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= LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient = [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]<br> [[Noi/NOTES/2014-12-5|'''Continued from 2014-12-05''']]<br> * Conditions: ** Probe set: MONOD V5 set A (LMS) ** Probe:target ratio: 150:1 for LMS probe set. ***Please note that I used very high amount of WGBS libraries for BSPP capture up to 600ng. This resulted in a very high amount of BSPP required for capture. I therefor reduced probe to target ratio from 200:1 to 125:1. The main reason is I do not have enough LMS BSPP to capture 50 samples with 600ng DNA each. For experiment I did for Illumina (Li Liu), I used 400ng of WGBS libraries, so I had enough probes to capture that amount. In order to be able to capture all unique molecule of ctDNA, I tried to increase WGBS libraries as much as possible. Ideally, Dr. Zhang wanted 1ug for capture. ** Sample: *** WGBS libraries of ctDNA from cancer patients prepared by KAPA Hyper Prep Kit 600ng. *** WGBS libraries of cfDNA from normal control (re-amplified) prepare by using RRBS protocol but skipping DNA digestion/fragmentation 600ng ==== Probe calculation ==== '''Probe set Probe size Probe:target ratio''' LMS 73,574 150:1 From 2014-12-08 exp., 400ng required 108ng (200:1). So 600ng (150:1) requires ~100ng of LMS probes * Since the probe concentration a little low (8.25ng/ul), I will set up capture reaction in 300ul. === NOTE: WHAT ARE DIFFERENT FROM EXP. on 2014-12-08 (ILLUMINA, for Li Liu) === * Template 600ng instead of 400ng * Probe: target ratio is 125:1 instead of 200:1 * Capture reaction 30ul instead of 25ul === Precedures === * I took 600ng of WGBS libraries in 1.5mL LoBind tube. Rationale: total volume of 600ng of WGBS libraries is too large to fit in 30ul capture reaction. * Speedvac at 60C for ~20-30min to dry up DNA in the tube. * Resuspend each tube with 14.88ul H2O which is the maximum volume of WGBS libraries to fit in 30ul capture reaction. ** Make sure that we resuspend DNA on the bottom of the tube very well. * Transfer 14.88ul of DNA to 95-well plate ** I re-organized the location of each sample on the well based on sample ID (small - large) * Aliquot 15.12ul of Probe & Buffer Mix in below table to each well * Total volume is 30ul. * Mix well, seal with plastic seal and spin down the plate * Layer with 30ul mineral oil * Seal with Aluminum seal and spin down the plate again. * Incubate the plate on thermocycler following program below {| {{table}} border = 1 | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 600ng (ul)''' |- | NC-1||13.73||43.69 |- | NC-2||16.00||37.50 |- | NC-3||16.93||35.43 |- | NC-5||13.07||45.92 |- | NC-6||15.33||39.13 |- | NC-7||13.73||43.69 |- | NC-8||15.87||37.82 |- | NC-9||16.53||36.29 |- | NC-12||14.80||40.54 |- | NC-13||13.19||45.50 |- | NC-14||14.93||40.18 |- | NC-15||13.60||44.12 |- | NC-16||13.87||43.27 |- | NC-17||13.47||44.55 |- | NC-18||14.67||40.91 |- | NC-19||13.47||44.55 |- | NC-20||12.79||46.92 |- | NC-21||12.31||48.75 |- | NC-22||16.00||37.50 |- | NC-23||13.60||44.12 |- | NC-25||11.97||50.11 |- | NC-27||14.67||40.91 |- | NC-29||12.96||46.30 |- | NC-30||15.47||38.79 |} <br> {| {{table}} border = 1 | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 600ng (ul)''' |- | PCP-9||26.13||22.96 |- | PCP-4||21.73||27.61 |- | PCP-8||19.07||31.47 |- | PCP-6||20.13||29.80 |- | PCP-7||22.80||26.32 |- | PCP-2||22.13||27.11 |- | PCP-3||21.47||27.95 |- | PCP-5||21.47||27.95 |- | 6P-1||26.00||23.08 |- | 6P-8||24.80||24.19 |- | 6P-10||22.27||26.95 |- | 6P-9||20.27||29.61 |- | 6P-5||24.67||24.32 |- | 6P-2||19.60||30.61 |- | 6P-3||19.87||30.20 |- | 6P-4||19.20||31.25 |- | 7P-8||22.80||26.32 |- | 7P-3||19.07||31.47 |- | 7P-10||25.33||23.68 |- | 7P-7||29.60||20.27 |- | 7P-6||26.93||22.28 |- | 7P-1||14.67||40.91 |- | 7P-2||19.73||30.41 |- | 7P-5||18.00||33.33 |} * In 96-well plate, I re-organized the sample ID from small to large like from PCP2, PCP-3, PCP4,.... ==== Probe & Buffer Mix ==== {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1X''' | align="center" style="width:80px;background:#f0f0f0;"|'''51X''' |- | MONOD V5A (LMS) 8.25ng/ul||12.12||618.18 |- | 10X Ampligase Buffer||3.00||153.00 |- | WGBS library (600ng)||14.88|| |- | Total||30.00|| |} '''Program''' -> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 23h -> 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 === 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 150ul''' |- | Hemo KlenTaq||100||%||20||%||30.00 |- | AmpLigase||5||U/ul||0.5||U/ul||15.00 |- | dNTP||1||mM||100||uM||15.00 |- | 10x AmpLigase Buffer||10||x||1||x||15.00 |- | H2O||||||||||75.00 |- | Total||||||||||150.00 |} # 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 18.5ul of 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. # Re-seal a 96-well plate with aluminum-seal. # Continue to incubate at 55 ºC for 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 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. This time I want to compare between the two enzymes, Phusion Flash High-Fidelity MM and KAPA HiFi Hotstart ReadyMix. ==== Quick PCR ==== * I randomly picked few samples from normal controls and cancer patient samples for a quick test PCR, including NC-1, NC-2, NC-3 NC-4, PCP-2, PCP-3, PCP-4, PCP-5, ^T1 from previous exp. as positive control and NTC. {| {{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 Hi-Fidelity MM or KAPA HiFi Hotstart ReadyMix ||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'' * Note that I just realized that KAPA HiFi Hotstart ReadyMix requires 2-3min for hot start activation. I paused the machine for 1 min a9 98C during denaturing step. I was not sure if this will result in not-fully active DNA polymerase for KAPA. :- In this experiment, we stopped at 19 cycles :- Loaded 5ul of PCR product in 6% TBE gel without purification (Run at 250 volts in 0.5X TBE buffer for 25min) [[File:ZhangLab_2 2014-12-21 17hr 59min_PhusionFlash-quickPCR-LMS-WGBS-5ul.jpg| 450px]] [[File:ZhangLab_2 2014-12-21 17hr 59min_KAPAHiFi-quickPCR-LMS-WGBS-5ul.jpg| 450px]] * The pattern of PCR products was consistent between the two enzymes. However, Phusion Flash Hi-Fidelity showed stronger intensity, so I will keep using this enzyme for the future. This should be note that a lower signal from KAPA HiFi Hotstart ReadyMix might be due to too short denaturation. However, I