Revision as of 17:54, 11 September 2014 by >Noi
Preparation of WGBS libraries of ctDNA/cfDNA from Illumina (no DNA fragmentation step)
Calendar
Sample info.
Re-quantification of cfDNA by Qubit dsDNA HS assay
Sample
|
Conc. By Qubit (ng/ul)
|
Amount (ng)
|
cfDNA |
7.09 |
49.63
|
NORM1-02 P1 |
1.66 |
33.2
|
NORM1-03 P1 |
0.77 |
15.3
|
COLO2-03 |
1.11 |
22.2
|
- One sample from Xuyu (received 2014-08-12). I added more TE buffer and re-quantified concentration (7.09ng/ul). The sample was labeled cdDNA without additional further information.
- Note that Alan has taken 10ng of each sample for WGBS library prep . I have no time to wait until the library prep done, so I perform experiment in parallel using 5ng of input DNA to make sure that I can continue BSPP capture and get the library ready for Illumina by this Friday.
1) End-repair/dA-tailing
- Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG:dT (final conc. 1:0.1:0.1:0.1mM) in total volume 20ul
Zhang lab ID
|
Conc. (ng/ul)
|
Volume for 5ng
|
Exp ID
|
H2O
|
Klenow,exo-:dAdCdG Mix
|
Total
|
Exp ID
|
cfDNA |
7.09 |
0.71 |
cf1 |
9.29 |
10.00 |
20.00 |
cf1
|
NORM1-02 P1 |
1.66 |
3.01 |
cf2 |
6.99 |
10.00 |
20.00 |
cf2
|
NORM1-03 P1 |
0.77 |
6.49 |
cf3 |
3.51 |
10.00 |
20.00 |
cf3
|
COLO2-03 |
1.11 |
4.50 |
cf4 |
5.50 |
10.00 |
20.00 |
cf4
|
NTC |
0.00 |
0.00 |
NTC |
10.00 |
10.00 |
20.00 |
NTC
|
Klenow exo- and dA:dG:dC:dT reaction mix
Components
|
1x rxn
|
5.5x rxn
|
10X Tango buffer |
2.00 |
11.00
|
dA:dC:dG:dT (20:2:2:2mM) |
1.00 |
5.50
|
Klenow fragment, exo- (5U/ul) |
1.00 |
5.50
|
H2O |
6.00 |
33.00
|
Total |
10.00 |
|
- - Add H2O to each tube
- - Add 5ng of DNA to each tube
- - Add 10ul of Klwnow, exo- and dA:dC:dG:dT 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
|
5.5x rxn
|
dA-tailed DNA |
20.00 |
0.00
|
10X Tango buffer |
0.50 |
2.50
|
HC T4 DNA ligase (30units/ul) |
1.00 |
5.00
|
10mM ATP |
1.25 |
6.25
|
H2O |
1.25 |
6.25
|
Total |
24.00 |
|
Exp ID
|
Sample ID
|
TruSeq Index
|
cf1 |
cfDNA |
1
|
cf2 |
NORM1-02 P1 |
2
|
cf3 |
NORM1-03 P1 |
4
|
cf4 |
COLO2-03 |
5
|
NTC |
NTC |
6
|
- - 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, 18h (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 tube 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
- In previous experiment, I worried about losing small ligated DNA ~200bp, so I used 1.25X AMPure beads to purify 1st round PCR. In this library prep, I want to generate WGBS library for BSPP capture. I want the inser size at least 200bp for capture, so I can remove all the ligate DNA below 200bp by using 1X AMPure beads.
1st round PCR (fix 12 cycles)
File:ZhangLab 2 2014-09-10 12hr 45min 1stPCR cfDNA1-4 12cycles 2ulPCR.jpg
2nd round PCR (fix 12 cycles)
OPTIMIZE PCR cycle number
File:ZhangLab 2 2014-09-10 13hr 19min 2ndPCR cfDNA 6-7cycles.jpg
- It seemed to be too saturated for 6 and 7 cycles. I the tried 4 and 5 cycles and also reduce 1st DNA template in PCR reaction from 30% to 20%
File:ZhangLab 2014-09-10 2ndPCR cfDNA 4-5cycles 2ul-in-10.jpg
2nd round PCR (large volume)
- From gel image above, the amplification for sample cf1 and cf4 were still saturated, so I ran 3 cycles for these two samples and 4 cycles for sample cf2 and cf3 in total 170ul (mix before split into 3x of 55ul)
Components
|
Conc
|
unit
|
Final conc.
|
unit
|
Volume (ul)
|
6 rxn mix
|
Purified 1st round DNA |
|
|
|
|
34.00 |
|
5X Phusion HF buffer |
5 |
X |
1 |
X |
34.00 |
204.00
|
dNTP mix |
10 |
mM |
0.25 |
mM |
4.25 |
25.50
|
TruS_F/R |
10 |
uM |
0.3 |
uM |
5.10 |
30.60
|
50X SYBR |
50 |
X |
0.4 |
X |
1.36 |
8.16
|
Phusion HF |
2 |
unit/ul |
|
|
1.70 |
10.20
|
H2O |
|
|
|
|
89.59 |
537.54
|
Total |
|
|
|
|
170.00 |
|
- - Aliquot 136 + 34ul DNA template
- - Mix well. Split 3X of 55ul for each condition
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [cf1 &cf4: 3X, cf2 & cf3: 4 X] cycles --> 72C for 2min
- - Perform one round of 1x volume AMPure bead purification because the intensity of adaptor dimer wwas not very high and elute in ~65ul EB buffer
- - Verify libraries by loading 1ul in 6% TBE gel. The 4 samples of 6-P-2 - 6-P-5 WGBS libraries that will be included for BSPP capture were also verified.
File:ZhangLab 2 2014-09-11 01hr 14min PAGE-verify cf1-4 6P-2-5.jpg