Noi/NOTES/2014-9-8

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Preparation of WGBS libraries of ctDNA/cfDNA from Illumina (no DNA fragmentation step)[edit]

Calendar
Sample info.

Re-quantification of cfDNA by Qubit dsDNA HS assay[edit]

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
  • 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[edit]

  • 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[edit]

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[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 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[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 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[edit]

  • 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)[edit]

Components Conc unit Final conc./amount unit Volume (ul) 6.5x rxn mix
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 5.00 32.50
dNTP mix 10 mM 0.25 mM 1.25 8.13
TruS_F/R 10 uM 0.3 uM 1.50 9.75
50X SYBG 50 X 0.4 X 0.40 2.60
PfuTurbo Cx 2.5 Unit/ul 1 unit 1.00 6.50
H2O 10.85 70.53
Total 50.00
  • Aliquot 20ul + 30ul BIS-DNA
  • Mix well
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 cycles --> 72C for 2min
File:ZhangLab 2 2014-09-10 12hr 45min 1stPCR cfDNA1-4 12cycles 2ulPCR.jpg

Discussion[edit]

  • Based on this result, I think that in the future to generate RRBS library or WGBS from ctDNA ~5ng, I do not need to do 2nd round PCR. In stead of using all 30ul of bis adaptor-ligated DNA for 50ul PCR, I can reduce amount of template and increase few more PCR cycles to get more PCR yields. In some application that do not require hight amount of libraries, I can stop PCR between 12-15 cycle based on qPCR curve. To make more amount of libraries for some downstream experiments, using lower concentration of template and increasing PCR cycle should be good enough. This can cut off bead purification step and save time. I can also try this on 1ng input.
  • However, for this experiment, I ran the gel test in the same time of bead purification. I need to go to 2nd PCR, but with fewer cycle number compare to previous experiment. I reduce the amount of template to 20% in PCR instead of 30% from total ~40ul eluted DNA from 1st round PCR.

2nd round PCR[edit]

OPTIMIZE PCR cycle number[edit]

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)[edit]

  • 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
- Quantify concentration of each library by Qubit dsDNA HS assay using 1ul of library
Sample Conc. in the Qubit Unit uL used Dilution Conc. (ng/ul) Yield in 60ul (ng)
cf1 41.6 ng/mL 1 200 8.33 499.80
cf2 25.4 ng/mL 1 200 5.07 304.20
cf3 47.2 ng/mL 1 200 9.43 565.80
cf4 48 ng/mL 1 200 9.60 576.00