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

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

Ligation reaction mix

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

  • 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

  • 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)

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

OPTIMIZE PCR cycle number

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