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]

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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[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