Dinh/NOTES/2010-9-23
DMR220K Normalization Capture[edit]
- gDNA template: bis-cvt BJ-iPS (145.5ng/ul, Nanodrop)
Calculations[edit]
Probe/Target Ratio | 100 | 100 | 100 |
Probes size | 6500 | 27000 | 55000 |
Template (ng) | 200 | 200 | 200 |
Human gDNA MW (g/mole) | 1.82E+012 | 1.82E+012 | 1.82E+012 |
Human gDNA(moles) | 1.10E-019 | 1.10E-019 | 1.10E-019 |
Probes required (moles) | 1.10E-017 | 1.10E-017 | 1.10E-017 |
Probes MW (g/mole) | 2.03E+008 | 8.45E+008 | 1.72E+009 |
Amount probes (ng) | 2.23 | 9.27 | 18.88 |
Probes Subset Information[edit]
- Probes were prepared by DD and ZL.
- We re-quantified the probes and got higher concentration, the issue was probably not shaking in SYBR Gold solution long enough.
Subset ID | Size | Conc(ng/ul) | Vol(ul) | Est. Efficiency Coefficient |
DMR.s1 | 6500 | 20.14 | 12 | 1106.55 |
DMR.s2 | 6500 | 25.32 | 12 | 597.53 |
DMR.s3 | 55000 | 37.42 | 12 | 226.35 |
DMR.s4 | 55000 | 52.65 | 13 | 56.42 |
DMR.s5 | 55000 | 61.93 | 13 | 11.68 |
DMR.s6 | 27000 | 69.03 | 19 | 1.26 |
DMR.s7 | 27000 | 44.27 | 19 | 1 |
File:ZhangLab 2 2010-09-23 DMR220 subset quant.png
Multiplexing Setup[edit]
- We calculated two multipliers for the multiplexing setup
- A. Using subset 4 as baseline 1 and the ratios of the estimated efficiencies of other probe sets as multiplier.
- B. For 1-3, use 10x of the multiplier in A, and for 5-7, use 0.5x of the multiplier in A.
Probes subset ID | Multiplier A | Amount ProbesxMultiplier | Vol Req.(ul) | Multiplier B | Amount ProbesxMultiplier | Vol Req.(ul) |
DMR.s1 | 0.05 | 0.11 | 0.01 | 0.51 | 1.14 | 0.06 |
DMR.s2 | 0.09 | 0.21 | 0.01 | 0.94 | 2.11 | 0.08 |
DMR.s3 | 0.25 | 4.71 | 0.13 | 2.49 | 47.07 | 1.26 |
DMR.s4 | 1 | 18.88 | 0.36 | 1 | 18.88 | 0.36 |
DMR.s5 | 4.83 | 91.22 | 1.47 | 2.42 | 45.61 | 0.74 |
DMR.s6 | 44.78 | 415.09 | 6.01 | 22.39 | 207.55 | 3.01 |
DMR.s7 | 56.42 | 523.01 | 11.81 | 28.21 | 261.51 | 5.91 |
Experimental Setup[edit]
- We prepared the reactions in PCR tubes
- Buffer used is 10x Ampligase Buffer
- First 7 reactions: Capture with individual probe set.
Dilution factor | Individual Capture Rxn | Probes | Template | Buffer | H2O | Total |
0.01 | DMR.s1 | 11.08 | 1.37 | 2 | 5.54 | 20 |
0.01 | DMR.s2 | 8.81 | 1.37 | 2 | 7.81 | 20 |
1 | DMR.s3 | 0.5 | 1.37 | 2 | 16.12 | 20 |
0.1 | DMR.s4 | 3.59 | 1.37 | 2 | 13.04 | 20 |
0.1 | DMR.s5 | 3.05 | 1.37 | 2 | 13.58 | 20 |
0.1 | DMR.s6 | 1.34 | 1.37 | 2 | 15.28 | 20 |
0.1 | DMR.s7 | 2.09 | 1.37 | 2 | 14.53 | 20 |
- Multiplex A: Capture with mixed probes.
Dilution factor | Multiplex A | Probes vol | Template | Buffer | H2O | Total |
0.01 | DMR.s1 | 0.56 | ||||
0.01 | DMR.s2 | 0.83 | ||||
0.1 | DMR.s3 | 1.26 | ||||
0.1 | DMR.s4 | 3.59 | ||||
1 | DMR.s5 | 1.47 | ||||
1 | DMR.s6 | 6.01 | ||||
1 | DMR.s7 | 11.81 | ||||
25.54 | 1.37 | 3 | 0.09 | 30 |
- Multiplex B: Capture with mixed probes.
