Noi/NOTES/2014-7-31

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Preparation of WGBS libraries of small fragment DNA in plasma samples (TEST)

Calendar

  • Rationale: Will be added
  • Aim: To generate WGBS libraries of ctDNA without fragmentation (either enzymatic or shearing) to exclude all large fragment DNA derived from lysed blood cell in sequencing library.
  • I chose two samples of PC-P samples which have total DNA more than 10ng to test the protocol
    • PC-P-1 (conc. 0.473 ng/ul, total amount 14.19ng)
    • PC-P-2 (conc. 0.852 ng/ul, total amount 25.56ng)

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
Sample Conc. (ng/ul) Volume for 1ng H2O Klenow,exo-:dA:dC:dG Mix Total
PC-P-1 0.47 2.11 7.69 10.20 20.00
PC-P-2 0.85 1.17 8.63 10.20 20.00
NTC 0.00 0.00 9.80 10.20 20.00

Klenow exo- and dA:dG:dC reaction mix

Components 1x rxn 3.50x rxn
10X Tango buffer 2.00 7.00
dA:dC:dG (20:2:2mM) 1.00 3.50
Klenow fragment, exo- (5U/ul) 1.00 3.50
H2O 6.20 21.70
Total 10.20
- Add H2O to each tube
- Add 1ng of DNA to each tube
- Add 10.2ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA. 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