Dinh/Dinh 2014/NOTES/2014-3-11

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Revision as of 00:03, 11 October 2014 by >Dinh (→‎Capture PCR)
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HOTSPOTS 460K Capture

Capture calculations

Size of Probes 90000 units
gDNA 150 ng
Human (MW) 1.82E+012 g/moles
1 Probe (MW) 31360.1 g/moles
Template 8.23E-011 nmoles
Probes(100:1) 8.23E-009 nmoles
Amount Probes needed 23.23 ng
Size of Probes 6553 units
gDNA 150 ng
Human (MW) 1.82E+012 g/moles
1 Probe (MW) 31360.1 g/moles
Template 8.23E-011 nmoles
Probes(100:1) 8.23E-009 nmoles
Amount Probes needed 1.69 ng
 Human MW = (3x109 bp)*(607.4 gmoles-1/bp) + 157.9 gmoles-1
 1 probe MW = (103 bp)*(303.7 gmoles-1)+ 79 gmoles-1
 Amount probes = (1 probe MW) * (Size of probes) * (Moles of probes required)

Prepared probes

  • After each probes prep. I pool all the prepared probes together by subset to quantify.
  • As of 04/04/2014:
Subset Dilution QUBIT(ng/mL) Conc(ng/uL)
A 100 107 10.7
B 100 71.2 7.12
C 100 86.1 8.61
D 100 150 15
E 100 85.1 8.51
F 100 53.5 5.35

Test capture - 4 reactions

Pool subset probes for 4 reactions

Subset Dilution QUBIT(ng/mL) Conc(ng/uL) Volume(uL) Amount for 4.5 capture (ng) Subset Size
A 100 81.2 8.12 12.88 104.55 90000
B 100 46.4 4.64 22.53 104.55 90000
C 100 84.8 8.48 12.33 104.55 90000
D 100 113 11.3 9.25 104.55 90000
E 100 84.7 8.47 12.34 104.55 90000
F 100 97.4 9.74 0.78 7.61 6553
  • Pool probes for 4.5 captures and aliquot 15.58 uL per capture.

Capture setup

  • I used up all the bisulfite converted gDNA because the converted DNA cannot be kept for long period of times.
Tube ID Converted sample Amount sample (ng) Volume sample (uL) Volume probes (uL) Volume of 10x ampligase buffer Volume water (uL) Total volume
1. 54 Ctrl H1 p54 Control TeSR 193.90 7.00 16.00 3.00 4.00 30
2. Act H1 p54 Activin-A 362.00 10.00 16.00 3.00 1.00 30
3. 47 Ctrl H1 p47 Control TeSR 144.95 6.50 16.00 3.00 4.50 30
4. Bmp H1 p47 BMP-4 205.60 8.00 16.00 3.00 3.00 30

Capture program

1. 95°C    30s
2. 94.8°C  30s
-0.2°C per 30s 198 times
3. 55°C    20h 
ADD 2.5 uL SLN mix
4. 55°C    19.5h
5. 94°C    2m
ADD 2.0 uL ExoI/ExoIII mix
6. 37°C    2h
7. 90°C    5m
8. 4°C     forever

Capture PCR

  • Amplified 4 uL in 25 uL reaction using KAPA 2xMM with AmpR6.3Ind17
 REAGENT                1x        5.5x
 Capture template     4.00 uL    --
 10 uM AmpF6.4Sol     0.50 uL   2.75 uL
 10 uM AmpF6.3RInd17  0.50 uL   2.75 uL
 2xKAPA Master Mix   12.50 uL  68.75 uL
 Nuclease free H2O    7.50 uL  41.25 uL
  • TBE Gel:
 File:HOTSPOTS capture1 test.jpg

Test capture subset A

Capture setup

Tube ID Converted sample Amount sample (ng) Volume sample (uL) Volume probes (uL) Volume of 10x ampligase buffer Volume water (uL) Total volume
GM-A-1-55 GM12878 150.0 2.400 6.00 2.00 9.60 20.00
GM-A-5-56.4 GM12878 150.0 2.400 6.00 2.00 9.60 20.00
GM-A-8-58.8 GM12878 150.0 2.400 6.00 2.00 9.60 20.00
GM-A-12-60 GM12878 150.0 2.400 6.00 2.00 9.60 20.00

