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= DNA methylation assay for Blueprint project = * [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] * Continued from: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-5-30]] * I aliquoted ~40ul of HAPMAP gDNA sample from the plate (well ID A1, B1 -> actual IDs will be added later). I used 2 IDs just in case the amount from plate#1 is not enough for all tests, and I wanted to save DNA in the second plate for other experiments if necessary. * I again did DNA concentration measurement using both Nanodrop and dsDNA Qubit assay to get the final decision that for BSPP capture experiment, which methods I should use to get the most accurate result as the yield after bisulfite conversion matters for capture efficiency. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''A260''' | align="center" style="background:#f0f0f0;"|'''A280''' | align="center" style="background:#f0f0f0;"|'''260/280''' | align="center" style="background:#f0f0f0;"|'''260/230''' | align="center" style="background:#f0f0f0;"|'''Conc. By Nanodrop (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Conc. by Qubit (ng/ul)''' |- | HAPMAP-A1||3.827||1.915||2||1.46||191.36||71.60 |- | HAPMAP-B1||3.667||1.934||1.9||1.29||183.37||88.10 |} * From the table above, it's clear that Nanodrop gave the overestimated concentration as I saw before. This should be due to the impurities in gDNA samples. To avoid the variation of impurities of gDNA sample from batch to batch, I will stick to Qubit measurement. === Experimental plan I === * I will perform bisulfite conversion using different amount of gDNA to see the recovery rate I can get (250, 500, 750ng) * I need to use 2 sample for the test since I don't have enough gDNA from plate1 == Bisulfite conversion == * I did bisulfite converssion by using '''EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo''' and followed the protocol of the kit * I did two replicates for each amount ==== Bisulfite conversion set up ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' |- | HAPMAP-A1||71.60||3.49||16.51||130.00||150.00||250.00 |- | HAPMAP-A1||71.60||6.98||13.02||130.00||150.00||500.00 |- | HAPMAP-B1||88.10||5.68||14.32||130.00||150.00||500.00 |- | HAPMAP-B1||88.10||8.51||11.49||130.00||150.00||750.00 |} ''' Program '''<br> 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold * Eluted bisulfite converted DNA form the bead with 20ul H2O * I used 2ul for ssDNA Qubit assay. The volume left after assay ~13ul. Next time I may use only 1ul for Qubit assay to save DNA sample for capture, since 1ul pipetting is accurate enough and the concentration is in the measurable range. ==== ssDNA Qubit assay result ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Start amount (ng)''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yield in 15ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Yield left after <br>Qubit (13ul)''' | align="center" style="background:#f0f0f0;"|'''% Recovery in 15ul''' |- | 1) HAPMAP-A1||250||6.07||91.05||78.91||36.42 |- | 2) HAPMAP-A1||250||5.89||88.35||76.57||35.34 |- | 3) HAPMAP-A1||500||13.70||205.50||178.10||41.10 |- | 4) HAPMAP-A1||500||12.50||187.50||162.50||37.50 |- | 5) HAPMAP-B1||500||15.90||238.50||206.70||47.70 |- | 6) HAPMAP-B1||500||17.00||255.00||221.00||51.00 |- | 7) HAPMAP-B1||750||25.90||388.50||336.70||51.80 |- | 8) HAPMAP-B1 ||750||25.20||378.00||327.60||50.40 |- | BSPP_Blueprint||NA||25.40||1651.00||NA||NA |} * Note: I measure the BSPP_Blueprint probe using ssDNA Qubit assay as well. The concentration was about half of the concentration by PAGE quantification. This makes sense because the low mass ladder always showed a smear in TBU. This