Noi/NOTES/2014-12-5: Difference between revisions
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==== Image of Bioanalyzer By Li ==== | ==== Image of Bioanalyzer By Li ==== | ||
[[File:Beadpurified-WGBSLib-BA.png| | [[File:Beadpurified-WGBSLib-BA.png|800px]] |
Revision as of 22:29, 9 December 2014
WGBS library prep and BSPP capture of cfDNA from Illumina
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
- 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 20ul of bisulfite converted DNA to strip tube to be easy to handle during PCR step
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 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.
- 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
- 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
- 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
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
File:Beadpurified-WGBSLib-BA.png