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= Preparation of WGBS of cfDNA from plasma of cancer patients using KAPA Hyper Prep kit = [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']] * As I mentioned before in previous experiment that the first batch of WGBS libraries from cfDNA of normal and cancer patients were generated by RRBS method without DNA fragmentation. This might lead to the incomplete of libraries as RRBS protocol uses only Klenow fragment, eco- but lack T4 DNA Pol and T4 PNK enzyme. Dr. Zhang suggested to generated WGBS libraries of cfDNA from cancer patients using KAPA Hyper Prep Kit as I did test experiment on 2014-10-24. * I also increased DNA input as much as possible for each sample to be able to capture all individual unique molecule from cancer cells in the DNA pool of cfDNA. {| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample conc. (ng/uL)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Yields (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amont left (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount used in WGBS/ KAPAHyper''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O to 25ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''End repair/dA-tailing rxn mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' |- | PCP-2||0.852||30||25.56||22.06||20||23.47||PCP-2||1.53||5||30.00 |- | PCP-3||1.360||30||40.80||37.30||20||14.71||PCP-3||10.29||5||30.00 |- | PCP-4||0.340||30||10.20||6.70||6.5||19.12||PCP-4||5.88||5||30.00 |- | PCP-5||0.813||30||24.39||20.89||20||24.60||PCP-5||0.40||5||30.00 |- | PCP-6||0.767||30||23.01||19.51||15||19.56||PCP-6||5.44||5||30.00 |- | PCP-7||0.644||30||19.32||15.82||15||23.29||PCP-7||1.71||5||30.00 |- | PCP-8||0.524||30||15.72||12.22||10||19.08||PCP-8||5.92||5||30.00 |- | PCP-9||0.238||30||7.14||3.64||3.5||14.71||PCP-9||10.29||5||30.00 |- | |||||||||||||||||||| |- | 6P-1||0.200||30||6||2.50||2.5||12.50||6P-1||12.50||5||30.00 |- | 6P-2||1.100||30||33||29.50||20||18.18||6P-2||6.82||5||30.00 |- | 6P-3||1.570||30||47.1||43.60||20||12.74||6P-3||12.26||5||30.00 |- | 6P-4||0.889||30||26.67||23.17||20||22.50||6P-4||2.50||5||30.00 |- | 6P-5||0.487||30||14.61||11.11||10||20.53||6P-5||4.47||5||30.00 |- | 6P-8||0.226||30||6.78||3.28||3.2||14.16||6P-8||10.84||5||30.00 |- | 6P-9||0.408||30||12.24||8.74||8.5||20.83||6P-9||4.17||5||30.00 |- | 6P-10||0.333||30||9.99||6.49||6.4||19.22||6P-10||5.78||5||30.00 |- | |||||||||||||||||||| |- | 7P-1||0.692||30||20.76||17.26||15||21.68||7P-1||3.32||5||30.00 |- | 7P-2||1.14||30||34.2||30.7||20||17.54||7P-2||7.46||5||30.00 |- | 7P-3||0.208||30||6.24||2.74||2.5||12.02||7P-3||12.98||5||30.00 |- | 7P-5||1.11||30||33.3||29.8||20||18.02||7P-5||6.98||5||30.00 |- | 7P-6||0.233||30||6.99||3.49||3.4||14.59||7P-6||10.41||5||30.00 |- | 7P-7||0.229||30||6.87||3.37||3.3||14.41||7P-7||10.59||5||30.00 |- | 7P-8||0.167||30||5.01||1.51||1.5||8.98||7P-8||16.02||5||30.00 |- | 7P-10||0.203||30||6.09||2.59||2.5||12.32||7P-10||12.68||5||30.00 |- | NTC||NA||NA||NA||NA||NA||NA||NTC||25.00||5||30.00 |} == End repair & dA-tailing == ==== End repair & dA-tailing reaction mix ==== * I prepared 5ul of End repair & dA-tailing reaction mix for each reaction * Maximum volume for End-repair/A-tailing = 25ul {| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:80px;background:#f0f0f0;"|'''half rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''26 rxn''' |- | End