Noi/NOTES/2014-7-31

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

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[edit]

  • 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[edit]

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[edit]

  • Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul

Ligation reaction mix[edit]

Components 1x rxn 3x rxn
dA-tailed DNA 20.00 0.00
10X Tango buffer 0.50 1.50
HC T4 DNA ligase (30units/ul) 1.00 3.00
10mM ATP 1.25 3.75
H2O 1.25 3.75
Total 24.00
Sample TruSeq Index
PC-P-1 21
PC-P-2 22
NTC 22
- 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
- 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

4) Bisulfite conversion[edit]

  • 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 1 tubes of complete CT Conversion Reagent by adding 850ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1000rpm
- Solution should be clear
- Spin down the tube
- Add 125ul 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
- Add 601ul Binding Buffer to each column (with 10ng of tRNA in each 601ul Binding Buffer)
- 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 30ul 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[edit]

  • Minor changes: Since 1ng of DNA input was not fragmented even there is a fraction of small fragments, I increase 1st round PCR from 12 cycles to 14 cycles to increase the amplified template.
  • To avoid losing small fragments below 200bp, I purified 1st round PCR with 1.25X AMPure bead. I tried not to increase the beads up to 1.5X or 2X since I did not want to get more adaptor dimers contamination in the next round PCR. I should have tested doing single round PCR for ~15-20 cycles and see if I can get sequencing libraries without 2nd round PCR.

1st round PCR (fix 14 cycles)[edit]

Components Conc unit Final conc./amount unit Volume (ul) 4.5 rxn mix
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 5.00 22.50
dNTP mix 10 mM 0.25 mM 1.25 5.63
TruS_F/R 10 uM 0.3 uM 1.50 6.75
50X SYBG 50 X 0.4 X 0.40 1.80
PfuTurbo Cx 2.5 Unit/ul 1 unit 1.00 4.50
H2O 10.85 48.83
Total 50.00
- Aliquot 20 + 30ul BIS-DNA template
- Mix well
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 14 cycles --> 72C for 2min
- Purify with 1.25X AMPure beads (incubate DNA with beads 30min, the rest steps are the same as previous experiment)
- Elute with 42ul EB Buffer
- Continue to 2nd round PCR. No PAGE verification.

2nd round PCR[edit]

OPTIMIZE PCR cycle number[edit]

Components Conc unit Final conc. unit Volume (ul) 17 rxn mix
Purified 1st round DNA 3.00 0.00
5X Phusion HF buffer 5 X 1 X 2.00 34.00
dNTP mix 10 mM 0.25 mM 0.25 4.25
TruS_F/R 10 uM 0.3 uM 0.30 5.10
50X SYBR 50 X 0.4 X 0.08 1.36
Phusion HF 2 unit/ul 0.10 1.70
H2O 4.27 72.59
Total 10.00
- Aliquot 28 + 12ul DNA template
- Mix well. Split 4X of 9ul
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [8X, 9X, 10X, 12X] cycles --> 72C for 2min
- Added 3ul 6X loading dye to each strip tube & mix well
- Load 6ul PCR/dye mix in 6% TBe gel and run at 250 volts for 21 min
  • I planned to optimize PCR cycle number between 10-16 cycles by doing PCR in small volume (~10ul). However, based on qPCR curves saturated vary quick at ~8x. so I stopped reaction at 8X, 9X, 10X, and 12X cycles.
  • It looked like I should test PCR cycles at lower cycle number. I repeat experiment with the same condition at 6X and 7X cycles.
File:ZhangLab 2 2014-08-01 noMspIRRBS 6-7cycles.jpg File:ZhangLab 2 2014-08-01 noMspIRRBS 8-9cycles.jpg

File:ZhangLab 2 2014-08-01 noMspIRRBS 10-12cycles.jpg
1: PC-P-1
2: PC-P-2
3: NTC
4: NTC PCR