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

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:- Resuspend with 21ul EB buffer
:- Resuspend with 21ul EB buffer
:- Transfer 20ul of eluted adaptor-ligated DNA to new 8-tube strip
:- 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
:- Continue to bisulfite conversion. Optional: Adaptor-ligated DNA can be stored at -20C before bisulfite conversion



Revision as of 18:16, 7 December 2014

WGBS library prep and BSPP capture of cfDNA from Illumina

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Aims

  • 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

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

End repair & dA-tailing

  • 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

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

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

Adaptor ligation reaction mix

  • 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

  • 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

  • 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

Amplification

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.
  • 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 KAPA HiFi Uracil+ Hot Start Ready Mix 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 KAPA HiFi Uracil+ 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.
  • For Illumina, the cost is not the limitation, so I suggest Li to do one round qPCR by increasing amplification volume and increase cycle number. This should save a lot of time.