Noi/NOTES/2014-12-16

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LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient

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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

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
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

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

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
  1. 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.
  2. 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.
  3. Swirl pipette 10 times to mix the capture reaction without moving the plate from thermal cycler.
  4. Re-seal a 96-well plate with aluminum-seal.
  5. Continue to incubate at 55 ºC for 20 hours.
  6. Denature DNA at 94 ºC for 2 minutes.

Exonuclease digestion

  1. 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
  2. Add 2 μL of exonuclease I/III mix to each well with multichannel pipette
  3. Mix the reaction by swirling pipette around the well 10 times.
  4. Incubate reaction at 37 ºC for 2 hours.
  5. Heat inactivate exonuclease I/III at 94 ºC for 5 minutes.
  6. 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.
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.