Noi/NOTES/2013-7-19

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DNA methylation assay for Blueprint project

Bisulfite conversion

  • I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit
Sample IDs WellIDs Conc. (ng/ul) Volume (ul) H2O (ul) CT conversion reagent (ul) Total volume (ul) Total amount (ng)
1 A1 37.71 13.26 6.74 130.00 150.00 500.00
2 B1 39.43 12.68 7.32 130.00 150.00 500.00
3 C1 50.57 9.89 10.11 130.00 150.00 500.00
4 D1 34.86 14.34 5.66 130.00 150.00 500.00
5 E1 56.00 8.93 11.07 130.00 150.00 500.00
6 F1 44.29 11.29 8.71 130.00 150.00 500.00
7 G1 73.71 6.78 13.22 130.00 150.00 500.00
8 H1 29.71 16.83 3.17 130.00 150.00 500.00
9 A2 40.29 12.41 7.59 130.00 150.00 500.00
10 B2 33.43 14.96 5.04 130.00 150.00 500.00
11 C2 44.86 11.15 8.85 130.00 150.00 500.00
12 D2 52.86 9.46 10.54 130.00 150.00 500.00
**13 E2 11.34 9.00 11.00 130.00 150.00 102.09
*14 F2 36.86 9.00 11.00 130.00 150.00 331.71
**15 G2 26.74 9.00 11.00 130.00 150.00 240.69
*16 H2 50.00 9.00 11.00 130.00 150.00 450.00
17 A3 34.00 14.71 5.29 130.00 150.00 500.00
18 B3 31.14 16.06 3.94 130.00 150.00 500.00
19 C3 26.46 18.90 1.10 130.00 150.00 500.00
*20 D3 20.29 20.00 0.00 130.00 150.00 405.71
*21 E3 18.23 20.00 0.00 130.00 150.00 364.57
22 F3 29.43 16.99 3.01 130.00 150.00 500.00
23 G3 47.71 10.48 9.52 130.00 150.00 500.00
24 H3 32.57 15.35 4.65 130.00 150.00 500.00
25 A4 29.14 17.16 2.84 130.00 150.00 500.00
*26 B4 19.60 20.00 0.00 130.00 150.00 392.00
27 C4 24.31 20.00 0.00 130.00 150.00 486.29
28 D4 23.66 20.00 0.00 130.00 150.00 473.14
29 E4 47.43 10.54 9.46 130.00 150.00 500.00
30 F4 76.86 6.51 13.49 130.00 150.00 500.00
31 G4 40.00 12.50 7.50 130.00 150.00 500.00
32 H4 32.29 15.49 4.51 130.00 150.00 500.00
33 A5 (12878) 58.00 8.62 11.38 130.00 150.00 500.00
34 B5 (12878) 59.00 8.47 11.53 131.00 151.00 500.00
 Program 
1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold
  • Eluted bisulfite converted DNA form the bead with 20ul H2O
  • I used 1ul for ssDNA Qubit assay (200x dilution). The volume left after assay ~15ul.

ssDNA Qubit assay result

Sample IDs WellIDs Start amount (ng) Conc. (ng/ul) Yield in 17ul (ng) Yield left after
Qubit (15ul)
% Recovery in 17ul
1 A1 500.00 21.00 357.00 315.00 71.40
2 B1 500.00 24.40 414.80 366.00 82.96
3 C1 500.00 19.10 324.70 286.50 64.94
4 D1 500.00 22.60 384.20 339.00 76.84
5 E1 500.00 20.60 350.20 309.00 70.04
6 F1 500.00 21.20 360.40 318.00 72.08
7 G1 500.00 22.00 374.00 330.00 74.80
8 H1 500.00 22.20 377.40 333.00 75.48
9 A2 500.00 20.60 350.20 309.00 70.04
10 B2 500.00 24.00 408.00 360.00 81.60
11 C2 500.00 19.06 324.02 285.90 64.80
12 D2 500.00 18.78 319.26 281.70 63.85
13 E2 102.09 2.06 35.02 30.90 34.30
14 F2 331.71 8.08 137.36 121.20 41.41
15 G2 240.69 6.50 110.50 97.50 45.91
16 H2 450.00 12.32 209.44 184.80 46.54
17 A3 500.00 11.56 196.52 173.40 39.30
18 B3 500.00 12.24 208.08 183.60 41.62
19 C3 500.00 11.44 194.48 171.60 38.90
20 D3 405.71 7.92 134.64 118.80 33.19
21 E3 364.57 8.10 137.70 121.50 37.77
22 F3 500.00 11.38 193.46 170.70 38.69
23 G3 500.00 13.96 237.32 209.40 47.46
24 H3 500.00 19.72 335.24 295.80 67.05
25 A4 500.00 21.20 360.40 318.00 72.08
26 B4 392.00 23.00 391.00 345.00 99.74
27 C4 486.29 23.80 404.60 357.00 83.20
28 D4 473.14 31.40 533.80 471.00 112.82
29 E4 500.00 20.80 353.60 312.00 70.72
30 F4 500.00 10.46 177.82 156.90 35.56
31 G4 500.00 47.00 799.00 705.00 159.80
32 H4 500.00 56.00 952.00 840.00 190.40
33 A5 (12878) 500.00 24.00 408.00 360.00 81.60
34 B5 (12878) 500.00 24.60 418.20 369.00 83.64
  • There are few sample have %recovery >100%. I repeat measurement again and got the same concentration. This is possible that I used too small volume when assay gDNA concentration, and there is some error of pipetting.
  • Most of sample start with the same amount of gDNA (500ng) have roughly close concentration of ssDNA after bisulfite conversion.

