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Alice:Whole Genome Bisulfite Sequencing Lab Notes/WGBS
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====Sequencing library preparation==== *In order to be consistent with WGBS data, I will use IQ MM for PCR instead of KAPA *The primer design of first round and second round PCR can be found here: **briefly, we first design primers that span part (or entire) region of the DMR identified. Then we add part of the Y-adaptor onto the end of both primers. **It is the complementary part of the Y-adaptor that will be added onto the end of both primers, then we can use PCR_F and PCR_N2R for 2nd round PCR **add in front of all Forward primer: 5' GCGAGAAGGCTAG 3' **add in front of all Reverse primer: 5' GCGAGAAGGCTAG 3' *sample info: {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''sample name''' | align="center" style="background:#f0f0f0;"|'''N2 barcode''' |- | 1||NB1||PCR_N2R10 |- | 2||SCNTB7||PCR_N2R11 |- | 3||SCNTB8||PCR_N2R12 |- | 4||SCNTB9||PCR_N2R13 |- | 5||SCNTB10||PCR_N2R14 |- | 6||SCNTB11||PCR_N2R15 |- | 7||SCNTB13||PCR_N2R16 |- | 8||B6P1-1||PCR_N2R17 |- | 9||B6P1-2||PCR_N2R18 |- | 10||miPSD1||PCR_N2R19 |- | 11||miPSD3||PCR_N2R20 |- | 12||miPSD8||PCR_N2R21 |- | 13||ZN_MEF||PCR_N2R22 |- | 14||TB_MEF||PCR_N2R23 |- | 15||2eiPS2P5||PCR_N2R24 |} '''PCR''' {| {{table}} | Width="200pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''9 rxn mix (9 primer pairs)''' |- | bis-cvt DNA||1.00||0.00 |- | 10uM Me-Enhancer primer F||1.00||0.00 |- | 10uM Me-Enhancer primer R||1.00||0.00 |- | 2x iQ MM (Biorad)||25.00||188 |- | H2O||21.00||160 |- | Total||50.00||375 |} '''PCR program''' *after two failed runs with 98C as denaturing temperature, final program used for big batch processing: 95C 2min --> (95C 15sec -> 58C 20sec -> 72C 30sec) x 26-28 cycles --> 72C 3min --> 15C Hold *combine the PCR amplicon of different targets into the same tube for the same sample, then perform Qiaquick column purification and elute in 33ul H2O *so now there are 15 samples (each with all 9 targets combined into one tube) to run 2nd PCR '''2nd PCR with Illumina primers''' {| {{table}} | Width="200pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''15 rxn mix''' |- | bis-cvt DNA targets pooled||10.00||- |- | 10uM PCR F||1.00||16.00 |- | 10uM PCR R_N2 barcoded||1.00||- |- | 2x iQ MM (Biorad)||25.00||380 |- | H2O||21.00||315 |- | Total||60.00|| |} '''PCR program''' 95C 2min --> (95C 15sec -> 60C 30sec -> 72C 30sec) x 32 cycles --> 72C 3min --> 15C Hold *Qiaquick column purification and elute in 42ul H2O each (15 total) *loaded 2ul of purified samples for PAGE gel validation *PAGE gel results: samples 1,2,4,9,13 had high yield, while the rest of them had very concentrations *so I did the pooling based on eye-balling the gel results: **3ul of the samples with high yield (sample 1, 2, 4, 9, 13) **12ul of the samples with relative low yield (sample 10, 11, 14) **15ul of the samples with low yield (sample 3, 5, 6, 7, 8, 12, 15) *after pooling, used the total volume ~156ul for gel size selection (~120-220bp bands) *ethanol precipitation and elute in total 60ul H2O (blue color due to ~3ul of glycoblue per tube, total of 18ul, was added during first step) *gel validation *Results: the sequences seem to be over 90% junk reads that mapped to other organisms. **Maybe due to difficulty to amplify non-coding region, and the primers include too many mismatches because of the Illumina primers attached to the end.
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