Revision as of 17:10, 28 October 2014 by >Noi
Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit & NEXTflex Cell Free DNA-Seq kit
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- REPEAT experiment of 2014-10-22
- KAPA Hyper Prep Kit Manual
- The two experiments were performed in parallel. I will list the procedures of KAPA Hyper Prep kit through adaptor ligation.
- I then will list the procedure of NEXTflex Cell Free DNA-Seq kit
- The down stream procedures, including bead purification of adaptor-ligated DNA, bisulfite conversion and amplification, were performed together and will be listed in the last part of this lab note.
KAPA Hyper Prep kit Experiment
Sample list
No. Sample IDs Conc. (ng/ul)
#1 NC-2 0.325
#2 NC-3 0.538
#3 6P-2 1.10
#4 6P-3 1.57
- Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally.
- KAPA Hyper Prep reaction set up (exactly in the instruction):
- - 50ul of fragmented DNA
- - 7ul of End repair & dA-tailing buffer
- - 3ul of End repair & dA-tailing enzyme mix
- - Total reaction: 60ul
End repair & dA-tailing
End repair & dA-tailing reaction mix
Components
|
Volume
|
half rxn
|
5.2x rxn
|
End repair/dA-tailing buffer |
7.00 |
3.50 |
18.20
|
End repair/dA-tailing enzyme mix |
3.00 |
1.50 |
7.80
|
Total |
10.00 |
5.00 |
|
Reaction set up
Sample IDs
|
Conc. (ng/ul)
|
Volume for 1ng (ul)
|
End repair/dA-tailing rxn mix
|
H2O (ul)
|
Total (ul)
|
NC-2 |
0.33 |
3.08 |
5.00 |
21.92 |
30.00
|
NC-3 |
0.54 |
1.86 |
5.00 |
23.14 |
30.00
|
6P-2 |
1.10 |
0.91 |
5.00 |
24.09 |
30.00
|
6P-3 |
1.57 |
0.64 |
5.00 |
24.36 |
30.00
|
NTC |
0.00 |
0.00 |
5.00 |
25.00 |
30.00
|
- - Mix well
- - 20C, 30min
- - 65C, 30min,
- - Hold 4C
- - Proceed to Adaptor ligation immediately
Adaptor ligation
Adaptor ligation reaction mix
Components
|
Volume
|
half rxn
|
5.2x rxn
|
Ligation buffer |
30.00 |
15.00 |
78.00
|
DNA ligase |
10.00 |
5.00 |
26.00
|
H2O |
8.00 |
4.00 |
20.80
|
Total |
48.00 |
24.00 |
|
Adaptor ligation reaction set up
Components
|
Volume (ul)
|
End repair & dA-tailing rxn product |
30.00
|
1:20 diluted adaptor (750nM) |
1.00
|
Adaptor ligation rxn mix |
24.00
|
Total |
55.00
|
- I used 1:20 diluted Truseq adaptor prepared in June 2014
KAPA Hyper Prep indexing
Sample IDs
|
Index
|
NC-2 |
11
|
NC-3 |
12
|
6P-2 |
21
|
6P-3 |
22
|
NTC |
11
|
- - Add 1ul of 1:20 diluted M-adaptor
- - Add 24ul of adaptor ligation reaction mix
- - Mix well
- - 20C, 15min
- - Continue to SPRI clean up immediately
- Final conc. of adaptor in 55ul reaction is ~13nM, which is very close to 14nM recommended by the kit.
NEXTflex Cell Free DNA Seq Kit
Sample list (the same as above)
No. Sample IDs Conc. (ng/ul)
#1 NC-2 0.325
#2 NC-3 0.538
#3 6P-2 1.10
#4 6P-3 1.57
- Note: I also reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally.
- NEXTflex Cell Free DNA Seq reaction set up (exactly in the instruction):
- - 32ul of cell-free DNA
- - 15ul of NEXTflex End-Repair & Adenylation Buffer mix
- - 3ul of NEXTflex End-Repair & Adenylation Enzyme mix
- - Total reaction: 50ul
End repair & Adenylation
End repair & Adenylation reaction mix
Components
|
Volume
|
half rxn
|
5.2x rxn
|
End repair & Adenylation buffer mix |
15.00 |
7.50 |
39.00
|
End repair & Adenylation enzyme mix |
3.00 |
1.50 |
7.80
|
Total |
18.00 |
9.00 |
|
Reaction set up
Sample IDs
|
Conc. (ng/ul)
|
Volume for 1ng (ul)
|
End repair/dA-tailing rxn mix
|
H2O (ul)
|
Total (ul)
|
NC-2 |
0.33 |
3.08 |
9.00 |
12.92 |
25.00
|
NC-3 |
0.54 |
1.86 |
9.00 |
14.14 |
25.00
|
6P-2 |
1.10 |
0.91 |
9.00 |
15.09 |
25.00
|
6P-3 |
1.57 |
0.64 |
9.00 |
15.36 |
25.00
|
NTC |
0.00 |
0.00 |
9.00 |
16.00 |
25.00
|
- - Mix the reaction well
- - 22C, 20min
- - 72C, 20min,
- - Hold 4C
- - Proceed to Adaptor ligation immediately
Adaptor ligation
- For this kit, it suggest to dilute the 25uM adaptor 1:8 or 9X (2777.78nM) and use 2.5ul in total reaction 100ul. The final conc. of adaptor in ligation reaction is 69.4nM, which is ~5 time more adaptor concentration suggested by KAPA Hyper Prep kit.
