Noi/NOTES/2014-11-4: Difference between revisions

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  2 = NP-SeqCap-pNormalSetA-Nov4
  2 = NP-SeqCap-pNormalSetA-Nov4
  3 = NP-SeqCap-pCancerSetA-Nov4
  3 = NP-SeqCap-pCancerSetA-Nov4
* I am not sure if the upper background will interfere sequencing. I might overloaded DNA. Anyway,next time, I might reduce PCR cycle number to 15 cycles.

Revision as of 22:13, 8 November 2014

SeqCap Epi probe pool (UMR v1) hybridization capture on normal and cancer patient WGBS libraries

Back to calendar
SeqCap Epi User Guide

  • Since the kit we purchased contains only SeqCap EZ HE-Oligo Kit A used to block the adaptor region also barcode adaptor of WGBS, I will pool only libraries that have the match barcode in set A

SeqCap EZ HE-Oligo Kit A oligo list

-HE Universal Oligo 1
-HE Index 2 Oligo
-HE Index 4 Oligo
-HE Index 5 Oligo
-HE Index 6 Oligo
-HE Index 7 Oligo
-HE Index 12 Oligo
-HE Index 13 Oligo
-HE Index 14 Oligo
-HE Index 15 Oligo
-HE Index 16 Oligo
-HE Index 18 Oligo
-HE Index 19 Oligo

Part I: Re-amplification of WGBS libraries of normal plasma samples

Link to WGBS library prep of normal plasma sample

  • Most of normal plasma WGBS libraries have very low yield. I need to re-amplify to increase the yield and have some left for future experiment.
  • I did a test on few samples, including NC-7, NC-7, and NC-30, for number of cycle to re-amplify .

Template amount for 30ul run

Sample Conc. (ng/ul) Volume for 3.6ng H2O to 10ul
NC-7 0.47 7.66 2.34
NC-8 1.52 2.37 7.63
NC-30 6.46 0.56 9.44

PCR Mix

Components Conc unit Final conc. unit Volume (ul) 3.5 rxn mix
Purified 1st round DNA 10.00 0.00
5X Phusion HF buffer 5 X 1 X 6.00 21.00
dNTP mix 10 mM 0.25 mM 0.75 2.63
TruS_F/R 10 uM 0.3 uM 0.90 3.15
50X SYBR 50 X 0.4 X 0.24 0.84
Phusion HF 2 unit/ul 0.30 1.05
H2O 11.81 41.34
Total 30.00
- Aliquot 20ul + DNA template with adjusted H2O total 10ul
- Split 9ul 3X
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5,6,7 cycles --> 72C for 2min
- Added 2ul of 6X loading dye
- Loaded 6ul of PCR and dye mix in 6% TBE gel
File:ZhangLab 2 2014-11-03 13hr 46min test-reamp-wgbs.jpg
  • 6 cycles seemed to be over-amplified either based on gel image or qPCR curve (not shown). I will use the same PCR condition to amplify the rest of libraries with the same template concentration at 5 cycles.
Sample Conc. (ng/ul) Volume for 30ng (ul) Sample H2O to 80 ul
NC-1 0.41 73.17 NC-1 6.83
NC-2 1.39 21.58 NC-2 58.42
NC-3 1.8 16.67 NC-3 63.33
NC-5 0.47 63.83 NC-5 16.17
NC-6 4.25 7.06 NC-6 72.94
NC-7 0.47 63.83 NC-7 16.17
NC-8 1.52 19.74 NC-8 60.26
NC-9 3.72 8.06 NC-9 71.94
NC-12 2.99 10.03 NC-12 69.97
NC-13 1.64 18.29 NC-13 61.71
NC-14 1.28 23.44 NC-14 56.56
NC-15 1.29 23.26 NC-15 56.74
NC-16 1.33 22.56 NC-16 57.44
NC-17 3.86 7.77 NC-17 72.23
NC-18 2.63 11.41 NC-18 68.59
NC-19 0.47 63.83 NC-19 16.17
NC-20 0.4 75.00 NC-20 5.00
NC-21 0.41 73.17 NC-21 6.83
NC-22 2.71 11.07 NC-22 68.93
NC-23 1.76 17.05 NC-23 62.95
NC-24 0.39 76.92 NC-24 3.08
NC-27 6.88 4.36 NC-27 75.64
NC-29 3.59 8.36 NC-29 71.64
NC-30 6.46 4.64 NC-30 75.36

