Noi/NOTES/2014-7-31
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Preparation of WGBS libraries of small fragment DNA in plasma samples (TEST)[edit]
- 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
- 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