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