Noi/NOTES/2013-7-19: Difference between revisions

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* I will do two captures for positive control (12878) to see the consistency between the two technical replicates and one NTC
* 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.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample IDs'''
| align="center" style="background:#f0f0f0;"|'''WellIDs'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Volume required (ul)'''
| align="center" style="background:#f0f0f0;"|'''BP-O (ul)'''
| align="center" style="background:#f0f0f0;"|'''BP.norm-A (ul)'''
| align="center" style="background:#f0f0f0;"|'''BP.norm-B (ul)'''
| align="center" style="background:#f0f0f0;"|'''10X Ampligase<br>Buffer (ul)'''
| align="center" style="background:#f0f0f0;"|'''H2O (ul)'''
| align="center" style="background:#f0f0f0;"|'''Total vulome (ul)'''
| align="center" style="background:#f0f0f0;"|'''Total bis-cvt 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
|}

Revision as of 18:18, 20 July 2013

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 bis-cvt 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