Noi/NOTES/2011-5-3
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Library-free BSPP DMR330k capture (Probe set S1-S5 plus Exp1-Exp3)[edit]
sample list[edit]
- 12 samples from Harvard (Yeguang)
- Scr-1
- Sh3513-1
- Sh1-1
- Scr-2
- Sh3513-2
- Sh1-2
- Mock
- TET2-35#
- TET2-75#
- HMEC-Scr
- HMEC-Sh3513
- HMEC-Sh1
- PGP1F and PGP1 iPS samples
- PGP1F
- PGP1 CD1 iPS P16
- HCT 116 and HCT116 DKO
- hHCT116
- hHCT116 DKO
- Sample from Sergio (Salk Institute)
- MSCs P7
- MSCiPS #4 P14
- MSCiPS #8 P14
- H1 P47
- H9 P45
- repeat capture of the sample with low quality (Belmonte's lab)
- cAWS1
- Total 22 samples
- Note: PGP1 CD1 iPS P16 capture will have two replicates
Bisulfite conversion[edit]
- Note: Since we alway get low yield of bis-cvt DNA (possibly due to low quality gDNA), 1.5ug of gDNA will be used in 1 bisulfite conversion reaction and each sample will be done in duplicate.
Capture set up[edit]
Probe calculation[edit]
- Previos probe ratio used for PD patient set up
Subset ID | Multiplier C |
DMR.s1 | 0.05 |
DMR.s2 | 0.09 |
DMR.s3 | 0.25 |
DMR.s4 | 1.00 |
DMR.s5 | 4.83 |
DMR.Exp1 | 1.00 |
DMR.Exp2 | 1.00 |
DMR.Exp3 | 1.00 |
- Dr. Zhang suggested to use probe set DMR330k S1-S5 and Exp1-3 for capturing and also set Exp1-3 as efficient as S4 set.
- Probe normalization by qPCR
Probe set | Ct Mean | Ct Dev. | Ratio compared to s1 | Probes size | Adjusted Ratio | % of s1 |
S1 100:1 | 18.06 | 0.19 | 1.00 | 6500 | 1.00 | 100.00 |
S2 100:1 | 18.60 | 0.27 | 0.69 | 6500 | 0.69 | 68.78 |
S3 100:1 | 16.66 | 0.20 | 2.64 | 55000 | 0.31 | 31.19 |
Exp1 100:1 | 17.21 | 0.31 | 1.80 | 55000 | 0.21 | 21.30 |
Exp2 100:1 | 16.87 | 0.02 | 2.28 | 55000 | 0.27 | 26.96 |
Exp3 100:1 | 17.39 | 0.07 | 1.59 | 55000 | 0.19 | 18.80 |
- Probe verification and normalization by Q-PCR and PAGE analysis: [[1]]
- Adjust new ratio based on the result from Q-PCR
Subset ID | Probes size | Multiplier C | capture efficiency ratio to s1 in mutiplier C | capture efficiency ratio to S1 by qPCR | Adjusted Multiplier based on qPCR (Multiplier E) | Actual amount of probe required (ng), probe:target ratio, 100:1 | Multiplier E x amount probe required (ng) | Probe concentration (ng/ul) | Volume (ul) |
DMR.s1 | 6500 | 0.05 | 1.00 | 1.00 | 0.05 | 2.23 | 0.11 | 1.86 | 0.06 |
DMR.s2 | 6500 | 0.09 | 0.56 | 0.69 | 0.07 | 2.23 | 0.16 | 1.95 | 0.08 |
DMR.s3 | 55000 | 0.25 | 0.20 | 0.31 | 0.23 | 18.90 | 4.41 | 23.20 | 0.19 |
DMR.s4 | 55000 | 1.00 | 0.05 | xx | 1.00 | 18.90 | 18.90 | 24.80 | 0.76 |
DMR.s5 | 55000 | 4.83 | 0.01 | xx | 4.83 | 18.90 | 91.29 | 24.70 | 3.70 |