PCR Mix

Components Conc unit Final conc. unit Volume (ul) 25rxn
Purified 1st round DNA 80.00 0.00
5X Phusion HF buffer 5 X 1 X 50.00 1,250.00
dNTP mix 10 mM 0.25 mM 6.25 156.25
TruS_F/R 10 uM 0.3 uM 7.50 187.50
50X SYBR 50 X 0.4 X 2.00 50.00
Phusion HF 2 unit/ul 2.50 62.50
H2O 101.75 2,543.75
Total 250.00
- Aliquot 170ul to each tube + 80 ul of DNA template adjusted volume with H2O
- Split ~60ul 4X in 96-well plate
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5 cycles --> 72C for 2min
- Purified with 1X diluted AMPure bead (dilute 1:1 with 20% PEG/5N NaCl)
- Elute with 60ul EB Buffer.
  • NOTE: SeqCap experiment requires DNA resuspended in H2O not TE or EB buffer. However, in this case, I plan to pool the library and re-purify with 1 round of the bead agin to completely remove adaptor dimer background and resuspend the pooled libraries with small volume of H2O to reduce evaporation time that will be explained in SeqCap experiment.

Qubit dsDNA HS assay result

Sample Conc. in the Qubit Unit uL used Dilution Conc. Unit Conc. (ng/ul) Yield in 60ul
NC-1 56.4 ng/mL 1.5 133.3 56.4 ng/mL 7.52 451.20
NC-2 53.9 ng/mL 1.5 133.3 53.9 ng/mL 7.19 431.20
NC-3 58.6 ng/mL 1.5 133.3 58.6 ng/mL 7.81 468.80
NC-5 59.5 ng/mL 1.5 133.3 59.5 ng/mL 7.93 476.00
NC-6 49.3 ng/mL 1.5 133.3 49.3 ng/mL 6.57 394.40
NC-7 60.5 ng/mL 1.5 133.3 60.5 ng/mL 8.07 484.00
NC-8 43.2 ng/mL 1.5 133.3 43.2 ng/mL 5.76 345.60
NC-9 49.6 ng/mL 1.5 133.3 49.6 ng/mL 6.61 396.80
NC-12 34.9 ng/mL 1.5 133.3 34.9 ng/mL 4.65 279.20
NC-13 40.7 ng/mL 1.5 133.3 40.7 ng/mL 5.43 325.60
NC-14 34.9 ng/mL 1.5 133.3 34.9 ng/mL 4.65 279.20
NC-15 42.6 ng/mL 1.5 133.3 42.6 ng/mL 5.68 340.80
NC-16 39.6 ng/mL 1.5 133.3 39.6 ng/mL 5.28 316.80
NC-17 42.7 ng/mL 1.5 133.3 42.7 ng/mL 5.69 341.60
NC-18 37.2 ng/mL 1.5 133.3 37.2 ng/mL 4.96 297.60
NC-19 44.5 ng/mL 1.5 133.3 44.5 ng/mL 5.93 356.00
NC-20 48.8 ng/mL 1.5 133.3 48.8 ng/mL 6.51 390.40
NC-21 51.1 ng/mL 1.5 133.3 51.1 ng/mL 6.81 408.80
NC-22 36.6 ng/mL 1.5 133.3 36.6 ng/mL 4.88 292.80
NC-23 41.5 ng/mL 1.5 133.3 41.5 ng/mL 5.53 332.00
NC-24 49.4 ng/mL 1.5 133.3 49.4 ng/mL 6.59 395.20
NC-27 39.5 ng/mL 1.5 133.3 39.5 ng/mL 5.27 316.00
NC-29 41.8 ng/mL 1.5 133.3 41.8 ng/mL 5.57 334.40
NC-30 31.9 ng/mL 1.5 133.3 31.9 ng/mL 4.25 255.20


