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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] * Note: This page was the summary of WGBS prep to share with David Lin from Eppendorf = WGBS sequencing library preparation using KAPA protocol (from Eppendorf) and epMotion = * Note that I used the same total volume of reaction similar to the protocol from Eppendorf. This might be slightly different from the original protocol from KAPA kit, but the total amount of enzyme is the same. I also adjust the volume of adapters in ligation step, but total volume and ratio of adapter to A-tailed DNA are the same. '''Image of sheared gDNA (loaded 0.5ul)'''<br> <br> [[File:ZhangLab_2 2012-07-26 12hr 55min_PAGE-Quan-shearedDNA.png| 350px]] == Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters == == Sample list == '''Labeled''' '''Sample IDs''' 1 GM12891 2 GM12892 3 GM10861 4 GM11994 5 GM11995 6 GM18506 7 GM18507 8 GM18505 '''End repair''' Total reaction in 70ul DNA 50ul End repair reaction mix 20ul KAPA End repair mix 1x rxn 8x rxn mix 10X End repai buffer 7.00 56.00 KAPA End repair enzyme 5.00 40.00 H2O 8.00 64.00 ================================================== Total 20.00 160.00 Aliquot to column #1: 20ul 20C for 30min '''A-tailing''' Total reaction in 30ul A-tailing reaction mix 1x rxn 8x rxn mix KAPA 10X A-tailing buffer 3.00 24.00 KAPA A-tailing enzyme 3.00 24.00 H2O 24.00 192.00 ================================================== Total 30.00 240.00 Aliquot to column #2: 30ul 30C for 30min '''Adapter ligation''' Total reaction in 50ul Ligation reaction mix 40ul Methylation adapter 10ul Ligation reaction mix 1x rxn 8x rxn mix KAPA 5X Ligation buffer 10.00 80.00 KAPA DNA ligase 5.00 40.00 H2O 25.00 200.00 ================================================== Total 40.00 320.00 Aliquot to column #3: 40ul '''Methylation adapter preparation''' 1. 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 2. 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) Methylation adapter 2ulx 8rxn 16.00 H2O 8ulx8rxn 64.00 ================================================== Total 80.00 20C for 15min '''Reagent preparation''' '''Reagents''' '''1rxn''' '''Min volume in 30mL reservior''' AMPure beads 120ul ~1.1mL 20% PEG/2.5M NaCl 90+50 = 140ul ~1.4mL 80% EtOH 140x6 = 840ul ~10.0mL H2O for elution 23ul ~1.0mL ==epMotion run == '''Report file of epMotion run:''' epMotion Started:5:00pm, finished: 7:30pm (~3hr for 8 samples) * Note: ** After adapter ligation I eluted DNA with 23ul H2O, and transfered eluted DNA to 96-well plate (on the same plate, column #3) ** I checked adapter ligated DNA in 6% TBE gel (loaded 0.5 ul each, also load sheared DNA of sample #1 and #2 as control) [[File:ZhangLab_2 2012-07-27 20hr 33min_KAPA-adapter.jpg| 450px]] '''Labeled''' '''Sample IDs''' #1B = #1 before end repair #2B = #2 before end repair #1 GM12891 #2 GM12892 #3 GM10861 #4 GM11994 #5 GM11995 #6 GM18506 #7 GM18507 #8 GM18505 '''2012.07.30'''<br> # Took 1ul of Adapter ligated DNA and added to 8ul EB buffer -> for some validation, like qPCR or PAGE quantification to compare to the bisulfite conversion products '''qPCR to check if the end repair, A-tailing, and adapter ligation works''' {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''10 rxn mix''' |- | DNA template (1:9 diluted)||2.00||0.00 |- | 10uM PCR_F||1.00||10.00 |- | 10uM PCR_R.Ind1||1.00||10.00 |- | 2x iQ MM||25.00||250.00 |- | H2O||21.00||210.00 |- | Total||50.00||500.00 |} Program 95C 2min -> (95C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles 72C 3min 15C Hold # Run the gel check (the same labeled as above picture, but #1B, #1, #2B, #2, #3,....,#8) ## 2ul of 10x diluted adapter ligated DNA (the same amount that added to 50ul qPCR) ## 3ul of PCR product (out of 50ul) [[File:ZhangLab_2 2012-07-30 16hr 17min_adapterligated_qPCR.jpg| 400px]] # From the image above, I can see the amplification of all samples. This suggests that the reaction in the previous steps performed by epMotion worked very well. Even this result is not the final after bisulfite conversion, but it showed that the library prep before bisulfite conversion was success. = 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]] ** For bisulfite conversion in the deep well plate, I did it manually since we don't have TM 1000-8 pipette and discarding spnt multiple rounds with TM 300-8 pipette might be prone to contamination * Elute with elution buffer 40ul == 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| 700px]] <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> * Note: To validate the quality of sequencing library, Dr. Zhang suggested to do small scale sequencing before doing deep sequencing, so I pooled small amount of each sequencing library and performed PAGE-size selection. == PAGE quantification of 400-600bp fragments == * Cut the band around 400-600bp 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 PAGE quantification (also verify the size of sequencing libraries) and by dsDNA Qubit -> conc. 1.47ng/ul or 4.83nM * '''Library ID: NP_WGBS_HAPMAP1-8_July27''', sequenced on GAII '''HL128''' run. The eight libraries were spiked 1/10 in lane 1. == Mapping result of NP_WGBS_HAPMAP1-8 sequencing libraries == * Mapping 1st read to hg19 {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Total # of reads''' | align="center" style="background:#f0f0f0;"|'''Mappable reads''' | align="center" style="background:#f0f0f0;"|'''Mapping rate (%)''' | align="center" style="background:#f0f0f0;"|'''Bisulfite conversion rate (%)''' |- | GM10861**||7,419,387||583,598||7.87||98.70 |- | GM11994||687,322||604,611||87.97||99.30 |- | GM11995||774,373||689,816||89.08||99.10 |- | GM12891||715,640||631,161||88.20||98.50 |- | GM12892||638,358||553,167||86.65||99.30 |- | GM18505||810,806||717,095||88.44||99.40 |- | GM18506||586,667||513,178||87.47||99.30 |- | GM18507||686,850||606,360||88.28||99.10 |} * Note: Sample ID GM10861 has about 10times more sequencing read than other libraries. I think this was because it was loaded on the same lane with bacterial sequencing library, which used the same barcode.
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