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)
  1. Scr-1
  2. Sh3513-1
  3. Sh1-1
  4. Scr-2
  5. Sh3513-2
  6. Sh1-2
  7. Mock
  8. TET2-35#
  9. TET2-75#
  10. HMEC-Scr
  11. HMEC-Sh3513
  12. HMEC-Sh1
  • PGP1F and PGP1 iPS samples
  1. PGP1F
  2. PGP1 CD1 iPS P16
  • HCT 116 and HCT116 DKO
  1. hHCT116
  2. hHCT116 DKO
  • Sample from Sergio (Salk Institute)
  1. MSCs P7
  2. MSCiPS #4 P14
  3. MSCiPS #8 P14
  4. H1 P47
  5. H9 P45
  • repeat capture of the sample with low quality (Belmonte's lab)
  1. 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]]