P-5 54.3 ng/mL 2 100 5.43 60.00 325.80 100.00 225.80 NO
6-P-10 67.6 ng/mL 2 100 6.76 60.00 405.60 0.00 405.60 YES
6-P-8 77.1 ng/mL 2 100 7.71 60.00 462.60 0.00 462.60 YES
6-P-9 123 ng/mL 2 100 12.30 60.00 738.00 0.00 738.00 YES
7-P-1 38.2 ng/mL 2 100 3.82 60.00 229.20 0.00 229.20 NO
7-P-2 86.7 ng/mL 2 100 8.67 60.00 520.20 0.00 520.20 YES
7-P-3 56.5 ng/mL 2 100 5.65 60.00 339.00 0.00 339.00 NO
7-P-5 84 ng/mL 2 100 8.40 60.00 504.00 0.00 504.00 YES
7-P-6 78 ng/mL 2 100 7.80 60.00 468.00 0.00 468.00 YES
7-P-7 52.7 ng/mL 2 100 5.27 60.00 316.20 0.00 316.20 NO
7-P-8 80.5 ng/mL 2 100 8.05 60.00 483.00 0.00 483.00 YES
7-P-10 43.4 ng/mL 2 100 4.34 60.00 260.40 0.00 260.40 NO