Dilution factor | Multiplex B | Probes vol | Template | Buffer | H2O | Total |
0.01 | DMR.s1 | 5.65 | ||||
0.01 | DMR.s2 | 8.32 | ||||
1 | DMR.s3 | 1.26 | ||||
1 | DMR.s4 | 0.36 | ||||
1 | DMR.s5 | 0.74 | ||||
1 | DMR.s6 | 3.01 | ||||
1 | DMR.s7 | 5.91 | ||||
25.24 | 1.37 | 3 | 0.39 | 30 |
RT PCR[edit]
Reagent | Concentration | Vol (ul) | Final Concentration | 11x Master Mix |
Template | --- | 10 | ||
AmpR6.3IndX | 10uM | 2 | 0.2uM | 22 |
AmpF6.3Sol | 10uM | 2 | 0.2uM | |
Phusion HF 2x MM | 2x | 50 | 1x | 550 |
SYBR Green | 50x | 0.4 | 1x | 4.4 |
H2O | --- | 36 | 396 | |
TOTAL | 100.4 | 972.4 |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
Purified each reaction with 0.7x AMPure Beads.
File:ZhangLab 2 2010-10-07 DMR220KNorm.jpg 1 ul of each subset loaded. We combined s1-s7, A, and B to form the barcoded DMR library, then performed PAGE purification.
Q-PCR[edit]
Reagent | Concentration | Vol (ul) | Final Concentration | 10x Master Mix |
Template | --- | 3 | ||
AmpR6.3 | 100uM | 0.6 | 0.2uM | 6 |
AmpF6.3 | 100uM | 0.6 | 0.2uM | 6 |
Phusion HF 2x MM | 2x | 750 | 1x | 750 |
SYBR Green | 50x | 1.2 | 1x | 12 |
H2O | --- | 72 | 720 | |
TOTAL | 152.4 | 1524 |
Results
Subset | Mean CT | Ratio compared to s1 | Probes size | Adjusted Ratio | % of s1 |
s1 | 19.51 | 1 | 6500 | 1 | 100% |
s2 | 20.33 | 0.57 | 6500 | 0.57 | 57% |
s3 | 16.99 | 5.76 | 55000 | 0.68 | 68% |
s4 | 18.8 | 1.64 | 55000 | 0.19 | 19% |
s5 | 20.45 | 0.52 | 55000 | 0.06 | 6% |
s6 | 18.91 | 1.52 | 27000 | 0.36 | 36% |
s7 | 22 | 0.18 | 27000 | 0.04 | 4% |
PAGE We repeated the Q PCR but stopping at cycle no. 22. Then ran a page gel to see which products are being amplified. File:ZhangLab 2 2010-10-08 17hr 36min DMR220k S1-7 MixAB 21cycles.jpg
Discussion[edit]
NP, AL, and DD:
1) PAGE gel shows consensus with expectation. S1 and S2 are the most efficient at capturing targeted regions than the other subsets. S3 is efficient, and since it has 4x more probes, it seems to have the strongest band. Comparing S3 and S4, both have equal amount of probes, we see lower efficiency for S4 as expected. S5-S7 all have strong bands for non-specific and chimeric products. We suspect that due to the low capturing efficiency, the undesired products were amplified and showed up stronger on the gel. Note that non-specific and chimeric product shows up for all subsets, but less evident in multiplexed reaction A and B.
2) Q PCR may not be the best method to accurately quantify the relative capturing efficiency. Since all of the capture reactions contain non-specific and chimeric products, it is not possible to accurately quantify the amount of desired product in the reaction. If the ratio of undesired:desired product in the reaction is low, we will be able to get closer to the correct quantification. From Q PCR calculations we found that s6 seems to be the outlier, but if factoring the possible error rates, we cannot conclude that s6 is truly an outlier at this point. The overall trend of the Q PCR does show consensus with our expectations. [In the future, we might want to increase the amount of template and probes in the normalization capture as much as possible in order to have more accurate Q PCR result.]
3) We have validated that the low representation of capture target for s5,s6,s7 is not due to poor representation of these subsets during production PCR, since we also saw low amount of capture product from s5, s6, and s7 individual capture. The subset can be pooled in ratios and prepared together.
4) The multiplexed capture amplified better than the individual capture, this is due to better representation of the desired product in the PCR reaction (More probes capture more target). Hence, with the same amount of probes we are better off performing the capture with the pooled subset than with having multiple capture of each subsets. At the same time, we will require much less starting template gDNA.
5) Purifying with 0.7x AMPure beads can remove chimeric products. Size selection after PCR might not be necessary, however, we did perform size selection after pooling the samples together since there was a significant amount of non-specific product for s5,s6, and s7.
Conclusion[edit]
- The QPCR result for subset #6 didn't seem to agree with the PAGE gel, but the PAGE result seems to be more convincing than the QPCR result because it seems like a lot of the non-specific products have been amplified. The overall trend of our experimental result did agree with the expected capture efficiency. However, the pooling ratio for different probe subsets could be adjusted based on the end sequencing result.