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= LMS + CRC BSPP capture on WGBS libraries prepared from ctDNA of CRC patient (Illumina) = [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]<br> [[Noi/NOTES/2014-12-5|'''Continued from 2014-12-05''']]<br> * Conditions: ** Probe set: MONOPD V5 set A (LMS) + CRC from Illumina ** Probe:target ratio: 200:1 for LMS probe set, 500:1 for CRC probe set from Illumina ** Sample: WGBS libraries prepared by KAPA Hyper Prep Kit [[Noi/NOTES/2014-12-5|'''(2014-12-05)''']] amount 400ng ==== Probe calculation (Example) ==== '''Probe set Probe size Probe:target ratio''' LMS 73,574 200:1 CRC 1,024 500:1 <br> {| {{table}} | align="center" style="width:220px;background:#f0f0f0;"|'''Probe:target''' | align="center" style="width:120px;background:#f0f0f0;"|'''200:1''' | align="center" style="background:#f0f0f0;"|'''''' |- | Probe size||73574|| |- | Template||400||ng |- | Human gDNA MW||1.95E+12||g/mol (3E+09 * 650Da/bp+157.9Da) |- | Human gDNA (400ng)||2.05128E-19||mol (400ng/1.9500E+12 g/mol) |- | Probe required (200:1)||4.10256E-17||mol (2.5641E-20 * 200) |- | Probe MW (73,574 probes, 110nt)||2.636E+09||g/mol (73,574 probes * (110nt * 325Da/nt + 79Da)) |- | Amount probe required||1.081E-07||g (2.051E-17mol * 2.636E+09g/mol) |- | Amount probe required||108||ng |- | |||| |- | CRC required (200:1)||1.50||ng |- | CRC required (500:1)||3.76||ng |} '''Probe set Amount required(ng) Conc. (ng/ul) Volume/rxn''' MONOD-V5A(LMS) 108.10 12.10 8.93 CRC 3.76 13.20 0.28 == BSPP capture == === Capture set up === ==== Things to do in order ==== * Set up capture reaction in total 25 μL in 8-tube strip (96-well plate if more samples are processed) with the following components: 400 ng of WGBS DNA, 200:1 of probe to target ratio and 1x Ampligase Buffer. # Calculate volume of WGBS libraries required for 400ng ## Adjust total volume to 50ul (could be any number) ## Evaporate WGBS libraries in 1.5mL LoBind tube to reduce total volume to fit 25ul capture reaction including probes and 10X Ampligase Buffer ## Resuspend evaporated WGBS libraries with H2O and transfer to 0.2mL 8-tube strip or 96-well plate based on number of sample # Calculate the amount of each probe set required. The amount is based on probe to target ratio and template amount. # Prepare probes and 10X Ampligase buffer mix ## Add probe/buffer mix to the tube with WGBS libraries ## Mix well (spinand layer with equal volume of mineral oil (25ul of mineral oil to 25ul of capture reaction) ## Spin down the tube again before incubate on thermocycler as the following program: 94 ºC for 30 s, gradually lower temperature to 55 ºC at 0.02 ºC/s in a thermocycler, and let hybridize at 55 ºC for 20 hours (This is requirement. Not recommend to incubate shorter than 20h) ==== WGBS libraries ==== * We require 400ng of WGBS library for BSPP capture. The total volume of 400ng is quite large and exceed 25ul which is the total volume of BSPP capture. I simply pipette 400ng of WGBS libraries to 1.5mL LoBind tube. Then add H2O to bring total volume to 50ul. {| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 70ul (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 400ul (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O to 50ul''' |- | KU_1||9.89||692.30||40.44||KU_1||9.56 |- | KU_2||14.90||1,043.00||26.85||KU_2||23.15 |- | KU_3||11.00||770.00||36.36||KU_3||13.64 |- | KU_4||14.30||1,001.00||27.97||KU_4||22.03 |- | KU_5||10.10||707.00||39.60||KU_5||10.40 |- | KU_6||12.70||889.00||31.50||KU_6||18.50 |- | KU_7||15.60||1,092.00||25.64||KU_7||24.36 |- | KU_8||9.22||645.40||43.38||KU_8||6.62 |- | KU_9||9.09||636.30||44.00||KU_9||6.00 |- | KU_10||8.17||571.90||48.96||KU_10||1.04 |} * I evaporated WGBS libraries by closing the lid of the tube and made a hole by poking with needle size G22 (3 hole/tube) and evaporate at 60C for ~15min. The left over volume was between 5-12ul. I think to make it simple, I just dry up the whole tube then resuspend with equal volume of H2O to fit the capture volume. In this experiment I adjust total volume of WGBS library to 12ul with H2O ==== Probe Buffer Mix ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:80px;background:#f0f0f0;"|'''11.5 