BSPP capture set up (plan)

Original set

  • Conc.: 25.4 ng/ul (by Qubit ssDNA assay)
Probe:target 500:1 '
Probe size 12000
Template 150 ng
Human gDNA MW 1.9500E+12 g/mol
Human gDNA (150ng) 7.6923E-20 mol (150ng/1.9500E+12 g/mol)
Probe required (500:1) 3.8462E-17 mol (7.6923E-20 mol * 500)
Probe MW (12,000 probes, 110nt) 4.2900E+08 g/mol (12000 probes * 110nt * 325Da/nt)
Amount probe required 1.6500E-08 g (4.2900E+08 g/mol * 3.8462E-17 mol)
Amount probe required 16.50 ng

Subset A

  • Conc.: 17.6ng/ul (by Qubit ssDNA assay)
Probe:target 500:1 '
Probe size 566 probes
Template 150 ng
Human gDNA MW 1.9500E+12 g/mol (3E+09 * 650Da/bp+157.9Da)
Human gDNA (200ng) 7.6923E-20 mol (200ng/1.9500E+12 g/mol)
Probe required (500:1) 3.8462E-17 mol (1.5385E-19mol * 500)
Probe MW (566 probes, 1100nt) 2.0235E+07 g/mol (566 probes * 110nt * 325Da/nt)
Amount probe required 7.7825E-10 g 2.0235E+07 g/mol * 3.8462E-17mol)
Amount probe required 0.78 ng
  • So for 1:5:5 (Original:A:B), amount probe required = 0.78ng * 5 = 8.89ng

Subset B

  • Conc.: 57.9ng/ul (by Qubit ssDNA assay)
Probe:target 500:1 '
Probe size 11397 probes
Template 150 ng
Human gDNA MW 1.9500E+12 g/mol (3E+09 * 650Da/bp+157.9Da)
Human gDNA (200ng) 7.6923E-20 mol (150ng/1.9500E+12 g/mol)
Probe required (500:1) 3.8462E-17 mol (1.5385E-19mol * 500)
Probe MW (11,397 probes, 1100nt) 4.0744E+08 g/mol (11397 probes * 110nt * 325Da/nt)
Amount probe required 1.5671E-08 g (4.0744E+08 g/mol * 3.8462E-17 mol)
Amount probe required 15.67 ng
  • So for 1:5:5 (Original:A:B), amount probe required = 0.15.67ng * 5 = 78.35ng

