Noi/NOTES/2012-5-24
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DMR 330k BSPP capture of samples from Harvard's group, ASM and ASCM in human cells project[edit]
- Will be using gDNA sent by Anwesha on 2012_05_24
Sample IDs | Conc. (ng.ul) by Nanodrop (from Harvard) |
Amount (ug) in 40ul |
GM12878 gDNA 4-5-12 | 64.52 | 2.58 |
P10E3 gDNA 4-5-12 | 37.86 | 1.51 |
DF2 gDNA 4-5-12 | 70.00 | 2.80 |
H0 gDNA 4-5-12 | 84.52 | 3.38 |
H7 gDNA 4-5-12 | 184.33 | 7.37 |
H14 gDNA 4-5-12 | 167.18 | 6.69 |
H16 gDNA 4-5-12 | 120.60 | 4.82 |
- After confirming concentration by Nanodrop, the conc. I got pretty close to the conc. they labeled.
Bisulfite conversion[edit]
Reagent prep Add the following reagents to CT Conversion Reagent (1tube) - H2O 900ul - M-Dissolving Buffer 50ul - M-Dilution Buffer 300ul Mix at room temperature with frequent vortexing or shaking for 15 minutes
Sample IDs | Conc. (ng/ul) | Volume for 1200ng | LabID ID | H2O | Total amount (ng) | CT conversion reagent (ul) | Total volume (ul) |
GM12878 gDNA 4-5-12 | 64.52 | 18.60 | GM | 1.40 | 1200.00 | 130.00 | 150.00 |
P10E3 gDNA 4-5-12 | 37.86 | 20.00 | P10E | 0.00 | 757.20 | 130.00 | 150.00 |
DF2 gDNA 4-5-12 | 70.00 | 17.14 | DF2 | 2.86 | 1200.00 | 130.00 | 150.00 |
H0 gDNA 4-5-12 | 84.52 | 14.20 | H0 | 5.80 | 1200.00 | 130.00 | 150.00 |
H7 gDNA 4-5-12 | 184.33 | 6.51 | H7 | 13.49 | 1200.00 | 130.00 | 150.00 |
H14 gDNA 4-5-12 | 167.18 | 7.18 | H14 | 12.82 | 1200.00 | 130.00 | 150.00 |
H16 gDNA 4-5-12 | 120.60 | 9.95 | H16 | 10.05 | 1200.00 | 130.00 | 150.00 |
GM12878+H0:: 1:1 | 9.3 (GM)+ 7.1 (H0) | GM+H0 | 3.60 | 1200.13 | 130.00 | 150.00 |
Program 98°C for 10 minutes 64°C for 2.5 hours 4°C storage up to 20 hours Elute with Elution buffer 23ul (final volume 20ul) Measure conc. with ssDNA Qubit assay (used 1ul of bis-cvt gDNA)
Qubit ssDNA assay[edit]
- Sample in assay 1ul, dilution 200x
Sample IDs | Concentration in the Qubit | ' | uL used | Dilution | Final conc.(ng/ul) | Yields in 20ul(ng) |
GM12878 | 88.50 | ng/mL | 1 | 200 | 17.70 | 354.00 |
P10E3 | 42.20 | ng/mL | 1 | 200 | 8.44 | 168.80 |
DF2 | 66.30 | ng/mL | 1 | 200 | 13.26 | 265.20 |
H0 | 76.40 | ng/mL | 1 | 200 | 15.28 | 305.60 |
H7 | 91.20 | ng/mL | 1 | 200 | 18.24 | 364.80 |
H14 | 93.10 | ng/mL | 1 | 200 | 18.62 | 372.40 |
H16 | 85.50 | ng/mL | 1 | 200 | 17.10 | 342.00 |
GM12878+H0:: 1:1 | 96.20 | ng/mL | 1 | 200 | 19.24 | 384.80 |
330k BSPP capture set up[edit]
Subset ID | Adjusted Multiplier F (2012_05_22) |
Actual amount of probe required (probe:target ratio, 100:1) |
Multiplier F x amount probe required |
Probe concentration (ng/ul) |
Volume (ul) | Volume for 10rxn (ul) | Subset ID |
DMR.s1 | 0.25 | 2.24 | 0.56 | 1.00 | 0.56 | 5.60 | DMR.s1 |