repair/dA-tailing buffer ||7.00||3.50||91.00 |- | End repair/dA-tailing enzyme mix||3.00||1.50||39.00 |- | Total||10.00||5.00|| |} :- Mix well :- 20C, 30min :- 65C, 30min, :- Hold 4C :- Proceed to Adaptor ligation immediately == Adaptor ligation == * Since I have different amount of DNA input, so I added different conc. of adaptor based on the range of input amount. * Dilute some adaptor to 1.5uM (10X) --> by mixing 3ul of adaptor with 27ul of 1X Stoffel Buffer :{| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Tube #''' | align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Stock 15uM''' | align="center" style="width:80px;background:#f0f0f0;"|'''1.11X Stoffel Buffer''' |- | #1||Ind_3||3||27 |- | #2||Ind_7||3||27 |- | #3||Ind_8||3||27 |- | #4||Ind_9||3||27 |- | #5||Ind_13||3||27 |- | #6||Ind_14||3||27 |- | #7||Ind_15||3||27 |- | #8||Ind_16||3||27 |- | #9||Ind_20||3||27 |- | #10||Ind_22||3||27 |- | #11||Ind_23||3||27 |- | #12||Ind_25||3||27 |- | #13||Ind_27||3||27 |} * Actually H2O can be used to dilute adaptor {| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''End repair/ dA-tailed DNA (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount (ng) used in WGBS/ KAPAHyper''' | align="center" style="width:80px;background:#f0f0f0;"|'''Adaptor stock (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | align="center" style="width:60px;background:#f0f0f0;"|'''Indx''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final Adaptor conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Ligation rxn mix (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total''' |- | PCP_2||30||20||15000||1||PCP_2||4||1||273||20||55 |- | PCP_3||30||20||15000||1||PCP_3||4||2||273||20||55 |- | PCP_4||30||6.5||1500||5||PCP_4||0||3||136||20||55 |- | PCP_5||30||20||15000||1||PCP_5||4||4||273||20||55 |- | PCP_6||30||15||15000||1||PCP_6||4||5||273||20||55 |- | PCP_7||30||15||15000||1||PCP_7||4||6||273||20||55 |- | PCP_8||30||10||1500||5||PCP_8||0||7||136||20||55 |- | PCP_9||30||3.5||1500||2.5||PCP_9||2.5||8||68||20||55 |- | 6P-1||30||2.5||1500||2.5||6P-1||2.5||9||68||20||55 |- | 6P-2||30||20||15000||1||6P-2||4||10||273||20||55 |- | 6P-3||30||20||15000||1||6P-3||4||11||273||20||55 |- | 6P-4||30||20||15000||1||6P-4||4||12||273||20||55 |- | 6P-5||30||10||1500||5||6P-5||0||13||136||20||55 |- | 6P-8||30||3.2||1500||2.5||6P-8||2.5||14||68||20||55 |- | 6P-9||30||8.5||1500||5||6P-9||0||15||136||20||55 |- | 6P-10||30||6.4||1500||5||6P-10||0||16||136||20||55 |- | 7P-1||30||15||15000||1||7P-1||4||18||273||20||55 |- | 7P-2||30||20||15000||1||7P-2||4||19||273||20||55 |- | 7P-3||30||2.5||1500||2.5||7P-3||2.5||20||68||20||55 |- | 7P-5||30||20||15000||1||7P-5||4||21||273||20||55 |- | 7P-6||30||3.4||1500||2.5||7P-6||2.5||22||68||20||55 |- | 7P-7||30||3.3||1500||2.5||7P-7||2.5||23||68||20||55 |- | 7P-8||30||1.5||1500||2.5||7P-8||2.5||25||68||20||55 |- | 7-P-10||30||2.5||1500||2.5||7P-10||2.5||27||68||20||55 |- | NTC||30||NA||1500||5||NTC||0||27||136||20||55 |} ==== Adaptor ligation reaction mix ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:80px;background:#f0f0f0;"|'''half rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''26x rxn''' |- | Ligation buffer||30.00||15.00||390.00 |- | DNA ligase||10.00||5.00||130.00 |- | Total||40.00||20.00|| |- | 1:20 diluted adaptor (714nM)||Varied||Varied|| |- | Total||110.00||55.00|| |}
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