caused the actual amount in each band was lower than it's supposed to be and the quantification will be overestimated. I will use conc. from Qubit for probe concentration as well. ==== Discussion about bisulfite conversion ==== * Will be added === Experimental plan II === * I will do BSPP capture by varying bisulfite converted gDNA amount to see the minimum amount of 12K BSPP probe set that work well for capture experiment. * I will also vary the probe: target ratio. As I know that the DMR330k BSPP probe set works well with the ratio ~100:1 - ~200:1, but I have adjusted the probe:target ratio in each set. In addition the DMR330K are muck more larger than the BSPP_Blueprint probe set. I may need to increase the probe:target ratio. * I will set up BSPP capture with template amount 50ng, 100ng, 150ng and 300ng (200-300ng is the amount that I usually used and worked well for previous experiment) * Each template amount will be captured with 200:1 and 500:1 probe:target. I actually should have done 1000:1 for comparison too. == BSPP capture set up == ==== Probe calculation (Example) ==== {| {{table}} | Probe:target||200:1|| |- | Probe size||12000|| |- | Template||50||ng |- | Human gDNA MW||1.9500E+12||g/mol (3E+09 * 650Da/bp+157.9Da) |- | Human gDNA (50ng)||2.5641E-20||mol (50ng/1.9500E+12 g/mol) |- | Probe required (200:1)||5.1282E-18||mol (2.5641E-20 * 200) |- | Probe MW (12,000 probes, 110nt)||4.2900E+08||g/mol (12000 probes * 110nt * 325Da/nt + 79Da)) |- | Amount probe required||2.2000E-09||g |- | Amount probe required||2.20||ng |}<br> {| border = 1 | align="center" style="background:#f0f0f0;"|'''Test #''' | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Amount (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume required (ul)''' | align="center" style="background:#f0f0f0;"|'''Probe amount<br>required (ng)''' | align="center" style="background:#f0f0f0;"|'''25.4ng/ul probe (ul)''' | align="center" style="background:#f0f0f0;"|'''2ng/ul probe (ul)''' | align="center" style="background:#f0f0f0;"|'''10X AmpLigase Buffer''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | BP-test # 1|| A1 tube1||6.07||50||8.24||2.20||NA||1.10||2.00||8.66||20.00 |- | BP-test # 2|| A1 tube2||5.89||50||8.49||5.50||NA||2.75||2.00||6.76||20.00 |- | BP-test # 3|| A1 tube 3||13.70||100||7.30||4.40||NA||2.20||2.00||8.50||20.00 |- | BP-test # 4|| A1 tube 4||12.50||100||8.00||11.00||NA||5.50||2.00||4.50||20.00 |- | BP-test # 5|| B1 tube 1||15.90||150||9.43||6.60||NA||3.30||2.00||5.27||20.00 |- | BP-test # 6|| B2 tube 2||17.00||150||8.82||16.50||NA||8.25||2.00||0.93||20.00 |- | BP-test # 7|| B3 tube 3||25.90||300||11.58||13.20||0.52||NA||2.00||5.90||20.00 |- | BP-test # 8|| B4 tube 4||25.20||300||11.90||33.00||1.30||NA||2.00||4.80||20.00 |} '''200:1 probe:target ratio''' BP-test # 1 BP-test # 3 BP-test # 5 BP-test # 7 '''500:1 probe:target ratio''' BP-test # 2 BP-test # 4 BP-test # 6 BP-test # 8 '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. ==== SLN mix solution ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume (ul), total 18ul''' |- | AmpliTaq Stoffel||10||U/ul||2||U/ul||3.60 |- | AmpLigase||5||U/ul||0.5||U/ul||1.80 |- | dNTP||1||mM||100||uM||1.80 |- | 10x AmpLigase Buffer||10||x||1||x||1.80 |- | H2O||||||||||9.00 |} == PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind1-8) == * I did PCR by adding 10ul of circularized DNA in total 50ul. I used smaller volume because the template amount should be less than the circularized DNA from 330k probe set and I actually did PCR test before (add 2ul of circularized DNA in total 25ul, and the curve shoot up at approx. cycle 14) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''8.5x rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||1.00||8.50 |- | 10uM AmpR6.3Indx (X=1-8)||1.00||0.00 |- | 2X KAPA SYBR MM||25.00||212.50 |- | H2O||13.00||110.50 |} * Aliquot 39ul, add 1ul of AmpR6.3Indx, 10ul of circularized DNA '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min '''qPCR curve''' [[File:PCRcurve_BlueprintAmp.png| 700px]] * I did the second qPCR by using the same condition as above and got the same result. * Purified all PCR products with 0.8 vol of AmPure beads (I diluted AMPure bead 1:1 ratio with 20%PEG/2.5M NaCl) * Eluted with 30ul H2O * Performed PAGE quantification by loading sample 3ul (1/10 of total purified PCR product). I need to load large volume to make sure I would see the band for PAGE quantification for library pooling. [[File:ZhangLab_2 2013-06-11 18hr 21min_beadpurified_BLUEPRINT.jpg| 500px]] * From the results above, I saw multiple bands from amplification, including ~200bp, 350bp, 400bp and a smear above 400bp band up to almost 500bp. * This result was different from the DMR330k probe capture (~375bp, gap size 180bp). Based on the probe design for this probe set with gap size between 200-280bp, I expected to get the fragments size ~ 400-480bp). * The smaller sizes ~200bp and ~350 should not be the expected product since the size seemed to be too small. I always saw very weak signal of these two bands in DMR330k probe set, so they should be the background. * The result clearly showed that increasing probe:target ratio significantly increased the capture efficiency. I should have tried 1000:1 ratio in this experiment. However, I believe to get higher efficiency when using 1000:1 ratio. I might use 1000:1 ratio for the real experiment. * Increasing template amount also increasing capture efficiency as well, but 50ng template may be too low for capture. Sequencing result should tell the quality of data and target coverage by varying these two factor. * There might be something wrong for 300ng , 500:1 sample since the amplification was poor and PAGE analysis agreed with qPCR curve. I suspected that SLN mix may not delivered right to the whole reaction since the tube of this samples was on the innermost of thermocycle and I couldn't add the SLN mix properly because of the lid. Next time, I should be more careful when add any reagent to the reaction. ''' 2013-06-13 ''' * I did PAGE quauntification on the fragments size 400-500bp. Since some samples have very low concentration, I combined them into two pools and did PAGE size-selection ** NP-BSPP-BP-TEST1-1-June6, 2013 Index 1,2,3,5,8 ** NP-BSPP-BP-TEST1-2-June6, 2013 Index 4,6,7 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 27ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Pool1 volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Pool2 volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Amount (ng)''' |- | BP-test # 1||AmpR6.3Ind1||0.03||0.83||27.00||NA||0.83 |- | BP-test # 2||AmpR6.3Ind2||0.22||5.91||27.00||NA||5.91 |- | BP-test # 3||AmpR6.3Ind3||0.75||20.36||19.89||NA||15.00 |- | BP-test # 4||AmpR6.3Ind4||1.87||50.40||NA||26.78||50.00 |- | BP-test # 5||AmpR6.3Ind5||1.21||32.76||12.36||NA||15.00 |- | BP-test # 6||AmpR6.3Ind6||2.88||77.65||NA||17.39||50.00 |- | BP-test # 7||AmpR6.3Ind7||2.33||62.86||NA||21.47||50.00 |- | BP-test # 8||AmpR6.3Ind8||0.57||15.44||27.00||||15.44 |} ''' PAGE size-selection '''<br> [[File:ZhangLab_2 2013-06-13 12hr 23min_PAGE-SS.jpg| 350px]] * Each pool was purified in 2-well of 6% 5-well TBE gel * Resuspended pool1 with 10ul H2O * Resuspended pool2 with 15ul H2O * Alan will do qPCR to quantify concentration ''' MiSeq: MiseqJun18: PE, 2x250bp ''' <br>
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