Part II: Pooling WGBS libraries

Normal control WGBS libraries

  • I pooled the re-amplified libraries as shown above
Sample Conc. (ng/ul) Volume for 100ng (ul) Sample Illumina Index
NC-2 7.19 13.91 NC-2 2
NC-3 7.81 12.80 NC-3 4
NC-5 7.93 12.61 NC-5 5
NC-6 6.57 15.21 NC-6 6
NC-7 8.07 12.40 NC-7 7
NC-12 4.65 21.49 NC-12 13
NC-13 5.43 18.43 NC-13 14
NC-14 4.65 21.49 NC-14 15
NC-15 5.68 17.61 NC-15 16
NC-17 5.69 17.56 NC-17 18
NC-18 4.96 20.16 NC-18 19
NC-21 6.81 14.68 NC-21 12
- Total volume: 198.34ul

Cancer patient WGBS libraries

Sample Conc. (ng/ul) Volume for 100ng (ul) Sample Illumina Index
PC-P-3 13.80 7.25 PC-P-3 2
PC-P-4 9.03 11.07 PC-P-4 4
PC-P-5 8.59 11.64 PC-P-5 5
PC-P-6 11.40 8.77 PC-P-6 6
PC-P-7 12.20 8.20 PC-P-7 7
6-P-1 9.58 10.44 6-P-1 13
6-P-2 12.60 7.94 6-P-2 14
6-P-3 6.16 16.23 6-P-3 15
6-P-4 10.40 9.62 6-P-4 16
6-P-10 6.76 14.79 6-P-10 18
6-P-8 7.71 12.97 6-P-8 19
7-P-2 8.67 11.53 7-P-2 12
- Total volume: 130.45ul
- Re-purified the two pools with 1X AMOPure bead
- Resuspended each library with 33ul H2O
- Measure conc. with Qubit dsDNA HS assay using 1ul of purified library pool. Total volume left ~32ul
- Normal WGBS library pool: conc. = 32.9ng/ul --> total amount in 32ul = 1052.8ng
- Cancer patient WGBS library pool: conc. = 30.80ng/ul --> total amount in 32ul = 985.6 ng
- The total amount of the two library pools is close to 1ug as recommended by User guide.
- Continue to SeqCap hybridization capture


Part III: SeqCap hybridization capture

  • Followed exactly the instruction describe in the User Guide page 22-24

Step 1. Preparing for hybridization

  1. Turn on a heat block to 95C. I use thermocycler to heat DNA mix at step 4.9 as I transfer the DNA mix from 1.5mL tube to 0.2ul PCR tube.


Step 2. Resuspend the SeqCap HE Universal and SeqCap HE Index Oligos

  • I prepare this step in the first time I open the kit
  • We have HE Oligo Index set A
  1. Spin the lyophilized oligo tubes, contained in the SeqCap HE-Oligo KitA briefly to allow the contents to pellet to the bottom of the tube.
  2. Add 120ul PCR-grade H2O to SeqCap HE Universal Oligo tube (1000uM final concentration).
  3. Add 10ul PCR-grade H2O to SeqCap HE Index Oligo tube (1000uM final concentration).
  4. Vortex the oligos plus H2O for 5s and spin down the resuspended oligo tube.
  5. After use, they should be stored at -15 to -25C


Step 3. Prepare the bisulfite-converted DNA sample library and HE oligos for hybridization

  1. Have ~1ug of the WGBS library pool in a Lo-bind 1.5 mL tube prepare above, including normal control WGBS library pool and cancer patient WGBS library pool
  2. Thaw the resuspended SeqCap HE Universal Oligos (1000uM) and the resuspended SeqCap HE Index Oligo (1000uM) that matches the DNA adaptor index in the WGBS library preparation
- In this experiment, I pooled all libraries using barcode adaptor match with all 12 indexes in HE-Oligo KitA (0.8ul each). I pool the same amount of the 12 indexes in kit A and use 1ul of the pooled oligos in step 4.3