DMR.s2 | 0.35 | 2.24 | 0.78 | 1.00 | 0.78 | 7.84 | DMR.s2 |
DMR.s3 | 1.15 | 18.90 | 21.74 | 21.15 | 1.03 | 10.28 | DMR.s3 |
DMR.s4 | 1.00 | 18.90 | 18.90 | 22.34 | 0.85 | 8.46 | DMR.s4 |
DMR.s5 | 4.83 | 18.90 | 91.29 | 29.00 | 3.15 | 31.48 | DMR.s5 |
DMR.Exp1 | 1.00 | 18.90 | 18.90 | 31.88 | 0.59 | 5.93 | DMR.Exp1 |
DMR.Exp2 | 1.00 | 18.90 | 18.90 | 24.11 | 0.78 | 7.84 | DMR.Exp2 |
DMR.Exp3 | 1.00 | 18.90 | 18.90 | 28.90 | 0.65 | 6.54 | DMR.Exp3 |
1.05uM Oligo suppressor-set1 | 0.125 | 1.25 | 1.05uM Oligo suppressor-set1 | ||||
1.05uM Oligo suppressor-set2 | 0.125 | 1.25 | 1.05uM Oligo suppressor-set2 |
Components | 1rxn (ul) | 10 rxn mix (ul) |
10X AmpLigase Buffer | 3.20 | 32.00 |
DMR330k probe mix (ul) | 8.40 | 84.00 |
105nM Oligo suppressor | 0.25 | 2.50 |
Total volume (ul) | 11.85 | 118.50 |
- The maximum volume of bis-cvt gDNA is 20.15ul in total volume 32ul
Sample IDs | Final conc. (ng/ul) | Volume for 300ng (ul) | H2O | Total amount of bis-cvt gDNA(ng) | Probe mix (ul) | 10X AmpLigase Buffer (ul) | 1.05uM Oligo suppressor set1&2 (ul) | Total volume (ul) |
GM12878 | 17.70 | 16.95 | 3.20 | 300.00 | 8.40 | 3.20 | 0.25 | 32.00 |
D3E10 | 8.44 | 20.15 | 0.00 | 170.07 | 8.40 | 3.20 | 0.25 | 32.00 |
DF2 | 13.26 | 20.15 | 0.00 | 267.19 | 8.40 | 3.20 | 0.25 | 32.00 |
H0 | 15.28 | 19.63 | 0.52 | 300.00 | 8.40 | 3.20 | 0.25 | 32.00 |
H7 | 18.24 | 16.45 | 3.70 | 300.00 | 8.40 | 3.20 | 0.25 | 32.00 |
H14 | 18.62 | 16.11 | 4.04 | 300.00 | 8.40 | 3.20 | 0.25 | 32.00 |
H16 | 17.10 | 17.54 | 2.61 | 300.00 | 8.40 | 3.20 | 0.25 | 32.00 |
GM12878+H0:: 1:1 | 19.24 | 15.59 | 4.56 | 300.00 | 8.40 | 3.20 | 0.25 | 32.00 |
- Add H2O to the striptube plate first then add bis-cvt gDNA - Prepare reaction mix in 0.5 mL tube by pipetting probe, oligo suppressor and 10x AmLigase buffer and mix well - Aliquot reaction mix 11.85ulto each tube and mix well - Layer the capture reaction with 40ul mineral oil - Spin down the tube and place in thermocycler with the following profile Program -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
SLN preparation[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 27ul |
AmpliTaq Stoffel | 10 | U/ul | 2 | U/ul | 5.4 |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 2.7 |
dNTP | 1000 | uM | 100 | uM | 2.7 |
10x AmpLigase Buffer | 10 | x | 1 | x | 2.7 |
H2O | 13.5 | ||||
Total | 27 |
ExoI/III preparation[edit]
Mix 9 ul each of ExoI and ExoIII
PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX No. 73-80)[edit]
- I tested if the capture works well by performing qPCR of few samples in small volume 25ul, sample GM, D3E10, DF2 and NTC
Components | 1 rxn | 4.2 rxn mix |
Captured template | 2.50 | 0.00 |
10uM AmpF6.4Sol | 0.50 | 2.10 |
