Noi/NOTES/2014-12-5: Difference between revisions

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:- Spin for 2min for second washing.
:- Spin for 2min for second washing.
:- Elute with 33ul M-Elution Buffer. The total volume left should be ~31-32ul
:- Elute with 33ul M-Elution Buffer. The total volume left should be ~31-32ul
:- Transfer 20ul of bisulfite converted DNA to strip tube to be easy to handle during PCR step
:- Transfer 30ul of bisulfite converted DNA to strip tube to be easy to handle during PCR step


== Amplification ==
== Amplification ==
=== Things to consider ===
=== Things to consider ===
* To prepare at least 500ng up to 1000ng amount  WGBS library from low input DNA, I alway do two-rounds of amplification. This is different from preparing WGBS libraries for sequencing which requires less than 100ng of each library for sequencing.  
* To prepare at least 500ng up to 1000ng amount  WGBS library from low input DNA, I alway do two-rounds of amplification. This is different from preparing WGBS libraries for sequencing which requires less than 100ng of each library for sequencing.  
* From my previous experiments, running qPCR in small volume (50ul) using all DNA from bisulfite conversion (input 1-10ng) was saturated with the yield less than 100ng). To be able to get almost up to 1ug of WGBS library, I need to set up the PCR volume up to 300ul (decreasing DNA template concentration) and amplify with more cycle number (12-18 cycles for input between 1ng-20ng, varied based on input DNA).  I have considered that using 2X KAPA HiFi HotStart Uracil+ ReadyMix for qPCR reaction 300ul is very expensive for a routine experiment and also to set up a protocol for large sample size. I then have tried to do two rounds of qPCR by using  2X KAPA HiFi HotStart Uracil+ ReadyMix in the first round qPCR in total reaction 50ul. I then do 1X bead purification and amplify the 2nd round PCR using other high efficient DNA Pol with proof reading activity like NEB Phusion HiFidelity of KAPA HiFi Hotstart Readymix. However, this two-step PCR is not convenient as well and AMPure bead is quite more expensive. I may need to judge between the cost and time between using one round and two-round PCR.  
* From my previous experiments, running qPCR in small volume (50ul) using all DNA from bisulfite conversion (input 1-10ng) was saturated with the yield less than 100ng). To be able to get almost up to 1ug of WGBS library, I need to set up the PCR volume up to 300ul (decreasing DNA template concentration) and amplify with more cycle number (12-18 cycles for input between 1ng-20ng, varied based on input DNA).  I have considered that using 2X KAPA HiFi HotStart Uracil+ ReadyMix for qPCR reaction 300ul is very expensive for a routine experiment and also to set up a protocol for large sample size. I then have tried to do two rounds of qPCR by using  2X KAPA HiFi HotStart Uracil+ ReadyMix in the first round qPCR in total reaction 50ul. I then do 1X bead purification and amplify the 2nd round PCR using other high efficient DNA Pol with proof reading activity like NEB Phusion HiFidelity or KAPA HiFi Hotstart Readymix. However, this two-step PCR is not convenient as well and AMPure bead is quite more expensive. I may need to judge between the cost and time between using one round and two-round PCR.  
* For Illumina, the cost is not a limitation, so I suggested Li to do one round qPCR by increasing amplification volume and increase cycle number. This should save a lot of time.
* For Illumina, the cost is not a limitation, so I suggested Li to do one round qPCR by increasing amplification volume and increase cycle number. This should save a lot of time.
==== Quick test PCR to monitor PCR cycle number ====
==== Quick test PCR to monitor PCR cycle number ====
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|}
|}
==== Image of Bioanalyzer By Li ====
==== Image of Bioanalyzer By Li ====
  [[File:Beadpurified-WGBSLib-BA.png|600px]]
  [[File:Beadpurified-WGBSLib-BA.png|800px]]

Latest revision as of 08:26, 18 March 2015

WGBS library prep and BSPP capture of cfDNA from Illumina[edit]

Back to calendar

Aims[edit]

  • To generate 10 WGBS libraries from ctDNA of colorectal cancer (CRC) patients from Illumina (received from Kansas University)
  • To capture WGBS libraries with MONOD v5 (LMS) probe set from Zhang's lab and CRC probe set from Illumina

Sample info[edit]

