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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]] ** 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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