10uM AmpR6.3Sol | 0.50 | 2.10 |
2x Kapa MM | 12.50 | 52.50 |
H2O | 9.00 | 37.80 |
Total | 25.00 | 105.00 |
- Aliquot 22.5ul to each tube and add 2.5ul of circularized DNA templates
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min File:2012 05 30 testqPCR HV.png
- Since the tested qPCR worked well, I did qPCR of each sample in duplicate
Components | 1x rxn | 18 rxn mix |
Captured template | 10.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 36.00 |
10uM AmpR6.3Indx (X=73-80) | 2.00 | 0.00 |
2x Kapa SYBR MM | 50.00 | 900.00 |
H2O | 36.00 | 648.00 |
Total | 100.00 | 1584.00 |
- Aliquot 88ul, add 2ul of 10uM AmpR6.3Indx and 10ul of circularized DNA template
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x12 -> 72C 3min File:2012 05 30 qPCR HV edit.png
Sample IDs | IndX |
GM12878 gDNA 4-5-12 | AmpR6.3.Ind73 |
P10E3 gDNA 4-5-12 | AmpR6.3.Ind74 |
DF2 gDNA 4-5-12 | AmpR6.3.Ind75 |
H0 gDNA 4-5-12 | AmpR6.3.Ind76 |
H7 gDNA 4-5-12 | AmpR6.3.Ind77 |
H14 gDNA 4-5-12 | AmpR6.3.Ind78 |
H16 gDNA 4-5-12 | AmpR6.3.Ind79 |
GM12878:H0:: 1:1 | AmpR6.3.Ind80 |
- Took 60ul of each duplicate (total volume 120ul) - Purified with 0.8volume AmPure beads - Eluted with 80ul EB buffer - Performed PAGE quantification by loading sample 1ul - I also measured concentration with Nanodrop to see if the two quantification give the same concentration or the same molar ratio between the samples File:ZhangLab 2 2012-05-30 17hr 20min PQ HV SeqLib.png - 1: GM12878 - 2: P10E3 - 3: DF2 - 4: H0 - 5: H7 - 6: H14 - 7: H16 - 8: GM12878:H0 1:1
Nanodrop measurement
Sample IDs | Conc. (ng/ul) | Yields (ng) in 80ul | Ratio to GM12878 sample |
GM12878 | 2.00 | 160.00 | 1.00 |
P10E3 | 0.70 | 56.00 | 0.35 |
DF2 | 3.00 | 240.00 | 1.50 |
H0 | 3.60 | 288.00 | 1.80 |
H7 | 3.10 | 248.00 | 1.55 |
H14 | 2.50 | 200.00 | 1.25 |
H16 | 4.50 | 360.00 | 2.25 |
GM12878:H0:: 1:1 | 5.20 | 416.00 | 2.60 |
PAGE quantification
Sample ID | Conc. (ng/ul) | Yields (ng) in 80ul | Ratio to GM12878 sample | Ratio of PAGE:Nanodrop |
GM12878 | 3.77 | 301.51 | 1.00 | 1.88 |
P10E3 | 2.03 | 162.17 | 0.54 | 2.90 |
DF2 | 5.51 | 440.42 | 1.46 | 1.84 |
H0 | 7.49 | 599.20 | 1.99 | 2.08 |
H7 | 5.42 | 433.83 | 1.44 | 1.75 |
H14 | 3.46 | 276.75 | 0.92 | 1.38 |
H16 | 5.64 | 451.17 | 1.50 | 1.25 |
GM12878:H0 1:1 | 5.51 | 440.87 | 1.46 | 1.06 |
- From the ratio above, it showed that samples GM, P10E3, DF2, H0 and H7 tend to give the related ratio between Nanodrop measurement and PAGE quantification, but there were variation in sample H14, H16, and HM:H0 1:1. I will use the concentrations from PQ and will compare the ratio of number of reads after sequencing to see the accuration by this method.
- Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-31