Capture program

1. 95°C    30s
2. 94.8°C  30s
-0.2°C per 30s 174 times -> 60°C
3. Gradient: 55/56.4/58.8/60 °C    20h 
ADD 2.5 uL SLN mix
4. Gradient: 55/56.4/58.8/60 °C    20h 
5. 94°C    2m
ADD 2.0 uL ExoI/ExoIII mix
6. 37°C    2h
7. 90°C    5m
8. 4°C     forever

Capture PCR

  • 4 uL of capture was used to amplify. Two step PCR with 8 cycles first step, and 10 cycles second step = 18 cycles of PCR total for all samples.
  • TBE Gel:
File:HOTSPOTS captureA test.jpg
File:HOTSPOTS captureA test repeat.job
Gel might be slightly shifted or 471 bp calculation is wrong? The expected band seems to be greater than 500bp. 
The best annealing temperature appears to be 56.4 degC. This temperature should apply to all the subsets since the probes were randomly assigned to subsets.
  • Will try capture subset B, C, D, E, and F at 56.4 degC. Also, I will amplify 19 or 20 cycles to hopefully increase yield of capture amplicons.

Test capture for A,B,C,D,E,F at 56.4 degC

Capture setup

Tube ID Converted sample Amount sample (ng) Volume sample (uL) Volume probes (uL) Volume of 10x ampligase buffer Volume water (uL) Total volume
GM-A-56.4 GM12878 150.0 2.4 6.00 2.00 9.60 20.00
GM-B-56.4 GM12878 150.0 2.4 10.00 2.00 5.60 20.00
GM-C-56.4 GM12878 150.0 2.4 6.00 2.00 9.60 20.00
GM-D-56.4 GM12878 150.0 2.4 4.00 2.00 11.60 20.00
GM-E-56.4 GM12878 150.0 2.4 6.00 2.00 9.60 20.00
GM-F-56.4 GM12878 150.0 2.4 0.50 2.00 15.10 20.00

Capture program

1. 95°C    30s
2. 94.8°C  30s
-0.2°C per 30s 174 times -> 60°C
3. 56.4°C    20h 
ADD 2.5 uL SLN mix
4. 56.4°C    20h 
5. 94°C    2m
ADD 2.0 uL ExoI/ExoIII mix & 10 uL water
6. 37°C    2h
7. 90°C    5m
8. 4°C     forever

Capture PCR

  • TBE Gel for capture at 56.4 (sample conditions), amplified 4 uL for 20 cycles.
File:HOTSPOTS captureA E newTemp.jpg
  • It is still unclear if the expected product was formed. There is a band slightly above 400 bp and another band slightly above 500bp.
  • B,E appears to perform the best (highest concentration of probes)
  • qPCR plot
 File:HOTSPOTS capturePCR A-E.bmp
  • Repeat PCR on A,C,D,and F for 21 cycles, and re-do gel with 8 ul loaded this time. I can now see the band at 471 (below 500bp and above 400bp). However, the capture is still inefficient. There are several ways to improve it, first, I can increase the amount of samples because here I've only used 150 ng while Noi usually use 200 ng. I can try with 300 ng. Also, F capture did not work because I either didn't amplify enough or need to use more probes. It appears that the quality of the individual subsets do vary substantially, but I'm not sure if this variation would be consistent across multiple captures. Also, even though annealing temperature 56.4 degC worked here, I wonder if there is a difference between 56, 56.4, and 56.8, so I might try to repeat using subset A at those temperatures.
 File:HOTSPOTS capture56.4PCR repeat

Test capture 56-57°C, 300 ng template, subset C only

  • Noi performed bisulfite conversion for GM12878, concentration by Qubit is 31.1ng/uL.
  • I used subset C here, which had the poorest efficiency from capture at 56.4 degC. I repeat the capture here for C because I did not expect subset C to perform worse than A,B,D, and E because the probes were randomly assigned to subsets. At least if I can find the proper annealing temperature for C, then it should also work fine for A,B,D, and E.