DMR.Exp1 | 55000 | 1.00 | 0.05 | 0.21 | 0.23 | 18.90 | 4.44 | 31.88 | 0.14 |
DMR.Exp2 | 55000 | 1.00 | 0.05 | 0.27 | 0.19 | 18.90 | 3.50 | 24.11 | 0.15 |
DMR.Exp3 | 55000 | 1.00 | 0.05 | 0.19 | 0.27 | 18.90 | 5.03 | 28.90 | 0.17 |
Bisulfite conversion[edit]
Sample ID | concentration (ng/ul) | New code | volume for 1500ng (ul) | H20 (ul) | Total amount for bis-cvt (ng) | CT conversion reagent (ul) | Total volume (ul) |
Scr-1 | 117.10 | T1 | 12.81 | 7.19 | 1500.00 | 130.00 | 150.00 |
Sh3513-1 | 116.50 | T2 | 12.88 | 7.12 | 1500.00 | 130.00 | 150.00 |
Sh1-1 | 80.30 | T3 | 18.68 | 1.32 | 1500.00 | 130.00 | 150.00 |
Scr-2 | 235.00 | T4 | 6.38 | 13.62 | 1500.00 | 130.00 | 150.00 |
Sh3513-2 | 313.00 | T5 | 4.79 | 15.21 | 1500.00 | 130.00 | 150.00 |
Sh1-2 | 142.00 | T6 | 10.56 | 9.44 | 1500.00 | 130.00 | 150.00 |
Mock | 117.00 | T7 | 12.82 | 7.18 | 1500.00 | 130.00 | 150.00 |
TET2-35# | 113.00 | T8 | 13.27 | 6.73 | 1500.00 | 130.00 | 150.00 |
TET2-75# | 138.00 | T9 | 10.87 | 9.13 | 1500.00 | 130.00 | 150.00 |
HMEC-Scr | 215.00 | T10 | 6.98 | 13.02 | 1500.00 | 130.00 | 150.00 |
HMEC-Sh3513 | 246.00 | T11 | 6.10 | 13.90 | 1500.00 | 130.00 | 150.00 |
HMEC-Sh1 | 51.30 | T12 | 20.00 | 0.00 | 1026.00 | 130.00 | 150.00 |
PGP1F | 250.00 | S13 | 6.00 | 14.00 | 1500.00 | 130.00 | 150.00 |
PGP1 CD1 iPS P16 | 31.40 | S14 | 20.00 | 0.00 | 628.00 | 130.00 | 150.00 |
hHCT116 | 40.90 | S16 | 20.00 | 0.00 | 818.00 | 130.00 | 150.00 |
hHCT116 DKO | 250.00 | S17 | 6.00 | 14.00 | 1500.00 | 130.00 | 150.00 |
MSCs P7 | 184.80 | S18 | 8.12 | 11.88 | 1500.00 | 130.00 | 150.00 |
MSCiPS #4 P14 | 181.40 | S19 | 8.27 | 11.73 | 1500.00 | 130.00 | 150.00 |
MSCiPS #8 P14 | 105.40 | S20 | 14.23 | 5.77 | 1500.00 | 130.00 | 150.00 |
H1 P47 | 54.60 | S21 | 20.00 | 0.00 | 1092.00 | 130.00 | 150.00 |
H9 P45 | 24.50 | S22 | 20.00 | 0.00 | 490.00 | 130.00 | 150.00 |
cAWS1 | 130.00 | S23 | 11.54 | 8.46 | 1500.00 | 130.00 | 150.00 |
- Note: cAWS1, and PGP1 CD1 iPS did 4bis-cvt reactions
Qubit ss-DNA assay[edit]
sample ID | Sample code | Conc. In Qubit (ng/mL) | Final conc. (ng/ul) | Volume (ul) | Yields (ng) |
Scr-1 | T1 | 366.00 | 73.20 | 30.00 | 2196.00 |
Sh3513-1 | T2 | 369.00 | 73.80 | 30.00 | 2214.00 |
Sh1-1 | T3 | 233.00 | 46.60 | 30.00 | 1398.00 |
Scr-2 | T4 | 102.00 | 20.40 | 30.00 | 612.00 |
Sh3513-2 | T5 | 116.00 | 23.20 | 30.00 | 696.00 |
Sh1-2 | T6 | 164.00 | 32.80 | 30.00 | 984.00 |
Mock | T7 | 282.00 | 56.40 | 30.00 | 1692.00 |
TET2-35# | T8 | 212.00 | 42.40 | 30.00 | 1272.00 |
