Revision as of 19:26, 2 April 2014 by >Dinh
HOTSPOTS 460K Capture
Capture calculations
Size of Probes
|
90000
|
units
|
gDNA |
150 |
ng
|
Human (MW) |
1.82E+012 |
g/moles
|
1 Probe (MW) |
31360.1 |
g/moles
|
Template |
8.23E-011 |
nmoles
|
Probes(100:1) |
8.23E-009 |
nmoles
|
Amount Probes needed |
23.23 |
ng
|
Size of Probes
|
6553
|
units
|
gDNA |
150 |
ng
|
Human (MW) |
1.82E+012 |
g/moles
|
1 Probe (MW) |
31360.1 |
g/moles
|
Template |
8.23E-011 |
nmoles
|
Probes(100:1) |
8.23E-009 |
nmoles
|
Amount Probes needed |
1.69 |
ng
|
Human MW = (3x109 bp)*(607.4 gmoles-1/bp) + 157.9 gmoles-1
1 probe MW = (103 bp)*(303.7 gmoles-1)+ 79 gmoles-1
Amount probes = (1 probe MW) * (Size of probes) * (Moles of probes required)
Prepared probes
Subset
|
Dilution
|
QUBIT(ng/mL)
|
Conc(ng/uL)
|
A |
100 |
81.2 |
8.12
|
B |
100 |
46.4 |
4.64
|
C |
100 |
84.8 |
8.48
|
D |
100 |
113 |
11.3
|
E |
100 |
84.7 |
8.47
|
F |
100 |
97.4 |
9.74
|
Test capture - 4 reactions
Pool subset probes for 4 reactions
Subset
|
Dilution
|
QUBIT(ng/mL)
|
Conc(ng/uL)
|
Volume(uL)
|
Amount for 4.5 capture (ng)
|
Subset Size
|
A |
100 |
81.2 |
8.12 |
12.88 |
104.55 |
90000
|
B |
100 |
46.4 |
4.64 |
22.53 |
104.55 |
90000
|
C |
100 |
84.8 |
8.48 |
12.33 |
104.55 |
90000
|
D |
100 |
113 |
11.3 |
9.25 |
104.55 |
90000
|
E |
100 |
84.7 |
8.47 |
12.34 |
104.55 |
90000
|
F |
100 |
97.4 |
9.74 |
0.78 |
7.61 |
6553
|
- Pool probes for 4.5 captures and aliquot 15.58 uL per capture.
Capture setup
- I used up all the bisulfite converted gDNA because the converted DNA cannot be kept for long period of times.
Tube ID
|
Converted sample
|
Amount sample (ng)
|
Volume sample (uL)
|
Volume probes (uL)
|
Volume of 10x ampligase buffer
|
Volume water (uL)
|
Total volume
|
1. 54 Ctrl |
H1 p54 Control TeSR |
193.90 |
7.00 |
16.00 |
3.00 |
4.00 |
30
|
2. Act |
H1 p54 Activin-A |
362.00 |
10.00 |
16.00 |
3.00 |
1.00 |
30
|
3. 47 Ctrl |
H1 p47 Control TeSR |
144.95 |
6.50 |
16.00 |
3.00 |
4.50 |
30
|
4. Bmp |
H1 p47 BMP-4 |
205.60 |
8.00 |
16.00 |
3.00 |
3.00 |
30
|
Capture program
1. 95°C 30s
2. 94.8°C 30s
-0.2°C per 30s 198 times
3. 55°C 20h
ADD 2.5 uL SLN mix
4. 55°C 19.5h
5. 94°C 2m
ADD 2.0 uL ExoI/ExoIII mix
6. 37°C 2h
7. 90°C 5m
8. 4°C forever
Capture PCR
- Amplified 4 uL in 25 uL reaction using KAPA 2xMM with AmpR6.3Ind17
REAGENT 1x 5.5x
Capture template 4.00 uL --
10 uM AmpF6.4Sol 0.50 uL 2.75 uL
10 uM AmpF6.3RInd17 0.50 uL 2.75 uL
2xKAPA Master Mix 12.50 uL 68.75 uL
Nuclease free H2O 7.50 uL 41.25 uL
File:HOTSPOTS capture1 test.jpg
Test capture subset A
Capture setup
Tube ID
|
Converted sample
|
Amount sample (ng)
|
Volume sample (uL)
|
Volume probes (uL)
|
Volume of 10x ampligase buffer
|
Volume water (uL)
|
Total volume
|
GM-A-1-55 |
GM12878 |
150.0 |
2.400 |
6.00 |
2.00 |
9.60 |
20.00
|
GM-A-5-56.4 |
GM12878 |
150.0 |
2.400 |
6.00 |
2.00 |
9.60 |
20.00
|
GM-A-8-58.8 |
GM12878 |
150.0 |
2.400 |
6.00 |
2.00 |
9.60 |
20.00
|
GM-A-12-60 |
GM12878 |
150.0 |
2.400 |
6.00 |
2.00 |
9.60 |
20.00
|
Capture program
1. 95°C 30s
2. 94.8°C 30s
-0.2°C per 30s 174 times -> 60°C
3. Gradient: 55/56.4/58.8/60 °C 20h
ADD 2.5 uL SLN mix
4. Gradient: 55/56.4/58.8/60 °C 20h
5. 94°C 2m
ADD 2.0 uL ExoI/ExoIII mix
6. 37°C 2h
7. 90°C 5m
8. 4°C forever
Capture PCR
- 4 uL of capture was used to amplify. Two step PCR with 8 cycles first step, and 10 cycles second step = 18 cycles of PCR total for all samples.