Subject# Sample_ID Plasma volume (ul) Size (bp) by BA Total Yield (ng) by BA
KUCC BRCF 011921 10.A13.3 964 187 14
KUCC BRCF 005881 9.B7.36 952 178 13.2
KUCC BRCF 007388 9.C28.47 876 173 11.9
KUCC BRCF 008966 9.I19.36 882 185 4.8
KUCC BRCF 008282 9.G9.25 670 191 9
KUCC BRCF 008116 9.F20.56 924 197 5.1
KUCC BRCF 007855 9.H5.58 914 223 8.6
KUCC BRCF 008472 9.H3.25 1,000 219 4.6
KUCC BRCF 008527 9.H7.69 1,150 237 11.1
KUCC BRCF 008794 9.I7.3 770 249 9.57
  • Each sample has volume ~22ul
  • Each sample has different amount of DNA and we want to use up all DNA for WGBS library prep. To make it simple for qPCR monitoring after adaptor ligation and bisulfite conversion, I re-named the sample ID and run based on the input DNA from low to high.
Subject# Sample_ID Total Yield (ng)by BA Zhang lab ID
KUCC BRCF 008472 9.H3.25 4.6 KU_1
KUCC BRCF 008966 9.I19.36 4.8 KU_2
KUCC BRCF 008116 9.F20.56 5.1 KU_3
KUCC BRCF 007855 9.H5.58 8.6 KU_4
KUCC BRCF 008282 9.G9.25 9 KU_5
KUCC BRCF 008794 9.I7.3 9.57 KU_6
KUCC BRCF 008527 9.H7.69 11.1 KU_7
KUCC BRCF 007388 9.C28.47 11.9 KU_8
KUCC BRCF 005881 9.B7.36 13.2 KU_9
KUCC BRCF 011921 10.A13.3 14 KU_10

Part I: WGBS library preparation using KAPA Hyper Prep Kit[edit]

End repair & dA-tailing[edit]

  • 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 reaction mix[edit]

Components Volume half rxn 11.2x rxn
End repair/dA-tailing buffer 7.00 3.50 39.20
End repair/dA-tailing enzyme mix 3.00 1.50 16.80
H2O 6.00 3.00
Total 10.00 8.00


Reaction set up[edit]

Sample IDs Zhang lab ID Volume End repair/dA-tailing rxn mix H2O (ul) Total (ul)
9.H3.25 KU_1 22.00 8 0.00 30.00
9.I19.36 KU_2 22.00 8 0.00 30.00
9.F20.56 KU_3 22.00 8 0.00 30.00
9.H5.58 KU_4 22.00 8 0.00 30.00
9.G9.25 KU_5 22.00 8 0.00 30.00
9.I7.3 KU_6 22.00 8 0.00 30.00
9.H7.69 KU_7 22.00 8 0.00 30.00
9.C28.47 KU_8 22.00 8 0.00 30.00
9.B7.36 KU_9 22.00 8 0.00 30.00
10.A13.3 KU_10 22.00 8 0.00 30.00
NTC NTC 0.00 8 25.00 33.00
- Add 8ul of End repair/dA-tailing rxn mix to each tube in 8-tube strip
- Transfer 22ul of ctDNA to each rxn tube.
- Mix well (spin down & pulse-vortexing 10X & spin down)
- 20C, 30min
- 65C, 30min,
- Hold 4C
- Proceed to Adaptor ligation immediately

Adaptor ligation[edit]

Adaptor ligation reaction mix[edit]

  • I prepare 10X diluted TruSeq adaptor (conc. after dilution = 1500nM) Index 1-10 by mixing 2ul of 15uM TruSeq adaptor with 18ul H2O.
Components Volume half rxn 11.2x rxn
End-repaired/A-tailed DNA 60.00 30.00 0.00
Ligation buffer 30.00 15.00 168.00
DNA ligase 10.00 5.00 56.00
Total 100.00 50.00 20ul/rxn
Diluted adaptor (1.5uM) 10.00 5.00
Total 110.00 55.00
- Add 5ul of 1.5uM diluted Truseq adaptor to each tube
- Final conc. of adaptor in 55ul ligation reaction is ~136nM
- Add 20ul of ligation reaction mix to each tube
- Mix well (spin down & pulse-vortexing 10X & spin down)
- 20C, 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

- Aliquot AMPure bead in 1.5mL tube and leave at RT

- Add 55ul AMPure (RT) bead Mix by pipetting 10x
- Incubate at RT for 15min
- Transfer to sit on magnet for 5min
- Discard spnt
- Wash twice with 200ul freshly prepared 80% EtOH
- After adding 80% EtOH, wait for 30sec (only the 1st time wash) before pipetting up and down 5X and discard spnt
- Dry the bead for 5min
- Resuspend with 21ul EB buffer
- Transfer 20ul of eluted adaptor-ligated DNA to new 8-tube strip
- Before continue to bisulfite conversion, we can check if ligation works by running PCR using 1ul of adaptor-ligated DNA in small volume. (I skipped this step)
- Continue to bisulfite conversion. Optional: Adaptor-ligated DNA can be stored at -20C before bisulfite conversion

Bisulfite conversion[edit]

  • Bisulfite conversion kit: EZ DNA Methylation-Lightning Kit (column-based method) , cat# D5030 or D5031
  • EZ-96 DNA Methylation-Lightning™ MagPrep (magnetic bead-based method) is another option for large sample set
  • I followed the instruction of bisulfite conversion exact the same as describe in the manual.
  • Few things to note:
- Incubate with desulfonation buffer for 18min.
- Discard spnt after binding step and first washing.
- Spin for 2min for second washing.
- Elute with 33ul M-Elution Buffer. The total volume left should be ~31-32ul
- Transfer 30ul of bisulfite converted DNA to strip tube to be easy to handle during PCR step