- Truseq adaptor concentration is 15uM. I diluted it to 2777.78nM by mixing 2ul of 15uM adaptor with 8.8ul H2O and use 1.25ul in total 50ul of ligation reaction.
Adaptor ligation reaction set up
NEXTflex indexing
Sample IDs
|
Index
|
NC-2 |
6
|
NC-3 |
7
|
6P-2 |
8
|
6P-3 |
10
|
NTC |
6
|
- The Enzyme mix is already in buffer, so I
- - Add 1.25ul of 2777.7nM TruSeq adaptor
- - Add 23.75ul of NEXTflex Ligase enzyme mix directly to dA-tailed DNA
- - Mix well
- - 22C, 15min
- - Continue to SPRI clean up immediately
1X AMPure bead purification
- The instruction suggested to use 0.8X of AMPure bead for clean up, but I use 1X of AMPure bead
- KAPA Hyper prep kit: does 1 round of 1X AMPure bead purification
- NEXTflext: does 2 round of 1X AMPure bead purification. I guess due to the high amount of adaptor in ligation reaction (5X more than KAPA Hyper)
- Aliquot AMPure bead in 1.5mL tube
- - Add 55ul (for KAPA Hyper) or 50ul (for NEXTflex) AMPure bead. Mix by pipetting 10x
- - Sit for 15min (NEXTflex suggested 5min)
- - Transfer to sit on magnet for 5min
- - Wash twice with 200ul freshly prepared 80% EtOH
- - Dry the bead for 3-5min
- - Resuspend with 21ul EB buffer
- - For NEXTflex, I resuspended with 50ul EB and repeat 1X vol AMPure bead purification following the procedures above.
- - Continue to bisulfite conversion immediately
Bisulfite conversion
- I performed bisulfite conversion using the same procedure following manufacturer's instruction of EZ DNA Methylation-Lightning kit and elute with 32ul elution buffer.
Prep
- - Aliquot 6.12mL of binding buffer & mix with 10.2ul of 10ng/ul tRNA
- - Aliquot 5.1mL of Washing buffer
- - Aliquot 2.1mL of Desulfonation buffer
- - Aliquot 340ul of Elution buffer
Amplification
- Since from the previous experiment, I saw very low amplification by doing 1 round of PCR at high cycle number. I would follow the old optimize protocol by doing 2 round PCR. the amplification was lower than the WGBS using Thermoscientific system I did before. I would increase 1 round PCR to 15 cycle instead of 12cycles. Do 1X AMPure bead purification and continue to 2nd round PCR.
- I use amplification primers in our lab designed for TruSeq adaptor.
1st round PCR
Components
|
Conc
|
unit
|
Final conc./amount
|
unit
|
Volume (ul)
|
11.5x rxn mix
|
Bis-cvt DNA |
|
|
|
|
30.00 |
0.00
|
10X Reaction buffer |
10 |
X |
1 |
X |
5.00 |
57.50
|
dNTP mix |
10 |
mM |
0.25 |
mM |
1.25 |
14.38
|
TruS_F/R |
10 |
uM |
0.3 |
uM |
1.50 |
17.25
|
50X SYBG |
50 |
X |
0.4 |
X |
0.40 |
4.60
|
PfuTurbo Cx |
2.5 |
Unit/ul |
2.5 |
unit |
1.00 |
11.50
|
H2O |
|
|
|
|
10.85 |
124.78
|
Total |
|
|
|
|
50.00 |
|
- - Aliquot 20 + 30ul BIS-DNA template
- - Mix well
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 15 --> 72C for 2min
- - Took 3ul for PAGE verification with no bead purify
File:ZhangLab 2 2014-10-24 22hr 44min 1st-PCR-KAPA-NEXTflex-3ulPCR.jpg
- - Purify with 1X AMPure beads
- - Elute with 40ul EB Buffer
- - Continue to 2nd round PCR. No PAGE verification.
2nd round PCR (QUICK TEST to monitor PCR cycle number)
- I did a quick test in small volume by using 2ul of bead purified 1st round PCR in total 10ul. Then split into 2X of 4.5ul and stopped based on qPCR curve.
Components
|
Conc
|
unit
|
Final conc.
|
unit
|
Volume (ul)
|
12 rxn mix
|
Purified 1st round DNA |
|
|
|
|
2.00 |
0.00
|
5X Phusion HF buffer |
5 |
X |
1 |
X |
2.00 |
24.00
|
dNTP mix |
10 |
mM |
0.25 |
mM |
0.25 |
3.00
|
TruS_F/R |
10 |
uM |
0.3 |
uM |
0.30 |
3.60
|
50X SYBR |
50 |
X |
0.4 |
X |
0.08 |
0.96
|
Phusion HF |
2 |
unit/ul |
|
|
0.10 |
1.20
|
H2O |
|
|
|
|
5.27 |
63.24
|
Total |
|
|
|
|
10.00 |
|
- - Aliquot 8 + 2ul DNA template
- - Mix well.
