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= Bisulfite conversion using EZ-96 DNA Methylation-Lightning™ MagPrep = * Performed bisulfite conversion manually and followed the product's protocol: [[Media:EZ-96DNAMethylation-LightninMagPrep.pdf| EZ-96DNAMethylation-LightninMagPrep.pdf]] * Elute with elution buffer 40ul == ssDNA Qubit quantification == * Used 3ul of bis-cvt DNA for the assay (dilution 200/3) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''uL used''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 40ul''' |- | #1||17.2||ng/mL||3||66.67||1.15||45.87 |- | #2||4.4||ng/mL||3||66.67||0.29||11.73** |- | #3||12.3||ng/mL||3||66.67||0.82||32.80 |- | #4||65.6||ng/mL||3||66.67||4.37||174.93 |- | #5||32.9||ng/mL||3||66.67||2.19||87.73 |- | #6||7.16||ng/mL||3||66.67||0.48||19.09** |- | #7||48.3||ng/mL||3||66.67||3.22||128.80 |- | #8||30.6||ng/mL||3||66.67||2.04||81.60 |} * The yield after bisulfite conversion is relatively low when compare to initial amount of DNA. However, the DNA amount would be reduced in each step. From the image, bead purification did'n recover the fragments < ~200bp, so I don't expect to get very high after bisulfite conversion. The total yields I got after bisulfite conversion are varied as well, and the yield of some sample didn't correlated with the intensity before bisulfite conversion. However, I could not rely on to this image because it is the image of previous experiment. Next time I should collect sample for verification. * **= samples have very low yields --> If compare to the intensity and total yield after amplification with barcode primers, these concentrations by ssDNA Qubit quantification seemed to agree to the results below. == Amplification of bisulfite sequencing llibraries (Test) == * Test if the adapter ligation reaction worked before amplify the libraries with barcoded primers {| {{table}} | width="160pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="60pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''10 rxn mix''' |- | DNA template ||2.50||0.00 |- | 10uM PCR_F||0.50||5.00 |- | 10uM PCR_R.Ind1||0.50||5.00 |- | 2x iQ MM||12.50||125.00 |- | H2O||9.00||90.00 |- | Total||25.00||250.00 |} * Aliquot 22.5ul, add 2.5ul of bis-cvt DNA template (2.5 out of 40ul) '''Program''' 98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x15cycles -> 72C 3min -> 15CHold '''qPCR results'''<br> [[File:20120801_qPCR_adapterligated.png| 600px]] <br> * Loaded 2.5 ul of bis-cvt DNA (#1 & #4) and 2.5ul of amplified amplicons [[File:ZhangLab_2 2012-08-02 11hr 31min-HAPMAP-bis-test.jpg| 400px]] <br> == Amplification of bisulfite sequencing libraries with N2 barcoded primers == {| {{table}} border = 1 | width="160pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="60pt" align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''26rxn mix''' |- | DNA template||10.00||0.00 |- | 10uM PCR_F||2.00||52.00 |- | 10uM PCR_R.Indx (1-8)||2.00||0.00 |- | 2x iQ Supermix||50.00||1,300.00 |- | H2O||36.00||936.00 |} * Aliquot 88ul, add 2ul of 10uM PCR_R Ind (Ind1-8), 10ul of bis-cvt DNA template '''Program''' 98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x10cycles -> 72C 3min -> 15CHold {| {{table}} | width="80pt" align="center" style="background:#f0f0f0;"|'''Sample ID''' | width="70pt" align="center" style="background:#f0f0f0;"|'''Labeling''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | WGBS.GM12891||#1||PCR_R.N2Indx1 |- | WGBS.GM12892||#2||PCR_R.N2Indx2 |- | WGBS.GM10861||#3||PCR_R.N2Indx3 |- | WGBS.GM11994||#4||PCR_R.N2Indx4 |- | WGBS.GM11995||#5||PCR_R.N2Indx5 |- | WGBS.GM18506||#6||PCR_R.N2Indx6 |- | WGBS.GM18507||#7||PCR_R.N2Indx7 |- | WGBS.GM18505||#8||PCR_R.N2Indx8 |} '''qPCR results'''<br> [[File:20120801_qPCRWGBS1-8-1.png| 600px]] <br> * Purified sequencing libraries with 0.8x vol AMPure bead with epMotion (3 strips), eluted with 25ul EB for each well -> total volume at the end ~70ul '''Report file of epMotion run:'''[[Media:MasterReport_20120802_beadPure.txt]]<br> [[File:BeadPure_Aug2.png| 500px]] [[File:ZhangLab_2 2012-08-02 16hr 05min_bead-HAPMAP-WGBS.jpg| 400px]] <br> == PAGE quantification of 400-600bp fragments == * Dr. Zhang suggested to cut the band around 400-600bp because 250bp is too short for PE sequencing. * Perform PAGE size-selection of the size range 400-600bp for a small scale sequencing. Then pooled 60ng of each library and performed PAGE size-selection. [[File:ZhangLab_2 2012-08-02 16hr 05min_bead-HAPMAP-WGBS-400-600bp_PQ.jpg| 400px]]<br> {| {{table}} border = 1 | width="60pt" align="center" style="background:#f0f0f0;"|'''Lab IDs''' | align="center" style="background:#f0f0f0;"|'''Final conc. of 400-600bp (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total yields in 70ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 60ng (ul)''' |- | #1||13.50||945.28||4.44 |- | #2||9.66||675.94||6.21 |- | #3||11.24||786.94||5.34 |- | #4||13.64||955.09||4.40 |- | #5||13.85||969.55||4.33 |- | #6||8.95||626.85||6.70 |- | #7||12.40||867.78||4.84 |- | #8||11.29||790.44||5.31 |}<br> == PAGE size-selection == [[File:ZhangLab_2 2012-08-02 18hr 23min-PAGE-SS.jpg| 350px]] * Note: From the image, it seemed like I cut the gel a little smaller than 600bp * Resuspend with 12ul H2O each tube -> total volume 24ul. * Quantify concentration by dsDNA Qubit HS -> conc. 1.47ng/ul or 4.83nM [[File:ZhangLab_2 2012-08-28 14hr 52min_PQ_NP_WGBS_HAPMAP_Aug01.png| 200px]] - Concentration by PAGE quantification is 4.42ng/ul * '''Library ID: NP_WGBS_HAPMAP1-8_July27''', sequenced on GAII '''HL128''' run. The eight libraries were spiked 1/10 in lane 1.
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