TET2-75# | T9 | 227.00 | 45.40 | 30.00 | 1362.00 |
HMEC-Scr | T10 | 147.00 | 29.40 | 30.00 | 882.00 |
HMEC-Sh3513 | T11 | 140.00 | 28.00 | 30.00 | 840.00 |
HMEC-Sh1 | T12 | 122.00 | 24.40 | 30.00 | 732.00 |
PGP1F | S13 | 151.00 | 30.20 | 15.00 | 453.00 |
PGP1 CD1 iPS P16 | S14 | 103.00 | 20.60 | 60.00 | 1236.00 |
hHCT116 | S16 | 122.00 | 24.40 | 30.00 | 732.00 |
hHCT116 DKO | S17 | 302.00 | 60.40 | 30.00 | 1812.00 |
MSCs P7 | S18 | 73.00 | 14.60 | 30.00 | 438.00 |
MSCiPS #4 P14 | S19 | 164.00 | 32.80 | 30.00 | 984.00 |
MSCiPS #8 P14 | S20 | 226.00 | 45.20 | 30.00 | 1356.00 |
H1 P47 | S21 | 181.00 | 36.20 | 30.00 | 1086.00 |
H9 P45 | S22 | 60.60 | 12.12 | 30.00 | 363.60 |
cAWS1 | S23 | 60.50 | 12.10 | 60.00 | 726.00 |
- Dr. Zhang used to suggested to put more bis-cvt gDNA (about 400ng) in capture reaction, but use the same amount of probe
- To be consistent, Sergio's samples were captured only with DMR220k probes
- I used 15ul of elution buffer in each bis-cvt reaction, so the samples were a little bit diluted. To put 400ng of bis-cvt gDNA in captured reaction, the total volume was larger than usual (approx. 20-25ul).
Capture set up[edit]
Probe calculation[edit]
- Total number of sample (excluded Sergio's samples) = 18 plus 1 NTC = 19 so prepare for 20rxn
Subset ID | Adjusted Multiplier based on qPCR (Multiplier E) | Actual amount of probe required (probe:target ratio, 100:1) | Multiplier E x amount probe required | Probe concentration (ng/ul) | Volume (ul) | Volume for 20rxn (ul) |
DMR.s1 | 0.05 | 2.23 | 0.11 | 1.86 | 0.06 | 1.20 |
DMR.s2 | 0.07 | 2.23 | 0.16 | 1.95 | 0.08 | 1.66 |
DMR.s3 | 0.23 | 18.90 | 4.41 | 23.20 | 0.19 | 3.80 |
DMR.s4 | 1.00 | 18.90 | 18.90 | 24.80 | 0.76 | 15.24 |
DMR.s5 | 4.83 | 18.90 | 91.29 | 24.70 | 3.70 | 73.92 |
DMR.Exp1 | 0.23 | 18.90 | 4.44 | 31.88 | 0.14 | 2.78 |
DMR.Exp2 | 0.19 | 18.90 | 3.50 | 24.11 | 0.15 | 2.91 |
DMR.Exp3 | 0.27 | 18.90 | 5.03 | 28.90 | 0.17 | 3.48 |
105nM Oligo suppressor | 1.00 | 20.00 | ||||
Total volume | 6.25 | 124.99 |
Reaction mix[edit]
Components | 1rxn (ul) | 20rxn mix (ul) |
10X AmpLigase Buffer | 2.90 | 58.00 |
DMR330k probe mix (ul) | 5.25 | 105.00 |
105nM Oligo suppressor | 1.00 | 20.00 |
Total volume (ul) | 9.15 | 183.00 |
- Sergio's samples
Subset ID | Adjusted Multiplier based on qPCR (Multiplier E) | Actual amount of probe required (probe:target ratio, 100:1) | Multiplier E x amount probe required | Probe concentration (ng/ul) | Volume (ul) | Volume for 7rxn (ul) |
DMR.s1 | 0.05 | 2.23 | 0.11 | 1.86 | 0.06 | 0.42 |
