Noi/NOTES/2014-12-16: Difference between revisions
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>Noi mNo edit summary |
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** Make sure that we resuspend DNA on the bottom of the tube very well. | ** Make sure that we resuspend DNA on the bottom of the tube very well. | ||
* Transfer 14.88ul of DNA to 95-well plate | * Transfer 14.88ul of DNA to 95-well plate | ||
** I re-organized the location of each sample on the well based on sample ID | ** 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) |
Latest revision as of 18:42, 13 January 2015
LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient[edit]
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Continued from 2014-12-05
- 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[edit]
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)[edit]
- Template 600ng instead of 400ng
- Probe: target ratio is 125:1 instead of 200:1
- Capture reaction 30ul instead of 25ul
Precedures[edit]
- 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
Sample | Conc. (ng/ul) | 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 |
Sample | Conc. (ng/ul) | 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[edit]
Components | 1X | 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[edit]
Components | Stock conc. | Unit | Final conc. | Unit | 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[edit]
- 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[edit]
- 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[edit]
- 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.
Components | Stock conc. | Final conc. In PCR | 1x rxn | 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 File:ZhangLab 2 2014-12-21 17hr 59min KAPAHiFi-quickPCR-LMS-WGBS-5ul.jpg
- 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 File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel2.jpg File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel3.jpg File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel4.jpg File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel5.jpg File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel6.jpg
PAGE size selection & Sequencing library information[edit]
- 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[edit]
Tube ID: NP-BSPP_V5A_NC.p1-Dec16[edit]
Sequencing Name Sample ID ZhangLab Index Seq(RC) Conc. (ng/ul) 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[edit]
Sequencing Name Sample ID ZhangLab Index Seq(RC) Conc. (ng/ul) 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[edit]
Sequencing Name Sample ID ZhangLab Index Seq(RC) Conc. (ng/ul) 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[edit]
Sequencing Name Sample ID ZhangLab Index Seq(RC) Conc. (ng/ul) 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[edit]
Sequencing Name Sample ID ZhangLab Index Seq(RC) Conc. (ng/ul) 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[edit]
Sequencing Name Sample ID ZhangLab Index Seq(RC) Conc. (ng/ul) 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)