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= Preparation of WGBS libraries of small fragment DNA in cancer patient plasma samples (no DNA fragmentation step, REPEAT with dAdGdCdT for end repair) = [[noi:DMR220k_LabNotes|'''Back to calendar''']] * [[Noi/NOTES/2014-8-3|'''Continue from''']] * There is one sample change for sample in colon cancer in [[Noi/NOTES/2014-8-3#Colon_.28UCSD-006.29|2014-08-03 note.]] '''Sample 6-P-6 was replace with sample 6-P-10.''' == 1) End-repair/dA-tailing == * Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG:dT (final conc. 1:0.1:0.1:0.1mM) in total volume 20ul ====<u>Pancreas</u>==== {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng''' | align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | align="center" style="width:80px;background:#f0f0f0;"|'''Klenow,exo-:dAdCdG Mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total''' | align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' |- | PC-P-2||0.852||1.17||wPC-P-2||8.83||10.00||20.00||wPC-P-2 |- | PC-P-3||1.360||0.74||wPC-P-3||9.26||10.00||20.00||wPC-P-3 |- | PC-P-4||0.340||2.94||wPC-P-4||7.06||10.00||20.00||wPC-P-4 |- | PC-P-5||0.813||1.23||wPC-P-5||8.77||10.00||20.00||wPC-P-5 |- | PC-P-6||0.767||1.30||wPC-P-6||8.70||10.00||20.00||wPC-P-6 |- | PC-P-7||0.644||1.55||wPC-P-7||8.45||10.00||20.00||wPC-P-7 |- | PC-P-8||0.524||1.91||wPC-P-8||8.09||10.00||20.00||wPC-P-8 |- | PC-P-9||0.238||4.20||wPC-P-9||5.80||10.00||20.00||wPC-P-9 |} ====<u>Colon</u>==== {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng''' | align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | align="center" style="width:80px;background:#f0f0f0;"|'''Klenow,exo-:dAdCdG Mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total''' | align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' |- | 6-P-1||0.200||5.00||w6-P-1||5.00||10.00||20.00||w6-P-1 |- | 6-P-2||1.100||0.91||w6-P-2||9.09||10.00||20.00||w6-P-2 |- | 6-P-3||1.570||0.64||w6-P-3||9.36||10.00||20.00||w6-P-3 |- | 6-P-4||0.889||1.12||w6-P-4||8.88||10.00||20.00||w6-P-4 |- | 6-P-5||0.487||2.05||w6-P-5||7.95||10.00||20.00||w6-P-5 |- | 6-P-10||0.333||3.00||6-P-10||7.00||10.00||20.00||6-P-10 |- | 6-P-8||0.226||4.42||w6-P-8||5.58||10.00||20.00||w6-P-8 |- | 6-P-9||0.408||2.45||w6-P-9||7.55||10.00||20.00||w6-P-9 |} ====<u>Lung</u>==== {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng''' | align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | align="center" style="width:80px;background:#f0f0f0;"|'''Klenow,exo-:dAdCdG Mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total''' | align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' |- | 7-P-1||0.692||1.45||w7-P-1||8.55||10.00||20.00||w7-P-1 |- | 7-P-2||1.140||0.88||w7-P-2||9.12||10.00||20.00||w7-P-2 |- | 7-P-3||0.208||4.81||w7-P-3||5.19||10.00||20.00||w7-P-3 |- | 7-P-5||1.110||0.90||w7-P-5||9.10||10.00||20.00||w7-P-5 |- | 7-P-6||0.233||4.29||w7-P-6||5.71||10.00||20.00||w7-P-6 |- | 7-P-7||0.229||4.37||w7-P-7||5.63||10.00||20.00||w7-P-7 |- | 7-P-8||0.167||5.99||w7-P-8||4.01||10.00||20.00||w7-P-8 |- | 7-P-10||0.203||4.93||w7-P-10||5.07||10.00||20.00||w7-P-10 |- | NTC_1||0.000||0.00||ntc1||10.00||10.00||20.00||ntc1 |- | NTC_2||0.000||0.00||ntc2||10.00||10.00||20.00||ntc2 |- | PC-S_5||1.580||0.63||wPC-S-5||9.37||10.00||20.00||wPC-S-5 |- | PC-S_8||0.953||1.05||wPC-S-8||8.95||10.00||20.00||wPC-S8 |} * I also included 2 matched paired serum samples for pancreatic cancer patients (PC-S-5 and PC-S-8) to compare the level of small fragment DNA. I expected to get lower amount of DNA in the library from serum sample than plasma as the total amount of DNA in serum is derived from lysed WBC. This can also confirm that the library is generated from small fragment DNA. ==== <u>Klenow exo- and dA:dG:dC reaction mix</u> ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''28x