DMR.s2 | 0.07 | 2.23 | 0.16 | 1.95 | 0.08 | 0.58 |
DMR.s3 | 0.23 | 18.90 | 4.41 | 23.20 | 0.19 | 1.33 |
DMR.s4 | 1.00 | 18.90 | 18.90 | 24.80 | 0.76 | 5.33 |
DMR.s5 | 4.83 | 18.90 | 91.29 | 24.70 | 3.70 | 25.87 |
105nM Oligo suppressor | 1.00 | 7.00 | ||||
Total volume | 5.79 | 40.54 |
Reaction mix[edit]
Components | 1rxn (ul) | 7rxn mix (ul) |
10X AmpLigase Buffer | 4.00 | 28.00 |
DMR330k probe mix (ul) | 4.79 | 33.53 |
105nM Oligo suppressor | 1.00 | 7.00 |
Total volume (ul) | 9.79 | 68.53 |
Sample code | Cocn. (ng/ul) | Volume for 400ng(ul) | H2O (ul) | Probe mix (ul) | 10x Amligase buffer (ul) | 105nM Oligo suppressor (ul) | Total volum (ul) |
T1 | 73.20 | 5.46 | 14.39 | 5.25 | 2.90 | 1.00 | 29.00 |
T2 | 73.80 | 5.42 | 14.43 | 5.25 | 2.90 | 1.00 | 29.00 |
T3 | 46.60 | 8.58 | 11.27 | 5.25 | 2.90 | 1.00 | 29.00 |
T4 | 20.40 | 19.61 | 0.24 | 5.25 | 2.90 | 1.00 | 29.00 |
T5 | 23.20 | 17.24 | 2.61 | 5.25 | 2.90 | 1.00 | 29.00 |
T6 | 32.80 | 12.20 | 7.65 | 5.25 | 2.90 | 1.00 | 29.00 |
T7 | 56.40 | 7.09 | 12.76 | 5.25 | 2.90 | 1.00 | 29.00 |
T8 | 42.40 | 9.43 | 10.42 | 5.25 | 2.90 | 1.00 | 29.00 |
T9 | 45.40 | 8.81 | 11.04 | 5.25 | 2.90 | 1.00 | 29.00 |
T10 | 29.40 | 13.61 | 6.24 | 5.25 | 2.90 | 1.00 | 29.00 |
T11 | 28.00 | 14.29 | 5.56 | 5.25 | 2.90 | 1.00 | 29.00 |
T12 | 24.40 | 16.39 | 3.46 | 5.25 | 2.90 | 1.00 | 29.00 |
S13 | 30.20 | 13.25 | 6.60 | 5.25 | 2.90 | 1.00 | 29.00 |
S14-1 | 20.60 | 19.42 | 0.43 | 5.25 | 2.90 | 1.00 | 29.00 |
S14-2 | 20.60 | 19.42 | 0.43 | 5.25 | 2.90 | 1.00 | 29.00 |
S16 | 24.40 | 16.39 | 3.46 | 5.25 | 2.90 | 1.00 | 29.00 |
S17 | 60.40 | 6.62 | 13.23 | 5.25 | 2.90 | 1.00 | 29.00 |
S23 | 12.10 | 30.00 | 0.00 | 5.25 | 4.00 | 1.00 | 40.25 |
S18 | 14.60 | 27.40 | 2.81 | 4.79 | 4.00 | 1.00 | 40.00 |
S19 | 32.80 | 12.20 | 18.01 | 4.79 | 4.00 | 1.00 | 40.00 |
S20 | 45.20 | 8.85 | 21.36 | 4.79 | 4.00 | 1.00 | 40.00 |
S21 | 36.20 | 11.05 | 19.16 | 4.79 | 4.00 | 1.00 | 40.00 |
S22 | 12.12 | 30.00 | 0.21 | 4.79 | 4.00 | 1.00 | 40.00 |
S23 | 12.10 | 30.00 | 0.21 | 4.79 | 4.00 | 1.00 | 40.00 |
- Note: total amount of cAWS1 sample was lower than 400ng (approx. 360ng)
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 3ul SLN mix to sample with total volume 29ul, and add 4ul to sample with total volume 40ul (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX)[edit]
- Dr. Zhang designed new primer AmpF6.4 Sol compatible to PE sequencing primer to replace AmpF6.3 Sol: Media:Scheme of amplifiable oligos.docx
- After receiving new primer, one reaction was tested to see if the primer works.
