Noi/NOTES/2014-11-30

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Preparation of WGBS of cfDNA from plasma of cancer patients using KAPA Hyper Prep kit[edit]

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  • As I mentioned before in previous experiment that the first batch of WGBS libraries from cfDNA of normal and cancer patients were generated by RRBS method without DNA fragmentation. This might lead to the incomplete of libraries as RRBS protocol uses only Klenow fragment, eco- but lack T4 DNA Pol and T4 PNK enzyme. Dr. Zhang suggested to generated WGBS libraries of cfDNA from cancer patients using KAPA Hyper Prep Kit as I did test experiment on 2014-10-24.
  • I also increased DNA input as much as possible for each sample to be able to capture all individual unique molecule from cancer cells in the DNA pool of cfDNA.
Sample Sample conc. (ng/uL) Volume (ul) Yields (ng) Amont left (ng) Amount used in WGBS/ KAPAHyper Volume (ul) Sample H2O to 25ul End repair/dA-tailing rxn mix Total (ul)
PCP-2 0.852 30 25.56 22.06 20 23.47 PCP-2 1.53 5 30.00
PCP-3 1.360 30 40.80 37.30 20 14.71 PCP-3 10.29 5 30.00
PCP-4 0.340 30 10.20 6.70 6.5 19.12 PCP-4 5.88 5 30.00
PCP-5 0.813 30 24.39 20.89 20 24.60 PCP-5 0.40 5 30.00
PCP-6 0.767 30 23.01 19.51 15 19.56 PCP-6 5.44 5 30.00
PCP-7 0.644 30 19.32 15.82 15 23.29 PCP-7 1.71 5 30.00
PCP-8 0.524 30 15.72 12.22 10 19.08 PCP-8 5.92 5 30.00
PCP-9 0.238 30 7.14 3.64 3.5 14.71 PCP-9 10.29 5 30.00
6P-1 0.200 30 6 2.50 2.5 12.50 6P-1 12.50 5 30.00
6P-2 1.100 30 33 29.50 20 18.18 6P-2 6.82 5 30.00
6P-3 1.570 30 47.1 43.60 20 12.74 6P-3 12.26 5 30.00
6P-4 0.889 30 26.67 23.17 20 22.50 6P-4 2.50 5 30.00
6P-5 0.487 30 14.61 11.11 10 20.53 6P-5 4.47 5 30.00
6P-8 0.226 30 6.78 3.28 3.2 14.16 6P-8 10.84 5 30.00
6P-9 0.408 30 12.24 8.74 8.5 20.83 6P-9 4.17 5 30.00
6P-10 0.333 30 9.99 6.49 6.4 19.22 6P-10 5.78 5 30.00
7P-1 0.692 30 20.76 17.26 15 21.68 7P-1 3.32 5 30.00
7P-2 1.14 30 34.2 30.7 20 17.54 7P-2 7.46 5 30.00
7P-3 0.208 30 6.24 2.74 2.5 12.02 7P-3 12.98 5 30.00
7P-5 1.11 30 33.3 29.8 20 18.02 7P-5 6.98 5 30.00
7P-6 0.233 30 6.99 3.49 3.4 14.59 7P-6 10.41 5 30.00
7P-7 0.229 30 6.87 3.37 3.3 14.41 7P-7 10.59 5 30.00
7P-8 0.167 30 5.01 1.51 1.5 8.98 7P-8 16.02 5 30.00
7P-10 0.203 30 6.09 2.59 2.5 12.32 7P-10 12.68 5 30.00
NTC NA NA NA NA NA NA NTC 25.00 5 30.00

End repair & dA-tailing[edit]

End repair & dA-tailing reaction mix[edit]

  • I prepared 5ul of End repair & dA-tailing reaction mix for each reaction
  • Maximum volume for End-repair/A-tailing = 25ul
Components Volume half rxn 26 rxn
End repair/dA-tailing buffer 7.00 3.50 91.00
End repair/dA-tailing enzyme mix 3.00 1.50 39.00
Total 10.00 5.00
- Mix well
- 20C, 30min
- 65C, 30min,
- Hold 4C
- Proceed to Adaptor ligation immediately

Adaptor ligation[edit]

  • Since I have different amount of DNA input, so I added different conc. of adaptor based on the range of input amount.
  • Dilute some adaptor to 1.5uM (10X) --> by mixing 3ul of adaptor with 27ul of 1X Stoffel Buffer
Tube # TruSeq Index Stock 15uM 1.11X Stoffel Buffer
#1 Ind_3 3 27
#2 Ind_7 3 27
#3 Ind_8 3 27
#4 Ind_9 3 27
#5 Ind_13 3 27
#6 Ind_14 3 27
#7 Ind_15 3 27
#8 Ind_16 3 27
#9 Ind_20 3 27
#10 Ind_22 3 27
#11 Ind_23 3 27
#12 Ind_25 3 27
#13 Ind_27 3 27
  • Actually H2O can be used to dilute adaptor
Sample End repair/ dA-tailed DNA (ul) Amount (ng) used in WGBS/ KAPAHyper Adaptor stock (nM) Volume (ul) Sample H2O Indx Final Adaptor conc. (nM) Ligation rxn mix (ul) Total
PCP_2 30 20 15000 1 PCP_2 4 1 273 20 55
PCP_3 30 20 15000 1 PCP_3 4 2 273 20 55
PCP_4 30 6.5 1500 5 PCP_4 0 3 136 20 55
PCP_5 30 20 15000 1 PCP_5 4 4 273 20 55
PCP_6 30 15 15000 1 PCP_6 4 5 273 20 55
PCP_7 30 15 15000 1 PCP_7 4 6 273 20 55
PCP_8 30 10 1500 5 PCP_8 0 7 136 20 55
PCP_9 30 3.5 1500 2.5 PCP_9 2.5 8 68 20 55
6P-1 30 2.5 1500 2.5 6P-1 2.5 9 68 20 55
6P-2 30 20 15000 1 6P-2 4 10 273 20 55
6P-3 30 20 15000 1 6P-3 4 11 273 20 55
6P-4 30 20 15000 1 6P-4 4 12 273 20 55
6P-5 30 10 1500 5 6P-5 0 13 136 20 55
6P-8 30 3.2 1500 2.5 6P-8 2.5 14 68 20 55
6P-9 30 8.5 1500 5 6P-9 0 15 136 20 55
6P-10 30 6.4 1500 5 6P-10 0 16 136 20 55
7P-1 30 15 15000 1 7P-1 4 18 273 20 55
7P-2 30 20 15000 1 7P-2 4 19 273 20 55
7P-3 30 2.5 1500 2.5 7P-3 2.5 20 68 20 55
7P-5 30 20 15000 1 7P-5 4 21 273 20 55
7P-6 30 3.4 1500 2.5 7P-6 2.5 22 68 20 55
7P-7 30 3.3 1500 2.5 7P-7 2.5 23 68 20 55
7P-8 30 1.5 1500 2.5 7P-8 2.5 25 68 20 55
7-P-10 30 2.5 1500 2.5 7P-10 2.5 27 68 20 55
NTC 30 NA 1500 5 NTC 0 27 136 20 55

Adaptor ligation reaction mix[edit]

Components Volume half rxn 26x rxn
Ligation buffer 30.00 15.00 390.00
DNA ligase 10.00 5.00 130.00
Total 40.00 20.00
1:20 diluted adaptor (714nM) Varied Varied
Total 110.00 55.00