- - Split 4.5ul
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5, 6, and 7 cycles --> 72C for 2min
KAPA Hyper prep
- - Sample NC2 & NC3 stopped at 5X and 7X
- - Sample 6P2 & 6P3 stopped at 5X and 6X
NEXTflex
- - Sample NC2 & NC3 stopped at 5X and 7X
- - Sample 6P2 & 6P3 stopped at 5X and 7X
- - Verify all PCR product in 6% TBE gel
File:ZhangLab 2 2014-10-27 12hr 50min 2ndPCR-KAPA-NEXTflex-5x.jpg
File:ZhangLab 2 2014-10-27 12hr 50min 2ndPCR-KAPA-NEXTflex-6x-7x.jpg
- From the gel image, XXXXX
- - I do 2nd round PCR in the large volume by using all 1st round PCR as template. I reduce template amount of 6P samples by KAPA Hyper Prep kit to 10% and increase 2nd round PCR to 6 cycles. For the rest samples, I use 20% of DNA template from 1st round PCR and ran 2nd round PCR for 8 cycles.
2nd round PCR (use all 1st round PCR)
Mix 1: Low signal
- Sample: KAPA Hyper Prep, NC2 & NC3 all sample of NEXTflex
Components
|
Conc
|
unit
|
Final conc.
|
unit
|
Volume (ul)
|
6.2 rxn mix
|
Purified 1st round DNA |
|
|
|
|
36.00 |
0.00
|
5X Phusion HF buffer |
5 |
X |
1 |
X |
36.00 |
223.20
|
dNTP mix |
10 |
mM |
0.25 |
mM |
4.50 |
27.90
|
TruS_F/R |
10 |
uM |
0.3 |
uM |
5.40 |
33.48
|
50X SYBR |
50 |
X |
0.4 |
X |
1.44 |
8.93
|
Phusion HF |
2 |
unit/ul |
|
|
1.80 |
11.16
|
H2O |
|
|
|
|
94.86 |
588.13
|
Total |
|
|
|
|
180.00 |
|
- - Aliquot 144ul + 36ul of template
- - Split 50ul to each well of 8-tube strip
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 8cycles --> 72C for 2min
Mix 2: High signal
- Sample: KAPA Hyper Prep 6P2 & 6P3
Components
|
Conc
|
unit
|
Final conc.
|
unit
|
Volume (ul)
|
2.2 rxn mix
|
Purified 1st round DNA |
|
|
|
|
36.00 |
0.00
|
5X Phusion HF buffer |
5 |
X |
1 |
X |
72.00 |
158.40
|
dNTP mix |
10 |
mM |
0.25 |
mM |
9.00 |
19.80
|
TruS_F/R |
10 |
uM |
0.3 |
uM |
10.80 |
23.76
|
50X SYBR |
50 |
X |
0.4 |
X |
2.88 |
6.34
|
Phusion HF |
2 |
unit/ul |
|
|
3.60 |
7.92
|
H2O |
|
|
|
|
225.72 |
496.58
|
Total |
|
|
|
|
360.00 |
|
- - Aliquot 324ul + 36ul of template
- - Split 50ul to each well of 8-tube strip
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 6 cycles --> 72C for 2min
- - Do 1 round (before I do 2 rounds, but this experiment the adaptor dimer was much fainter than before) bead purification of all PCR product with 1X AMPure beads and elute with 50ul EB buffer.
- - Verified 2ul of bead purified sequencing library in 6% TBE gel
File:ZhangLab 2 2014-10-27 17hr 01min 2ul PAGEverify KAPAHyper NEXTflex WGBS.jpg
- - Took 1.5ul for Qubit dsDNA HS quantification
Qubit dsDNA HS quantification result
Sample
|
Conc. in the Qubit
|
Unit
|
uL used
|
Dilution
|
Conc. (ng/ul)
|
Yield in 46ul
|
NC-2_KAPA |
6.92 |
ng/mL |
1.5 |
133.33 |
0.92 |
42.44
|
NC-2_KAPA |
23.2 |
ng/mL |
1.5 |
133.33 |
3.09 |
142.29
|
6P2_KAPA |
137 |
ng/mL |
1.5 |
133.33 |
18.27 |
840.27
|
6P3_KAPA |
72.6 |
ng/mL |
1.5 |
133.33 |
9.68 |
445.28
|
NC2_NEXTflex |
3.86 |
ng/mL |
1.5 |
133.33 |
0.51 |
23.67
|
NC3_NEXTflex |
8.29 |
ng/mL |
1.5 |
133.33 |
1.11 |
50.85
|
6P2_NEXTflex |
80.5 |
ng/mL |
1.5 |
133.33 |
10.73 |
493.73
|
6P3_NEXTflex |
25.2 |
ng/mL |
1.5 |
133.33 |
3.36 |
154.56
|
- Compare the PCR cycle number and yield of these samples to the previous protocol using only Klenow fragment, eco-