Components | 1rxn | 2.1 rxn |
Captured template | 6.00 | 0.00 |
10uM AmpF6.4Sol | 1.00 | 2.10 |
10uM AmpR6.3 (IndX, X = 13) | 1.00 | 2.10 |
50x SYBG | 0.40 | 0.84 |
2x Phusion MM | 25.00 | 52.50 |
H2O | 16.60 | 34.86 |
Total | 50.00 | aliquot 44ul, add 6ul template |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x5 -> (98C 10s -> 72C 20s)x13 -> 72C 3min
File:PGP1-testAmpF64Sol.PNG
- After testing that the AmpF6.4Sol primer works, PCR was set up
- Total sample 24 (2 rxn/sample = 48) plus 1 NTC, so prepare 50rxn mix
Components | 1rxn | 50rxn mix |
Captured template | 12.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 100.00 |
10uM AmpR6.3 (IndX, X = 13) | 2.00 | 0.00 |
50x SYBG | 0.80 | 40.00 |
2x Phusion MM | 50.00 | 2500.00 |
H2O | 33.20 | 1660.00 |
Total | 100.00 | aliquot 88ul, add 12ul tmplate |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x5 -> (98C 10s -> 72C 20s)x13 -> 72C 3min
sample ID | Sample code | IndX |
Scr-1 | T1 | R6.3 Ind1 |
Sh3513-1 | T2 | R6.3 Ind2 |
Sh1-1 | T3 | R6.3 Ind3 |
Scr-2 | T4 | R6.3 Ind4 |
Sh3513-2 | T5 | R6.3 Ind5 |
Sh1-2 | T6 | R6.3 Ind6 |
Mock | T7 | R6.3 Ind7 |
TET2-35# | T8 | R6.3 Ind8 |
TET2-75# | T9 | R6.3 Ind9 |
HMEC-Scr | T10 | R6.3 Ind10 |
HMEC-Sh3513 | T11 | R6.3 Ind11 |
HMEC-Sh1 | T12 | R6.3 Ind12 |
PGP1F | S13 | R6.3 Ind13 |
PGP1 CD1 iPS P16 | S14-1 | R6.3 Ind14 |
PGP1 CD1 iPS P16 | S14-2 | R6.3 Ind15 |
hHCT116 | S16 | R6.3 Ind16 |
hHCT116 DKO | S17 | R6.3 Ind17 |
MSCs P7 | S18 | R6.3 Ind18 |
MSCiPS #4 P14 | S19 | R6.3 Ind19 |
MSCiPS #8 P14 | S20 | R6.3 Ind20 |
H1 P47 | S21 | R6.3 Ind21 |
H9 P45 | S22 | R6.3 Ind22 |
cAWS1 (220kS1-S5) | S23 | R6.3 Ind23 |
cAWS1 (330k) | S23 | R6.3 Ind24 |
NTC | R6.3 Ind24 |
Realtime-PCR curves[edit]
File:QPCR-TETsamples 2011 05 11.png File:QPCR-PGP1F PGP1 iPS 2011 05 11.png File:QPCR HCT116 HCT115-DKOsamples 2011 05 11.png File:QPCR sergiosample 2011 05 11-e.png
Ct values[edit]
Sample code | Average Ct | Stdev |
T1 | 12.04 | 0.11 |
T2 | 12.13 | 0.01 |
T3 | 12.79 | 0.06 |
T4 | 14.49 | 0.09 |
T5 | 12.93 | 0.01 |
T6 | 12.55 | 0.01 |
T7 | 12.14 | 0.04 |
T8 | 12.13 | 0.03 |
T9 | 12.30 | 0.11 |
T10 | 12.68 | 0.08 |
T11 | 12.63 | 0.06 |
T12 | 12.88 | 0.01 |
S13-PGP1F | 12.56 | 0.10 |
S14-1 PGP1 iPS-1 | 12.84 | 0.06 |