Capture setup

Tube ID Converted sample Amount sample (ng) Volume sample (uL) Volume probes (uL) Volume of 10x ampligase buffer Volume water (uL) Total volume
N-GM-C-3-56.1 GM12878 300.0 9.65 5.48 2.00 2.87 20.00
N-GM-C-6-56.4 GM12878 300.0 9.65 5.48 2.00 2.87 20.00
N-GM-C-9-56.9 GM12878 300.0 9.65 5.48 2.00 2.87 20.00

Capture program

1. 95°C    30s
2. 94.8°C  30s
-0.2°C per 30s 174 times -> 60°C
3. Gradient: 56.1/56.4/56.9 °C    20h 
ADD 2.5 uL SLN mix
4. Gradient: 56.1/56.4/56.9 °C    20h 
5. 94°C    2m
ADD 2.0 uL ExoI/ExoIII mix, add 10 uL water
6. 37°C    2h
7. 90°C    5m
8. 4°C     forever

Capture PCR

  • Amplified 4 uL for 21 (8 + 13) cycles as test PCR.
  • qPCR plot
 File:HOTSPOTS 56.1to56.9capture.bmp
  • gel image: loaded 10 uL in first 3 lanes, and 5 uL in last 3 lanes
    • I always get multiple bands. The top & clearest band is between 500-600bp. The expected band should be 471 bp, so that should be within the smear in between 400 and 500 bp.
    • Since I did not run this gel for long enough, I could not see a clear separation for the 471 bp band.
    • Capture at 56.1 degC produce the topmost band, whereas before with 55 degC, I was not able to see bands larger than 400bp. 56.4 degC appears to be the best annealing temperature.
 File:HOTSPOTS 56.1to56.9capture PCR.jpg
  • I might split capture product in two, each half will be amplified with a different barcode & perform gel cut for (1) the topmost band between 500-600bp, (2) the middle band between 400-500bp. The two libraries can be spiked in with Noi's MONOD samples for sequencing. Since capture with 56.4 and 56.9 degC are similar, I will pool them together to increase the amount of product.
  • Pool 56.4 and 56.9 degC reactions together and split to perform PCR (22 cycles) with Index Primers 28 and 29.
                       1x         7x
 Capture products     4.0 uL    28.0 uL
 10 uM forward        0.5 uL     3.5 uL
 10 uM reverse(28,29) 0.5 uL     3.5 uL
 KAPA 2X MM          12.5 uL    87.5 uL
 Water                7.5 uL    52.5 uL
 TOTAL               25.0 uL   175.0 uL
  • Size selection
 File:HOTSPOTS Indx28,Indx29 sizeselect.jpg

Test capture using full probes set

  • Perform capture using 460K probes in one reaction.
  • Amount sample will be 300 ng and annealing temperature will be set at 56.4 degC.
  • Vary probes to target ratio: 100:1, 50:1, 30:1

Prepare mixed probes

  • Mix probes for 100:1 ratio, 300 ng template, for 1.85 reactions.
Subset Dilution QUBIT(ng/mL) Conc(ng/uL) Volume(uL) Mass(ng) Subset Size
A 100 107 10.7 8.03 85.96 90000
B 100 71.2 7.12 12.07 85.96 90000
C 100 86.1 8.61 9.98 85.96 90000
D 100 150 15 5.73 85.96 90000
E 100 85.1 8.51 10.10 85.96 90000
F 100 53.5 5.35 1.17 6.26 6553

Capture setup

  • Aliquot probes & concentrate before adding buffer and other reagents.
Tube ID Converted sample Amount sample (ng) Volume sample (uL) Volume mixed probes (uL) Volume of 10x ampligase buffer Volume water (uL) Total volume
N-GM-100 GM12878 300.0 9.65 25.46 2.00 -17.11 20.00
N-GM-50 GM12878 300.0 9.65 12.73 2.00 -4.38 20.00
N-GM-30 GM12878 300.0 9.65 7.64 2.00 0.71 20.00