had no time to repeat experiment. * I continued to amplified the rest of captured DNA in large volume (200ul --> split 2X of 100ul reaction) for 20 cycles using Phusion Flash Hi-Fidelity. * I pooled 80ul from each well (total 160ul) and purified with 0.7X AMPure bead. * Resuspended with 60ul H2O and quantified by PAGE quantification. [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel1.jpg|450px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel2.jpg|450px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel3.jpg|450px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel4.jpg|475px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel5.jpg|450px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel6.jpg|450px]] == PAGE size selection & Sequencing library information == * Alan will do PAGE size selection * There are high variations of PAGE quantification between different gel, so I ask Alan to do size selection separately for samples quantified in different gel. There should be total 6 library pools. ==== WHERE I KEEP INDIVIDUAL SEQUENCING LIBRARIES ==== [[File:NP_photo 5-1.JPG| 400px]] ==== Tube ID: NP-BSPP_V5A_NC.p1-Dec16 ==== :{| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Sequencing Name''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''ZhangLab Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Seq(RC)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 70ng (ul)''' |- | NP-BSPP-V5A-Dec16-NC-1_41||NC-1||41||GGGAAC||5.23||13.39 |- | NP-BSPP-V5A-Dec16-NC-2_42||NC-2||42||GAAAAG||5.42||12.92 |- | NP-BSPP-V5A-Dec16-NC-3_43||NC-3||43||AGTGCC||9.44||7.42 |- | NP-BSPP-V5A-Dec16-NC-5_44||NC-5||44||GCATCC||4.74||14.77 |- | NP-BSPP-V5A-Dec16-NC-6_45||NC-6||45||ACTACG||5.54||12.64 |- | NP-BSPP-V5A-Dec16-NC-7_46||NC-7||46||CATTTC||4.44||15.76 |- | NP-BSPP-V5A-Dec16-NC-8_47||NC-8||47||CTCTCC||4.01||17.46 |- | NP-BSPP-V5A-Dec16-NC-9_48||NC-9||48||ACGTTA||5.49||12.76 |} Total 107.12 6X Dye 22.00 Sum 129.12 Per well 32.28 (4 wells) ==== Tube ID: NP-BSPP_V5A_NC.p2-Dec16 ==== :{| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Sequencing Name''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''ZhangLab Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Seq(RC)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 70ng (ul)''' |- | NP-BSPP-V5A-Dec16-NC-12_49||NC-12||49||CTGTGT||2.41||29.09 |- | NP-BSPP-V5A-Dec16-NC-13_50||NC-13||50||ACCTTT||2.14||32.66 |- | NP-BSPP-V5A-Dec16-NC-14_51||NC-14||51||TATCGC||2.70||25.91 |- | NP-BSPP-V5A-Dec16-NC-15_52||NC-15||52||GACACG||2.65||26.45 |- | NP-BSPP-V5A-Dec16-NC-16_53||NC-16||53||TTCTAC||2.60||26.95 |- | NP-BSPP-V5A-Dec16-NC-17_54||NC-17||54||ACGTCC||3.37||20.80 |- | NP-BSPP-V5A-Dec16-NC-18_55||NC-18||55||TCGACT||2.26||31.01 |- | NP-BSPP-V5A-Dec16-NC-19_56||NC-19||56||TCAGAC||3.55||19.72 |} Total 212.59 6X Dye 45.00 Sum 257.59 Per well 64.40 (4 wells) ==== Tube ID: NP-BSPP_V5A_NC.p3-Dec16 ==== :{| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Sequencing Name''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''ZhangLab Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Seq(RC)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' |- | NP-BSPP-V5A-Dec16-NC-20_57||NC-20||57||TCCTTC||5.50||18.17 |- | NP-BSPP-V5A-Dec16-NC-21_58||NC-21||58||CAGCAT||6.45||15.52 |- | NP-BSPP-V5A-Dec16-NC-22_59||NC-22||59||GATAGA||6.02||16.61 |- | NP-BSPP-V5A-Dec16-NC-23_60||NC-23||60||ACAGAT||6.10||16.38 |- | NP-BSPP-V5A-Dec16-NC-25_61||NC-25||61||CTCTAT||5.68||17.61 |- | NP-BSPP-V5A-Dec16-NC-27_62||NC-27||62||TTTAGC||5.85||17.08 |- | NP-BSPP-V5A-Dec16-NC-29_63||NC-29||63||CCTGGT||5.82||17.18 |- | NP-BSPP-V5A-Dec16-NC-30_64||NC-30||64||AGTTGG||7.19||13.90 |} Total 132.44 6X Dye 30.00 Sum 162.44 Per well 27.07 (4 wells) ==== Tube ID: NP-BSPP_V5A_PCP-Dec16 ==== :{| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Sequencing Name''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''ZhangLab Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Seq(RC)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 70ng (ul)''' |- | NP-BSPP-V5A-Dec16-PCP-2_65||PCP-2||65||TTCCTT||3.06||22.90 |- | NP-BSPP-V5A-Dec16-PCP-3_66||PCP-3||66||TGGAGG||2.32||30.13 |- | NP-BSPP-V5A-Dec16-PCP-4_67||PCP-4||67||GACGTG||2.55||27.46 |- | NP-BSPP-V5A-Dec16-PCP-5_68||PCP-5||68||GTTATG||3.56||19.65 |- | NP-BSPP-V5A-Dec16-PCP-6_69||PCP-6||69||ATATGG||1.99||35.16 |- | NP-BSPP-V5A-Dec16-PCP-7_70||PCP-7||70||GACTTC||2.25||31.16 |- | NP-BSPP-V5A-Dec16-PCP-8_71||PCP-8||71||TCTTTG||2.07||33.81 |- | NP-BSPP-V5A-Dec16-PCP-9_72||PCP-9||72||CTGCCA||4.03||17.36 |} Total 217.63 6X Dye 45.00 Sum 262.63 Per well 65.66 (4 wells) ==== Tube ID: NP-BSPP_V5A_6P-Dec16 ==== :{| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Sequencing Name''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''ZhangLab Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Seq(RC)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 80ng (ul)''' |- | NP-BSPP-V5A-Dec16-6P-1_73||6P-1||73||GGACTC||4.68||17.08 |- | NP-BSPP-V5A-Dec16-6P-2_74||6P-2||74||TGGCGA||3.99||20.05 |- | NP-BSPP-V5A-Dec16-6P-3_75||6P-3||75||CGACTT||6.79||11.77 |- | NP-BSPP-V5A-Dec16-6P-4_76||6P-4||76||CCTATT||4.62||17.31 |- | NP-BSPP-V5A-Dec16-6P-5_77||6P-5||77||ACGGGT||4.63||17.26 |- | NP-BSPP-V5A-Dec16-6P-8_78||6P-8||78||CGTGTT||5.58||14.35 |- | NP-BSPP-V5A-Dec16-6P-9_79||6P-9||79||CCAAGC||3.99||20.05 |- | NP-BSPP-V5A-Dec16-6P-10_80||6P-10||80||TGGTAA||4.87||16.42 |} Total 134.30 6X Dye 30.00 Sum 164.30 Per well 41.07 ==== Tube ID: NP-BSPP_V5A_7P-Dec16 ==== :{| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Sequencing Name''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''ZhangLab Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Seq(RC)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 70ng (ul)''' |- | NP-BSPP-V5A-Dec16-7P-1_81||7P-1||81||ACCTGG||3.42||20.48 |- | NP-BSPP-V5A-Dec16-7P-2_82||7P-2||82||CAAACG||1.60||43.62 |- | NP-BSPP-V5A-Dec16-7P-3_83||7P-3||83||GTGGTC||3.86||18.13 |- | NP-BSPP-V5A-Dec16-7P-5_84||7P-5||84||TCTTGT||3.22||21.74 |- | NP-BSPP-V5A-Dec16-7P-6_85||7P-6||85||TGCGGT||3.25||21.54 |- | NP-BSPP-V5A-Dec16-7P-7_86||7P-7||86||TACCCA||4.18||16.75 |- | NP-BSPP-V5A-Dec16-7P-8_87||7P-8||87||CGGAAT||3.48||20.13 |- | NP-BSPP-V5A-Dec16-7P-10_88||7P-10||88||ACATTC||3.69||18.98 |} Total 181.37 6X Dye 40.00 Sum 221.37 Per well 55.34 (4 wells)
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