rxn mix''' |- | MONOD V5A (LMS) (12.1 ng/ul)||8.93||102.70 |- | CRC (13.2ng/ul)||0.28||3.22 |- | 10X Ampligase Buffer||2.50||28.75 |- | H2O||1.29||14.84 |- | Total||13.00|| |} '''Program''' -> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2.5ul PLN mix (20%v/v Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP) --> '''No need to change program while adding PLN mix (keep the tube at 55C all the time)''' -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 5min -> 10C hold. --> '''Press proceed once''' before adding ExoI/III and waiting until temperature down to 37C before adding ExoI/III ==== Actual thermocycle program ==== :- 1: 95C 5min :- 2: 94.8C, 30s ::- -0,2C/cycle (increment -0.2C/cycles) :- 3: Go to 2 193 times :- 4: 55C forever :- 5: 94C, 2min :- 6: 37C, 2h :- 7: 94C, 5min :- 8: Hold at 4-10C forever or continue to PCR * Note: After adding PLN, the incubation time could be varied between 2-20h. === PLN (Polymerase/Ligase/ dNTP) mix solution Mix === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Stock conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Prepare volume (ul), total 38ul''' |- | Hemo KlenTaq||100||%||20||%||7.60 |- | AmpLigase||5||U/ul||0.5||U/ul||3.80 |- | dNTP||1||mM||100||uM||3.80 |- | 10x AmpLigase Buffer||10||x||1||x||3.80 |- | H2O||||||||||19.00 |} # Prepare PLN solution mix as follows: 20% (v/v) Hemo KlenTaq, 0.5 unit/μL Ampligase, 100 μM of dNTP mix, and 1x Ampligase Buffer. # Aliquot PLN mix to 8-tube strip and add 2.5 μL of KLN solution mix to each well with 10 μL multichannel pipette. Make sure that PLN mix is added under the mineral oil layer. # Swirl pipette 10 times to mix the capture reaction without moving the plate from thermal cycler. # Continue to incubate at 55 ºC for 4-20 hours. # Denature DNA at 94 ºC for 2 minutes. === Exonuclease digestion === # Prepare exonuclease I/III mix by mixing exonuclease I (20 units/μL) and exonuclease III (200 units/μL) in 1:1 ratio and aliquot equal volume to 8-tube strip # Add 2 μL of exonuclease I/III mix to each well with multichannel pipette # Mix the reaction by swirling pipette around the well 10 times. # Incubate reaction at 37 ºC for 2 hours. # Heat inactivate exonuclease I/III at 94 ºC for 5 minutes. # Continue to qPCR without any purification or store at 4C == Amplification == * I normally do a quick test PCR in small volume ~10 or 20ul with 10% v/v of captured DNA. This to monitor PCR cycle number and check if we get the right amplified product by running PCR product in 6% TBE gel without bead/colum purification. If the experiment has been optimized for a certain probe set, may not need to do quick qPCR check. ==== Quick PCR ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Stock conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc. In PCR''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''13x rxn''' |- | Captured template||100%||10%||2.00||0.00 |- | 10uM AmpF6.4Sol||10uM||0.2uM||0.40||5.20 |- | 10uM AmpR6.3.IndX 8||10uM||0.2uM||0.40||5.20 |- | 2X Phusion Flash High-Fidelity MM||2X||1X||10.00||130.00 |- | 50X SYBR||50X||0.4X||0.16||2.08 |- | H2O||||||7.04||91.52 |- | Total||||||20.00|| |} :- Aliquot 18ul + 2ul of captured DNA template * Note: In PCR, I included one of positive control (PTC) of captured templates from gDNA of tumor tissue sample to compare the size of amplified amplicons. ::- ''98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x10-15 cycles -> 72C 3min'' :- In this experiment, we stopped at 18 cycles :- Loaded 3ul of PCR product in 6% TBE gel without purification (Run at 250 volts in 0.5X TBE buffer for 25min). [[File:ZhangLab_2 2014-12-10 15hr 14min_quickPCR-capture-18x-3ul.jpg| 500px]] [[File:ZhangLab_2 2014-12-10 15hr 14min_quickPCR-capture-18x-3ul-gel2.jpg| 250px]] PTC: Positive control NTC: Negative control I am not pretty sure what is the larger fragment is. We use enzyme without strand displacement activity, but not sure if there is some activity still exist. :- From PAGE analysis, the pattern of amplified amplicon looked similar to positive control of capture on gDNA. This is a good sign! The intensity look a little faint