Summary of probes required for cpature

Probe set Conc. (ng/ul) Vmount required Volume (ul)/samples Volume for ~38x rxn
Original 25.40 16.50 0.65 24.69
Subset A 17.60 3.89 0.22 8.40
Subset B 57.90 78.35 1.35 51.42
  • I will do two captures for positive control (12878) to see the consistency between the two technical replicates and one NTC
  • I will save at least 2ul of bis-cvt DNA for bisulfite PCR if we need to do that. However, Dr. Zhang suggested us to ignore the recommended_4 site.
Sample IDs WellIDs Conc. (ng/ul) Volume required (ul) BP-O (ul) BP.norm-A (ul) BP.norm-B (ul) 10X Ampligase
Buffer (ul)
H2O (ul) Total vulome (ul) Total biscvt DNA (ng)
1 A1 21.00 7.14 0.65 0.22 1.35 2.00 8.64 20.00 150.00
2 B1 24.40 6.15 0.65 0.22 1.35 2.00 9.63 20.00 150.00
3 C1 19.10 7.85 0.65 0.22 1.35 2.00 7.93 20.00 150.00
4 D1 22.60 6.64 0.65 0.22 1.35 2.00 9.14 20.00 150.00
5 E1 20.60 7.28 0.65 0.22 1.35 2.00 8.50 20.00 150.00
6 F1 21.20 7.08 0.65 0.22 1.35 2.00 8.70 20.00 150.00
7 G1 22.00 6.82 0.65 0.22 1.35 2.00 8.96 20.00 150.00
8 H1 22.20 6.76 0.65 0.22 1.35 2.00 9.02 20.00 150.00
9 A2 20.60 7.28 0.65 0.22 1.35 2.00 8.50 20.00 150.00
10 B2 24.00 6.25 0.65 0.22 1.35 2.00 9.53 20.00 150.00
11 C2 19.06 7.87 0.65 0.22 1.35 2.00 7.91 20.00 150.00
12 D2 18.78 7.99 0.65 0.22 1.35 2.00 7.79 20.00 150.00
13 E2 2.06 13.00 0.65 0.22 1.35 2.00 2.78 20.00 26.78
14 F2 8.08 13.00 0.65 0.22 1.35 2.00 2.78 20.00 105.04
15 G2 6.50 13.00 0.65 0.22 1.35 2.00 2.78 20.00 84.50
16 H2 12.32 12.18 0.65 0.22 1.35 2.00 3.60 20.00 150.00
17 A3 11.56 12.98 0.65 0.22 1.35 2.00 2.80 20.00 150.00
18 B3 12.24 12.25 0.65 0.22 1.35 2.00 3.53 20.00 150.00
19 C3 11.44 13.00 0.65 0.22 1.35 2.00 2.78 20.00 148.72
20 D3 7.92 13.00 0.65 0.22 1.35 2.00 2.78 20.00 102.96
21 E3 8.10 13.00 0.65 0.22 1.35 2.00 2.78 20.00 105.30
22 F3 11.38 13.00 0.65 0.22 1.35 2.00 2.78 20.00 147.94
23 G3 13.96 10.74 0.65 0.22 1.35 2.00 5.04 20.00 150.00
24 H3 19.72 7.61 0.65 0.22 1.35 2.00 8.17 20.00 150.00
25 A4 21.20 7.08 0.65 0.22 1.35 2.00 8.70 20.00 150.00
26 B4 23.00 6.52 0.65 0.22 1.35 2.00 9.26 20.00 150.00
27 C4 23.80 6.30 0.65 0.22 1.35 2.00 9.48 20.00 150.00
28 D4 31.40 4.78 0.65 0.22 1.35 2.00 11.00 20.00 150.00
29 E4 20.80 7.21 0.65 0.22 1.35 2.00 8.57 20.00 150.00
30 F4 10.46 13.00 0.65 0.22 1.35 2.00 2.78 20.00 135.98
31 G4 47.00 3.19 0.65 0.22 1.35 2.00 12.59 20.00 150.00
32 H4 56.00 2.68 0.65 0.22 1.35 2.00 13.10 20.00 150.00
33 A5 (12878) 24.00 6.25 0.65 0.22 1.35 2.00 9.53 20.00 150.00
34 B5 (12878) 24.60 6.10 0.65 0.22 1.35 2.00 9.68 20.00 150.00
35 C5 (NTC) 0.00 0.00 0.65 0.22 1.35 2.00 15.78 20.00 0.00

Reaction mix

Components 1x rxn 38 rxn mix
10X Ampligase buffer 2.00 76.00
BP-O 0.65 24.70
BP.norm-A 0.22 8.36
BP.norm-B 1.35 51.30
Total 4.22 160.36
  • Aliquot ~18ul to 8-tube strip and add 4.22ul of reaction mix to the 96-well plate with bis-cvt DNA and H2O with multichannel pipette
  • Mix 15 time by pipetting
  • Add 40ul of mineral oil
  • Spin the plate and place on thermocycler