Revision as of 08:44, 13 January 2015 by >Noi
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
|
- 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.
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.