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[[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]<br> = WGBS sequencing library preparation using KAPA protocol (Eppendorf) and epMotion (Repeat!) = * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-27 * I have sheared gDNA from the previous batch left ~22ul (conc. ~40ng/ul based on conc. from the HAPMAP plate). I added 25ul H2O to each tube -> total volume 47ul, and will prepare end repair reaction mix in 23ul * Note: Since I start with sheared 880ng, I reduce the amount of enzymes in each step, but keep total volume the same (except ligation 50ul -> 40ul total) == End repair == {| {{table}} | width ="150pt" align="center" style="background:#f0f0f0;"|'''Total reaction''' | align="center" style="background:#f0f0f0;"|'''70.00''' | align="center" style="background:#f0f0f0;"|'''ul''' |- | DNA ||47.00||ul |- | End repair reaction mix ||23.00||ul |}<br> {| {{table}} | width ="150pt" align="center" style="background:#f0f0f0;"|'''KAPA End repair mix''' | width ="80pt" align="center" style="background:#f0f0f0;"|'''1x rxn''' | width ="80pt" align="center" style="background:#f0f0f0;"|'''8x rxn mix''' |- | 10X End repai buffer||7.00||56.00 |- | KAPA End repair enzyme||3.00||24.00 |- | H2O||13.00||104.00 |- | Total||23.00||184.00 |} * Aliquot to column #1: 23ul == A-tailing == A-tailing reaction mix: Total 30ul/reaction<br> {| {{table}} | width ="150pt" align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix''' | width ="80pt" align="center" style="background:#f0f0f0;"|'''1x rxn''' | width ="80pt" align="center" style="background:#f0f0f0;"|'''8x rxn mix''' |- | KAPA 10X A-tailing buffer||3.00||24.00 |- | KAPA A-tailing enzyme||2.00||16.00 |- | H2O||25.00||200.00 |- | Total||30.00||240.00 |} * Aliquot to column #2: 30ul == Adapter ligation == Adapter ligation mix: Total 40ul/reaction -> 10ul is methylation adapters {| {{table}} | width ="150pt" align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | width ="80pt" align="center" style="background:#f0f0f0;"|'''1x rxn''' | width ="80pt" align="center" style="background:#f0f0f0;"|'''8x rxn mix''' |- | KAPA 5X Ligation buffer||8.00||64.00 |- | KAPA DNA ligase||3.00||24.00 |- | H2O||19.00||152.00 |- | Total||30.00||240.00 |} * Aliquot to column #3: 30ul == Methylation adapter preparation == '''Alan's note:''' <br> # Use the methylated adapter with paired-end pcr primer pairs(PCR primer PE1.0 and PCR primer PE 2.0) provided in illumina's paired end genomic DNA sample prep kit # Use 10ul of methylated adapter to 5ug gDNA insert, if starting DNA amount is less than 5ug, titrate the volume of adapter reagent accordingly to maintain 10:1 ratio of DNA. Since I used 1ug of DNA, used 2ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same) Prepare adapter 2ulx 8rxn --> 16.00ul<br> H2O 8ulx8rxn --> 64.00ul<br> Total 80.00ul<br> == Reagent preparation == {| {{table}} | width ="150pt" align="center" style="background:#f0f0f0;"|'''Reagents''' | width ="80pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | width ="120pt" align="center" style="background:#f0f0f0;"|'''Min volume in 30mL reservior''' |- | AMPure beads||120ul||~1.1mL |- | 20% PEG/2.5M NaCl||90+50 = 140ul||~1.4mL |- | 80% EtOH||140x6times||~10mL |- | H2O for elution||22ul||~1mL |} ==epMotion run == '''Report file of epMotion run:''' [[Media:MasterReport_LibPrep_HAPMAPAug1.txt]]<br> [[File:LibPreP_HAPMAP_Aug1.png| 500px]] * Note: I used all adapter ligated DNA for bisulfite conversion, so I don't have the image showing DNA before and after end repair, A-tailing and adapter ligation. However I did most of the step the same as I did on July 27. '''Image of DNA before end repair and after adapter ligation performed on 2012.07.27 [[File:ZhangLab_2 2012-07-26 12hr 55min_PAGE-Quan-shearedDNA.png| 350px]] [[File:ZhangLab_2 2012-07-27 20hr 33min_KAPA-adapter.jpg| 400px]] = 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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