rxn''' |- | 10X Tango buffer||2.00||56.00 |- | dA:dC:dG:dT (20:2:2:2mM)||1.00||28.00 |- | Klenow fragment, exo- (5U/ul)||1.00||28.00 |- | H2O||6.00||168.00 |- | Total||10.00|| |} - Aliquot 32 of Klwnow, exo- and dA:dC:dG:dT reaction mix to 8-tube strip :- Add H2O to each tube :- Add 1ng of DNA to each tube :- Add 10ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA with multichannel pipette. Pipette few times :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid) :- 75C, 10min --> hold at 4C (heat lid on) * No AMPure bead purification<br> <br> == 2) Methylated adaptor ligation == * Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul {| {{table}} class = wikitable | align="center" style="width:80px;background:dodgerblue;"|'''Exp ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index''' | align="center" style="width:80px;background:dodgerblue;"|'''Exp ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index''' | align="center" style="width:80px;background:dodgerblue;"|'''Exp ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index''' | align="center" style="width:80px;background:dodgerblue;"|'''Exp ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index''' |- | wPC-P-2||1||w6-P-1||13||w7-P-1||11||ntc1||3 |- | wPC-P-3||2||w6-P-2||14||w7-P-2||12||ntc2||9 |- | wPC-P-4||4||w6-P-3||15||w7-P-3||21||wPC-S_5||23 |- | wPC-P-5||5||w6-P-4||16||w7-P-5||22||wPC-S_8||25 |- | wPC-P-6||6||w6-P-5||27||w7-P-6||3|||| |- | wPC-P-7||7||w6-P-10||18||w7-P-7||9|||| |- | wPC-P-8||8||w6-P-8||19||w7-P-8||23|||| |- | wPC-P-9||10||w6-P-9||20||w7-P-10||25|||| |} ==== <u>Ligation reaction mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''30x rxn''' |- | dA-tailed DNA||20.00||0.00 |- | 10X Tango buffer||0.50||15.00 |- | HC T4 DNA ligase (30units/ul)||1.00||30.00 |- | 10mM ATP ||1.25||37.50 |- | H2O||1.25||37.50 |- | Total||24.00|| |} :- Aliquot 13ul of ligation reaction mix to 8-tube strip :- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction :- Add 4ul of ligation reaction mix with multichannel pipette :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 16C, 20h (no heat lid) :- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on) :- Continue to bisulfite conversion == Bisulfite conversion == [[Noi/NOTES/2014-8-3#Bisulfite_conversion| '''Bisulfite conversion procedures''']]<br> == Amplification == * Repeat all procedure exactly the same as previous experiment on 2014-08-03 === 1st round PCR (fix 12 cycles) === {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''30 rxn mix''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||150.00 |- | dNTP mix||10||mM||0.25||mM||1.25||37.50 |- | TruS_F/R||10||uM||0.3||uM||1.50||45.00 |- | 50X SYBG||50||X||0.4||X||0.40||12.00 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00||30.00 |- | H2O||||||||||10.85||325.50 |- | Total ||||||||||50.00|| |} :- Aliquot 20 + 30ul BIS-DNA template :- Mix well :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 --> 72C for 2min'' :- Purify with 1.25X AMPure beads :- Elute with 42ul EB Buffer :- Continue to 2nd round PCR. No PAGE verification. === 2nd round PCR === === Quick test === {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''60 rxn mix''' |- | Purified 1st round DNA||||||||||3.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||2.00||120.00 |- | dNTP mix||10||mM||0.25||mM||0.25||15.00 |- | TruS_F/R||10||uM||0.3||uM||0.30||18.00 |- | 50X SYBR||50||X||0.4||X||0.08||4.80 |- | Phusion HF||2||unit/ul||||||0.10||6.00 |- | H2O||||||||||4.27||256.20 |- | Total||||||||||10.00|| |} :- Aliquot 14 + 6ul DNA template :- Mix well. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 7 and 8 cycles --> 72C for 2min''<br> :- Added 2ul 6X loading dye to each strip tube & mix well :- Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min. I loaded only PCR at 7 cycles first. - Gel1: LM PC-Pv(8) PC-S-5 PC-S-8<br> - Gel2: LM 6-P (8) NTC1 NTC2<br> - Gel3: LM 7-P (8) NTCPCR [[File:ZhangLab_2 2014-08-20 12hr 20min_test-2ndPCR-pat-7x_gel1.jpg| 450px]] [[File:ZhangLab_2 2014-08-20 12hr 20min_test-2ndPCR-pat-7x_gel2.jpg| 450px]] [[File:ZhangLab_2 2014-08-20 12hr 20min_test-2ndPCR-pat-7x_gel3.jpg| 450px]] * From quick test qPCR, I continue to do qPCR of the remaining 1st round PCR in a large volume ( 120 ul total) and reduce PCR cycle number to 6 cycles {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''27 rxn mix''' |- | Purified 1st round DNA||||||||||36.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||24.00||648.00 |- | dNTP mix||10||mM||0.25||mM||3.00||81.00 |- | TruS_F/R||10||uM||0.3||uM||3.60||97.20 |- | 50X SYBR||50||X||0.4||X||0.96||25.92 |- | Phusion HF||2||unit/ul||||||1.20||32.40 |- | H2O||||||||||51.24||1383.48 |- | Total||||||||||120.00|| |} :- Aliquot 84 + 36ul DNA template :- Mix well. Split 38ul X3 ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 6 cycles --> 72C for 2min''<br> :- I pool ~110ul PCR product from the two replicates, purify with 2 rounds of 1X AMPure beads (fist round elute with EB buffer 60ul. 2nd round elute with 65ul EB buffer. * Note that the band of PCR after reducing PCR cycle to 6 cycles was different from the gel images above as the band around 300bp was stronger and less smear from over-amplification. Some examples was shown in the gel image below. [[File:ZhangLab_2 2014-09-11 01hr 14min_PAGE-verify_cf1-4_6P-2-5.jpg| 500px]] * I will use Qbit dsDNA HS assay for library concentration quantification. === Qubit dsDNA HS quantification of WGBS libraries from cancer patient ctDNA === {| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:60px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:60px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Used for prev. exp (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount left (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''> 400ng''' |- | PC-P-2||88.1||ng/mL||2||100||8.81||60.00||528.60||0.00||528.60||YES |- | PC-P-3||138||ng/mL||2||100||13.80||60.00||828.00||0.00||828.00||YES |- | PC-P-4||90.3||ng/mL||2||100||9.03||60.00||541.80||0.00||541.80||YES |- | PC-P-5||85.9||ng/mL||2||100||8.59||60.00||515.40||0.00||515.40||YES |- | PC-P-6||114||ng/mL||2||100||11.40||60.00||684.00||0.00||684.00||YES |- | PC-P-7||122||ng/mL||2||100||12.20||60.00||732.00||0.00||732.00||YES |- | PC-P-8||103||ng/mL||2||100||10.30||60.00||618.00||0.00||618.00||YES |- | PC-P-9||115||ng/mL||2||100||11.50||60.00||690.00||0.00||690.00||YES |- | 6-P-1||95.8||ng/mL||2||100||9.58||60.00||574.80||0.00||574.80||YES |- | 6-P-2||126||ng/mL||2||100||12.60||60.00||756.00||100.00||656.00||YES |- | 6-P-3||61.6||ng/mL||2||100||6.16||60.00||369.60||100.00||269.60||NO |- | 6-P-4||104||ng/mL||2||100||10.40||60.00||624.00||100.00||524.00||NO |- | 6-P-5||54.3||ng/mL||2||100||5.43||60.00||325.80||100.00||225.80||NO |- | 6-P-10||67.6||ng/mL||2||100||6.76||60.00||405.60||0.00||405.60||YES |- | 6-P-8||77.1||ng/mL||2||100||7.71||60.00||462.60||0.00||462.60||YES |- | 6-P-9||123||ng/mL||2||100||12.30||60.00||738.00||0.00||738.00||YES |- | 7-P-1||38.2||ng/mL||2||100||3.82||60.00||229.20||0.00||229.20||NO |- | 7-P-2||86.7||ng/mL||2||100||8.67||60.00||520.20||0.00||520.20||YES |- | 7-P-3||56.5||ng/mL||2||100||5.65||60.00||339.00||0.00||339.00||NO |- | 7-P-5||84||ng/mL||2||100||8.40||60.00||504.00||0.00||504.00||YES |- | 7-P-6||78||ng/mL||2||100||7.80||60.00||468.00||0.00||468.00||YES |- | 7-P-7||52.7||ng/mL||2||100||5.27||60.00||316.20||0.00||316.20||NO |- | 7-P-8||80.5||ng/mL||2||100||8.05||60.00||483.00||0.00||483.00||YES |- | 7-P-10||43.4||ng/mL||2||100||4.34||60.00||260.40||0.00||260.40||NO |}
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