Capture PCR

  • Amplify 28 uL in 175 uL (split into 2 tubes)
 Indx33 100:1
 Indx35  50:1
 Indx37  30:1
  • qPCR plot
 File:HOTSPOTS GM12878 capture probeRatios.bmp
  • TBE gel
 File:HOTSPOTS GM12878 capture probeRatios.jpg

Capture of differentation samples

Capture setup

Tube ID Converted sample Amount sample (ng) Volume sample (uL) Volume probes (uL) Volume of 10x ampligase buffer Volume water (uL) Total volume Capture sample conc(ng/ul) Capture probe conc (ng/ul)
NEG (1) water 0.0 0.00 4.50 1.00 4.50 10.00 0.00 4.23
N-GM-50 (2) GM12878 46.5 1.50 4.50 1.50 7.50 15.00 3.10 2.82
N-GM-100 (3) GM12878 93.0 3.00 9.00 1.50 1.50 15.00 6.20 5.64
N-GM-300 (4) GM12878 341.0 11.00 15.00 3.00 1.00 30.00 11.37 4.7
MA-16 (5) H1 p54 Control TeSR 501.6 11.00 15.00 3.00 1.00 30.00 16.72 4.7
MA-17 (6) H1 p54 Activin-A 469.7 11.00 15.00 3.00 1.00 30.00 15.66 4.7
AP-13 (7) H1 p47 Control TeSR 314.6 11.00 15.00 3.00 1.00 30.00 10.49 4.7
AP-14 (8) H1 p47 BMP-4 308.0 11.00 15.00 3.00 1.00 30.00 10.27 4.7
ESC-d0-1 (9) hESC d0 465.3 11.00 15.00 3.00 1.00 30.00 15.51 4.7
ESC-d0-2 (10) hESC d0 482.9 11.00 15.00 3.00 1.00 30.00 16.10 4.7
ESC-d0-3 (11) hESC d0 409.2 11.00 15.00 3.00 1.00 30.00 13.64 4.7
DE-d3-1 (12) definitive endoderm d3 430.1 11.00 15.00 3.00 1.00 30.00 14.34 4.7
DE-d3-2 (13) definitive endoderm d3 413.6 11.00 15.00 3.00 1.00 30.00 13.79 4.7
DE-d3-3 (14) definitive endoderm d3 376.2 11.00 15.00 3.00 1.00 30.00 12.54 4.7
naïve ESC-1 (15) naïve ESC 1 413.6 11.00 15.00 3.00 1.00 30.00 13.79 4.7
naïve ESC-2 (16) naïve ESC 2 470.8 11.00 15.00 3.00 1.00 30.00 15.69 4.7

Low input qPCR, 20 cycles

  • Capture with 50 and 100 ng of template seem to yield the correct band. I might need to amplify for more cycles to increase the band intensity.

File:HOTSPOTS lowInput 50,100.bmp File:HOTSPOTS lowInput 50,100 gel.jpg

Differentiation samples qPCR, 20 cycles

  • I performed qPCR for 20 cycles on the samples #4-16, except for #13 and #15 were amplified with 21 cycles.
  • Each sample was amplified using 28 ul total volume of capture products in 16 v/v%.
  • Each sample was purified with 1 qiagen column, and eluted with 30 ul water.
  • Each sample was visualized by gel using 4 ul purified product to quantify prior to pooling
 Indx29 #13
 Indx30 #15
 Indx38 #4
 Indx39 #5
 Indx40 #6
 Indx41 #7
 Indx42 #8
 Indx43 #9
 Indx44 #10
 Indx45 #11
 Indx46 #12
 Indx47 #14
 Indx48 #16

File:HOTSPOTS differentiation Song,Sergio.bmp File:HOTSPOTS differentiation Song,Sergio gel29-41.jpg File:HOTSPOTS differentiation Song,Sergio gel42-48.jpg

  • Pool #1: 1:1 of each samples
 Indx29
 Indx38 -- DO not pool this library for sequencing because it is positive control, GM12878 sample
 Indx39
 Indx40
 Indx41
  • Pool #2: 1:1 of each samples
 Indx42
 Indx43
 Indx44
 Indx45
 Indx46
 Indx47
 Indx48
  • Final Pool: 4:7:1
 Pool #1
 Pool #2
 Indx49 - Repeat of NaiveESC1(capture #15 that failed)

Test 5 different polymerases for capture

  • Due to the high background from all my previous captures, I need to further optimize my captures. Also, it's important to have a very low failure rate for the capture in order to save precious Tet1-treated DNA.
  • For this test, I will continue to use the Ampligase buffer. If the polymerases do not work well in Ampligase buffer, I will switch to a different buffer next.
    • If using a different PCR buffer, I need to add NAD for Ampligase.
  • Also only subset E is used in this capture at 100:1 ratio.