Amplification[edit]

Things to consider[edit]

  • To prepare at least 500ng up to 1000ng amount WGBS library from low input DNA, I alway do two-rounds of amplification. This is different from preparing WGBS libraries for sequencing which requires less than 100ng of each library for sequencing.
  • From my previous experiments, running qPCR in small volume (50ul) using all DNA from bisulfite conversion (input 1-10ng) was saturated with the yield less than 100ng). To be able to get almost up to 1ug of WGBS library, I need to set up the PCR volume up to 300ul (decreasing DNA template concentration) and amplify with more cycle number (12-18 cycles for input between 1ng-20ng, varied based on input DNA). I have considered that using 2X KAPA HiFi HotStart Uracil+ ReadyMix for qPCR reaction 300ul is very expensive for a routine experiment and also to set up a protocol for large sample size. I then have tried to do two rounds of qPCR by using 2X KAPA HiFi HotStart Uracil+ ReadyMix in the first round qPCR in total reaction 50ul. I then do 1X bead purification and amplify the 2nd round PCR using other high efficient DNA Pol with proof reading activity like NEB Phusion HiFidelity or KAPA HiFi Hotstart Readymix. However, this two-step PCR is not convenient as well and AMPure bead is quite more expensive. I may need to judge between the cost and time between using one round and two-round PCR.
  • For Illumina, the cost is not a limitation, so I suggested Li to do one round qPCR by increasing amplification volume and increase cycle number. This should save a lot of time.

Quick test PCR to monitor PCR cycle number[edit]

  • I use 1ul of bisulfite converted DNA in 10ul reaction for a quick test and will use this condition for large volume amplification after obtaining optimal PCR cycle number
Components 1X 50ul rxn 10ul rxn 12X 10ul rxn
Adaptor ligated DNA 5.00 1.00 0.00
2X KAPA HiFi HotStart Uracil+ ReadyMix 25.00 5.00 60.00
10uM TruS F/R 1.50 0.30 3.60
50X SYBR 0.40 0.08 0.96
H2O 18.10 3.62 43.44
Total 50.00 10.00
- Aliquot 9ul + 1ul template
- Mix well
98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 8-18X --> 72C for 1min
- Load 3ul PCR product in 6% TBE gel to verify the amplified libraries
File:ZhangLab 2 2014-12-05 16hr 57min ILMN qPCR-quicktest.jpg

  • Note: qPCR curves of KU_2 and KU_6 shoot up later than other samples with the same input amount. KU_2 curve was very close to NTC, but it had good amplification at 18 cycles. The color (orange) appeared in this sample may interfere fluorescence reading.

Amplification in a large volume[edit]

  • After quick test qPCR and PAGE verification, I continued to amplify the rest of bisulfite converted DNA in total 300ul (split 50ul/tube) by adjusting PCR cycle number for some samples regarding gel intensity.
Components 1X 50ul rxn 1X 150 21 rxn
Adaptor ligated DNA 5.00 15.00 0.00
2X KAPA HiFi HotStart Uracil+ ReadyMix 25.00 75.00 1575.00
10uM TruS F/R 1.50 4.50 94.50
50X SYBR 0.40 1.20 25.20
H2O 18.10 54.30 1140.30
Total 50.00 150.00
- Aliquot 135ul + 15ul template (2X of 150ul -> then split 50ul/tube)
- Mix well
98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 12-18X --> 72C for 1min


Zhang lab ID Input ampiunt # of cycle
KU_1 4.6 15
KU_2 4.8 18
KU_3 5.1 15
KU_4 8.6 15
KU_5 9 14
KU_6 9.57 16
KU_7 11.1 14
KU_8 11.9 12
KU_9 13.2 12
KU_10 14 12
- Perform 1X of AMPure bead purification by pooling 100ul of PCR product of the same sample in each tube (3 tubes/sample)
- Elute each tube with 25ul H2O -> total volume of each sample is ~75ul.
- I aliquot 2ul for Li for BA measurement.
- I took 1ul for Qubit dsDNA HS assay.
- The total volume left for each sample should be ~70ul

Qubit quantification assay results[edit]

Sample Conc. in the Qubit Unit uL used Dilution Conc. (ng/ul) Amount in 70ul (ng)
KU_1 49.4 ng/mL 1 200 9.89 692.30
KU_2 74.4 ng/mL 1 200 14.90 1043.00
KU_3 54.8 ng/mL 1 200 11.00 770.00
KU_4 71.5 ng/mL 1 200 14.30 1001.00
KU_5 50.6 ng/mL 1 200 10.10 707.00
KU_6 63.5 ng/mL 1 200 12.70 889.00
KU_7 78.2 ng/mL 1 200 15.60 1092.00
KU_8 46.1 ng/mL 1 200 9.22 645.40
KU_9 45.5 ng/mL 1 200 9.09 636.30
KU_10 40.8 ng/mL 1 200 8.17 571.90

Image of Bioanalyzer By Li[edit]

File:Beadpurified-WGBSLib-BA.png