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)

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  • There is one sample change for sample in colon cancer in 2014-08-03 note. Sample 6-P-6 was replace with sample 6-P-10.

1) End-repair/dA-tailing

  • 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

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

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

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

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

  • 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

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

Bisulfite conversion

Bisulfite conversion procedures