- TBE Gel:
File:HOTSPOTS captureA test.jpg
File:HOTSPOTS captureA test repeat.job
Gel might be slightly shifted or 471 bp calculation is wrong? The expected band seems to be greater than 500bp.
The best annealing temperature appears to be 56.4 degC. This temperature should apply to all the subsets since the probes were randomly assigned to subsets.
- Will try capture subset B, C, D, E, and F at 56.4 degC. Also, I will amplify 19 or 20 cycles to hopefully increase yield of capture amplicons.
Test capture for A,B,C,D,E,F at 56.4 degC
Capture setup
Tube ID
|
Converted sample
|
Amount sample (ng)
|
Volume sample (uL)
|
Volume probes (uL)
|
Volume of 10x ampligase buffer
|
Volume water (uL)
|
Total volume
|
GM-A-56.4 |
GM12878 |
150.0 |
2.4 |
6.00 |
2.00 |
9.60 |
20.00
|
GM-B-56.4 |
GM12878 |
150.0 |
2.4 |
10.00 |
2.00 |
5.60 |
20.00
|
GM-C-56.4 |
GM12878 |
150.0 |
2.4 |
6.00 |
2.00 |
9.60 |
20.00
|
GM-D-56.4 |
GM12878 |
150.0 |
2.4 |
4.00 |
2.00 |
11.60 |
20.00
|
GM-E-56.4 |
GM12878 |
150.0 |
2.4 |
6.00 |
2.00 |
9.60 |
20.00
|
GM-F-56.4 |
GM12878 |
150.0 |
2.4 |
0.50 |
2.00 |
15.10 |
20.00
|
Capture program
1. 95°C 30s
2. 94.8°C 30s
-0.2°C per 30s 174 times -> 60°C
3. 56.4°C 20h
ADD 2.5 uL SLN mix
4. 56.4°C 20h
5. 94°C 2m
ADD 2.0 uL ExoI/ExoIII mix & 10 uL water
6. 37°C 2h
7. 90°C 5m
8. 4°C forever
Capture PCR
- TBE Gel for capture at 56.4 (sample conditions), amplified 4 uL for 20 cycles.
File:HOTSPOTS captureA E newTemp.jpg
- It is still unclear if the expected product was formed. There is a band slightly above 400 bp and another band slightly above 500bp.
- B,E appears to perform the best (highest concentration of probes)
- qPCR plot
File:HOTSPOTS capturePCR A-E.bmp
- Repeat PCR on A,C,D,and F for 21 cycles, and re-do gel with 8 ul loaded this time. I can now see the band at 471 (below 500bp and above 400bp). However, the capture is still inefficient. There are several ways to improve it, first, I can increase the amount of samples because here I've only used 150 ng while Noi usually use 200 ng. I can try with 300 ng. Also, F capture did not work because I either didn't amplify enough or need to use more probes. It appears that the quality of the individual subsets do vary substantially, but I'm not sure if this variation would be consistent across multiple captures. Also, even though annealing temperature 56.4 degC worked here, I wonder if there is a difference between 56, 56.4, and 56.8, so I might try to repeat using subset A at those temperatures.
File:HOTSPOTS capture56.4PCR repeat
Test capture 56-57°C, 300 ng template, subset C only
- Noi performed bisulfite conversion for GM12878, concentration by Qubit is 31.1ng/uL.
Capture setup
Tube ID
|
Converted sample
|
Amount sample (ng)
|
Volume sample (uL)
|
Volume probes (uL)
|
Volume of 10x ampligase buffer
|
Volume water (uL)
|
Total volume
|
N-GM-C-3-56.1 |
GM12878 |
300.0 |
9.65 |
5.48 |
2.00 |
2.87 |
20.00
|
N-GM-C-6-56.4 |
GM12878 |
300.0 |
9.65 |
5.48 |
2.00 |
2.87 |
20.00
|
N-GM-C-9-56.9 |
GM12878 |
300.0 |
9.65 |
5.48 |
2.00 |
2.87 |
20.00
|
Capture program
1. 95°C 30s
2. 94.8°C 30s
-0.2°C per 30s 174 times -> 60°C
3. Gradient: 56.1/56.4/56.9 °C 20h
ADD 2.5 uL SLN mix
4. Gradient: 56.1/56.4/56.9 °C 20h
5. 94°C 2m
ADD 2.0 uL ExoI/ExoIII mix, add 10 uL water
6. 37°C 2h
7. 90°C 5m
8. 4°C forever
Capture PCR