Revision as of 17:24, 11 September 2014 by >Noi
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