Noi/NOTES/2014-10-24: Difference between revisions

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* The instruction suggested to use 0.8X of AMPure bead for clean up, but I use 1X of AMPure bead
* 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
** KAPA Hyper prep kit: does 1 round of 1X AMPure bead purification
** NEXTflext: does 2 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
- Aliquot AMPure bead in 1.5mL tube
:- Add 55ul (for KAPA Hyper) or 50ul (for NEXTflex) AMPure bead. Mix by pipetting 10x
:- Add 55ul (for KAPA Hyper) or 50ul (for NEXTflex) AMPure bead. Mix by pipetting 10x
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:- Continue to bisulfite conversion immediately
:- Continue to bisulfite conversion immediately
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== Bisulfite conversion ==
== 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.
* I performed bisulfite conversion using the same procedure following manufacturer's instruction of EZ DNA Methylation-Lightning kit and elute with 32ul elution buffer.

Revision as of 15:53, 28 October 2014

Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit & NEXTflex Cell Free DNA-Seq kit

Link to calendar

  • 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.