S14-2 PGP1 iPS-2 | 12.94 | 0.00 |
S16 hHCT116 | 12.77 | 0.03 |
S17 hHCT116-DKO | 12.84 | 0.05 |
S18 MSC P7 | 12.97 | 0.08 |
S19 MSC iPS #4 P14 | 13.17 | 0.25 |
S20 MSC iPS #8 P14 | 14.19 | 0.21 |
S21 H1 P47 | 13.81 | 0.62 |
S22 H9 P45 | 15.18 | 0.83 |
S23 cAWS1 | 15.33 | 0.80 |
S23 cAWS1 330k | 15.08 | 1.10 |
- Purify seq lib with 0.8 volume Ampure beads, elute with 30ul EB each tube (total volume for each = 60ul)
PAGE quantification of amplified amplicon[edit]
File:ZhangLab 2 2011-05-11 19hr 25min Gel1.jpgFile:ZhangLab 2 2011-05-11 19hr 27min Gel2.jpgFile:ZhangLab 2 2011-05-11 19hr 37min Gel3.jpg
sample ID | Sample code | Final conc (ng/ul) | Yields in 60ul (ng) | Volume for 100ng (ul) |
Scr-1 | T1 | 15.80 | 947.89 | 6.33 |
Sh3513-1 | T2 | 12.95 | 777.11 | 7.72 |
Sh1-1 | T3 | 11.60 | 695.82 | 8.62 |
Scr-2 | T4 | 9.11 | 546.85 | 10.97 |
Sh3513-2 | T5 | 10.90 | 653.91 | 9.18 |
Sh1-2 | T6 | 13.44 | 806.29 | 7.44 |
Mock | T7 | 14.01 | 840.32 | 7.14 |
TET2-35# | T8 | 12.61 | 756.53 | 7.93 |
TET2-75# | T9 | 10.93 | 655.62 | 9.15 |
HMEC-Scr | T10 | 11.22 | 673.35 | 8.91 |
HMEC-Sh3513 | T11 | 11.88 | 713.03 | 8.41 |
HMEC-Sh1 | T12 | 10.75 | 645.22 | 9.30 |
PGP1F | S13 | 11.71 | 702.73 | 8.54 |
PGP1 CD1 iPS P16 | S14 | 10.97 | 658.16 | 9.12 |
PGP1 CD1 iPS P16 | S14-2 | 10.46 | 627.80 | 9.56 |
hHCT116 | S16 | 14.57 | 874.15 | 6.86 |
hHCT116 DKO | S17 | 13.81 | 828.50 | 7.24 |
MSCs P7 | S18 | 8.00 | 479.73 | 12.51 |
MSCiPS #4 P14 | S19 | 7.12 | 427.36 | 14.04 |
MSCiPS #8 P14 | S20 | 8.33 | 500.05 | 12.00 |
H1 P47 | S21 | 8.17 | 490.34 | 12.24 |
H9 P45 | S22 | 8.55 | 513.17 | 11.69 |
cAWS1 | S23 | 7.18 | 430.54 | 13.94 |
cAWS1 330k | S23 | 8.28 | 497.03 | 12.07 |
- Pooled 100ng from each library and performed PAGE size selection in two 6% TBE gels (5-wells)
File:ZhangLab 2 2011-05-11 22hr 03min PAGE size-selection library-free.jpg
- total volume of multiplexed library: 45ul
File:ZhangLab 2 2011-05-12 12hr 46min PQ Lib-freeSeqLib.jpg
- 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.:23.9ng/ul or 110nM (yields = 23.9ng/ul x 45ul = 1075ng)
- Sequencing: Sample ID: NP-Lib-free_TET_Sergio.Ind1-24-May3, 2011, HL094run
- Actual Sequencing ID in Sequencing REcord: NP-Lib_free_Ind1_24-May3
- Mapping data: [[2]]