at 3ul loading. I will increase one more cycle (19X) for large volume amplification. === PCR in large volume set up === ==== IMPORTANT NOTE: ==== * We try to amplified every captured molecules, so at least 2/3 of captured reaction should be amplified by PCR. * We can use any proofreading DNA polymerase for amplification such as 2XPhusion® High-Fidelity PCR Master Mix from NEB or 2X KAPA HiFi HotStart ReadyMix from KAPABiosystem. We do not use regular Taq-based DNA polymerase to avoid strand displacement during amplification and it will result in large amplified amplicons. <br> {| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''11.5x rxn''' |- | Captured template||20.00||0.00 |- | 10uM AmpF6.4Sol||4.00||46.00 |- | 10uM AmpR6.3.IndX 8||4.00||0.00 |- | 2X Phusion Flash MM||100.00||1,150.00 |- | 50X SYBR||1.60||18.40 |- | H2O||70.40||809.60 |- | Total||200.00|| |} :- Aliquot 176ul + 4ul of 10uM AmpR6.3 IndXX + 20ul template (avoid pipetting mineral oil, I always spin down the tube before pipetting) :- Mix well and split into 2X of 100ul :: ''98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x11 -> 72C 3min (total 19 cycles)'' * Use the same condition as in quick test PCR, but amplify in larger volume (prepare 200ul PCR/sample --> split to 2X of 100ul reaction with indexing barcode primers, reverse primer) # In 100ul PCR reaction, it consists of 10 μL of circularized DNA, 200 nM each of AmpF6.4Sol primers and AmpR6.3 indexing primer, and 1x Phusion Flash mastermix. # The PCR programs of Phusion Flas are 98 ºC for 30 s, 8 cycles of 98 ºC for 10 s; 58 ºC for 20 s; and 72 ºC for 20 s, 10-15 cycles of 98 ºC for 10 s and 72 ºC for 20 s, and final extension at 72 ºC for 3 minutes. Monitor qPCR during amplification and stop the reaction before the fluorescent curve reaches plateau to prevent over-amplification. # Verify the amplified amplicons in 12-well 6% TBE gel by loading 3 μL of PCR products and run at 250 V in 0.5x TBE buffer for 25 minutes. # Stain the gel with 1x SYBR Gold in 0.5x TBE buffer for 4 minutes and visualize in Gel Doc. # Pool 80ul of PCR product from each tube (total 160ul) and purified PCR products with 0.8x volume of AMPure magnetic beads using standard protocol by the manufacturer and elute with 50 μL TE buffer. # Measure concentration of purified PCR products with Bioanalyzer ## I took 3ul of bead purified sequencing libraries, but made some mistake by mixing with non-purified PCR product. I did not have gel image of bead purified sequencing libraries. ## I handed all bead purified sequencing libraries to Li for BA measurement and sequencing # Pool equimolar ratio of each library into single tube for PAGE size selection if there are high background in the libraries after bead purification. If the band is clear, we can easily re-purify with 0.8X AMPure bead and resuspend with small volume of TE buffer to increase concentration of library pool. [[File:Beadpurified-BSPP-BA-gel.png| 650px]] [[File:Beadpurified-BSPPLib.jpg| 600px]] === Information for sample tracking and BA measurement === {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Subject#''' | align="center" style="width:120px;background:#f0f0f0;"|'''Sample_ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample code''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Seq(RC)''' | align="center" style="width:80px;background:#f0f0f0;"|'''ng/ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''nM''' |- | KUCC BRCF 008472||9.H3.25||KU_1||#1||ATCACG||1.9||9.65 |- | KUCC BRCF 008966||9.I19.36||KU_2||#2||CGATGT||0.72||3.73 |- | KUCC BRCF 008116||9.F20.56||KU_3||#3||TTAGGC||0.53||2.7 |- | KUCC BRCF 007855||9.H5.58||KU_4||#4||TGACCA||1.55||7.92 |- | KUCC BRCF 008282||9.G9.25||KU_5||#5||ACAGTG||1.22||6.21 |- | KUCC BRCF 008794||9.I7.3||KU_6||#6||GCCAAT||0.93||4.74 |- | KUCC BRCF 008527||9.H7.69||KU_7||#7||CAGATC||0.96||4.91 |- | KUCC BRCF 007388||9.C28.47||KU_8||#8||ACTTGA||0.67||3.48 |- | KUCC BRCF 005881||9.B7.36||KU_9||#9||GATCAG||0.71||3.62 |- | KUCC BRCF 011921||10.A13.3||KU_10||#10||TAGCTT||1.1||5.7 |- | ||||NTC||#11||GGCTAC||0.07||0.36 |} ---- == Reagent list ==
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