Capture setup

Tube ID Converted sample Amount sample (ng) Volume sample (uL) Volume probes (uL) Volume of 10x ampligase buffer Volume water (uL) Total volume
(HK) HT-E-56.4-HemoKlenTag HT1080-1 300.0 7.21 2.70 2.00 8.09 20.00
(PT) HT-E-56.4-PfuTurboCX HT1080-1 300.0 7.21 2.70 2.00 8.09 20.00
(ST) HT-E-56.4-Stoffel HT1080-1 300.0 7.21 2.70 2.00 8.09 20.00
(KT) HT-E-56.4-KlenTherm HT1080-1 300.0 7.21 2.70 2.00 8.09 20.00
(TT) HT-E-56.4-TruncatedTaq HT1080-1 300.0 7.21 2.70 2.00 8.09 20.00
(-) HT-E-56.4-NoPolymerase HT1080-1 300.0 7.21 2.70 2.00 8.09 20.00

Capture program

1. 95°C    30s
2. 94.8°C  30s
-0.2°C per 30s 192 times -> 56.4°C
3. 56.4°C  20h 
ADD 2.5 uL Pol-LN mix
4. 56.4°C  20h 
5. 94°C    2m
ADD 2.0 uL ExoI/ExoIII mix
6. 37°C    2h
7. 90°C    5m
8. 4°C     forever

SLN set up

  • For a final volume of 2.5 uL to each tube: 0.1 uL of 5 U/uL Ampligase (final = 0.5 U/uL), 0.25 uL of 1 mM dNTPs, 0.25 uL of 10X Ampligase Buffer. I added 0.60 uL of LN mix to each first.
  • Added LN mix at 20 hour 56.4C incubation.
  • For PfuTurboCX Hotstart, I aliquoted 1.5 uL that was then incubated at 95C for 3 minutes. Then transferred 1.2 uL to the capture reaction, (tube - PT).
  • I will use 3 U of each polymerases:
Polymerase Concentration (U/ul) 3 U Vol (uL) Water (uL)
Stoffel 10 0.3 1.6
HemoKlentaq 5 0.6 1.3
KlenTherm 10 0.3 1.6
PfuTurboCX 2.5 1.2 0.7
Truncated Taq 5 0.6 1.3
No-Polymerase 0 0 1.9
  • After adding polymerases, I added water last.
  • After 20 hours, deactivated enzymes at 95C for 3 minutes.
  • Add 1 uL of Exo I and 1 uL of ExoIII. Incubate at 37C for 2 hours.
  • After ExoI/III, I performed amplification with Phusion Flash and saw a big smear in all my lanes. I believe this is due to the ExoI/III reaction, so I repeated ExoI&III digestion with new tubes of enzymes & repeated amplification.

Capture PCR

  • 1X Reaction:
 captured circles         4.00 uL (half of original capture+18uL water+1 uL ExoI + 1 uL ExoIII)
 PhusionFlash (2X)       20.00 uL 
 AmpF6.4Sol   (10uM)      0.80 uL
 AmpR6.3Indx12(10uM)      0.80 uL
 SybrGreen    (50x)       0.16 uL
 Water                   14.24 uL
  • PCR program:
 98C (10s) -> [ 98C (10s) -> 58C (30s) -> 72C (20s) ] - 8 cycles -> [ 98C (10s) -> 72C (20s) ] - 22 cycles -> 72C (3 min) 
  • qPCR plot:
  • TBE PAGE gel: (expected band at 471 bp)
 File:HOTSPOTS E capture test polymerases 1.jpg