Noi/NOTES/2014-10-24
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Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit & NEXTflex Cell Free DNA-Seq kit[edit]
- 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[edit]
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[edit]
End repair & dA-tailing reaction mix[edit]
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[edit]
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[edit]
Adaptor ligation reaction mix[edit]
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[edit]
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[edit]
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[edit]
End repair & Adenylation reaction mix[edit]
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[edit]
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[edit]
- 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[edit]
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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
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)[edit]
- 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)[edit]
Mix 1: Low signal[edit]
- 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[edit]
- 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[edit]
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 |
Comparison of the PCR cycle number and yield of these samples to the previous protocol using only Klenow fragment, exo-[edit]
Sample | 1st PCR | 2nd PCR | Total PCR cycles | Yield (ng) |
Klenow exo- | ||||
NC2 | 12 | 7 | 19 | 111.20 |
NC3 | 12 | 7 | 19 | 144.00 |
6P2 | 12 | 6 | 18 | 756.00 |
6P3 | 12 | 6 | 18 | 369.60 |
KAPA Hyper Prep kit | ||||
NC2 | 15 | 8 | 23 | 42.44 |
NC3 | 15 | 8 | 23 | 142.29 |
6P2 | 15 | 6 | 21 | 840.27 |
6P3 | 15 | 6 | 21 | 445.28 |
NEXTflex Cell Free DNA-Seq kit | ||||
NC2 | 15 | 8 | 23 | 23.67 |
NC3 | 15 | 8 | 23 | 50.85 |
6P2 | 15 | 8 | 23 | 493.73 |
6P3 | 15 | 8 | 23 | 154.56 |