Step 4. Prepare the hybridization samples

  1. Add 10ul of Bisulfite Capture Enhancer (found in the SeqCap Epi Accessary Kit) to a new Lo-bind 1.5 mL tube
  2. Add 1ug of WGBS library pool
  3. Add 1,000pmol SeqCap HE Universal Oligo 1 and 1,000pmol of the 1000uM SeqCap HE Index Oligo SetA as described above.
    1. This equal to 1ul of 1,000uM of oligos
  4. Close the tube and make a hole in the top of the tube's cap with an 20 gauge needle (I poked many holes)
  5. Dry the mixture of WGBS library pool/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos in a vacuum concentrator on high heat 60C for ~15-20min until it dry up. A little liquid left in the tube between 1-2ul should be fine to prevent over dry.
  6. Cover the holes in the tube's cap with a sticker
  7. To each tube, add
    1. 7.5ul of 2X Hybridization Buffer (vial 5)
    2. 3ul of Hybridization Component A (vial 6)
  8. Vortext the mixture for 10s and centrifuge at maximum speed for 10s. I transfer the contents in the tube to 0.2mL Lo-bind PCR tube
  9. Heat the mixture at 95C for 10min with a heat lid on thermocycler. During this time, I thaw a 4.5ul aliquot of SeqCap Epi probe pool in a 0.2mL PCR tube on ice and spend down
  10. Centrifuge the heated tubes at maximum speed for 10s at RT.
  11. Transfer the WGBS library pool/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail to the 4.5mL SeqCap Epi probe pool on ice.
  12. Vortext quickly for 3s and at maximum speed for 10s at RT.
  13. Incubate on a thermo cycler at 47C for 64-72h. Turn on the thermocycler 's heat lid and set to maintain at 57C (10C above the hybridization temperature).
  • NOTE: I overlaid the hybridization sample with 20ul mineral oil as Alice did before to prevent evaporation
The hybridization sample contains the following components:

Component                                  Solution Capture
Bisulfite Capture Enhancer                      10ug
WGBS libraries                                   1ug
Hybridization Enhancing Oligos               2,000pmol*
------------------------------------------------------- (All component above were heated up to dry)
2X Hybridization Buffer (vial 5)               7.50ul
Hybridization Component A (vial 6)             3.00ul
SeqCap Epi probe pool                          4.50ul
Total                                         15.00ul

* Composed of 50% (1,000pmol) SeqCap HE Universal Oligo & 50% (1,000pmol) of the appropriate SeqCap HE Index Oligo.
  • For this experiment, I incubate the hybridization sample for 65h.


Washing and recovering captured bisulfite-converted DNA samples

IMPORTANT NOTE

  • It is extremely important to maintain the water bath at 47C. I performed very few samples, so I used thermomixer and prepare all washing buffer in 1.5mL or 2mL tube
  • Equilibrate buffer at 47C for at least 2h before washing the captured Multiplex DNA sample. This should mean Stringent Wash Buffer and Wash Buffer I as they were used at 47C.


Step 1. Preparing Sequence Capture and Bead Wash Buffer

Concentrated Buffer Volume/sample (ul) H2O (ul) Total (ul) Volume for 2.1X H2O for 2.1X
10X Stringent Wash Buffer (vial 4) 40 360 400 84 756
10X Wash Buffer I (vial 1) 30 270 300 63 567
10X Wash Buffer II (vial 2) 20 180 200 42 378
10X Wash Buffer III (vial 3) 20 180 200 42 378
2.5X Bead Wash Buffer (vial 7) 200 300 500 420 630
  1. Dilute 10X Wash Buffer (I, II, III and Stringent) and 2.5X Bead Wash Buffer to 1X working solutions as table above.
    1. Warm 1X Wash Buffer I and 1X Stringent Wash Buffer at 47C on thermocycler at least 47C as describe above. I have a very short time to prepare, so I incubated only ~1h.
  • NOTE: All of Stringent Wash Buffer is used at 47C. Wash Buffer I will be used at 47C and RT (100ul at 47C and 200 at RT per rxn). I May need to aliquot accordingly.


Step 2. Preparing the Capture Beads

  • Step below for 2 captures
  1. Allow the capture beads to warm to RT for 30min prior to use.
  2. Mix the beads thoroughly by vortexing to 15s.
  3. Aliquot 100ul of beads for each capture into a 1.5mL tube. For 2 captures, aliquot 200ul. of beads.
  4. Place the tube with beads on magnet device for 1.5mL tube for 3-5min until the liquid is clear. Remove and discard the liquid.
  5. Add 400ul of Bead Wash Buffer (200ul/sample)
  6. Vortex the beads for 10s
  7. Place the tube back to the magnet. Remove the liquid once the liquid is clear.
  8. Repeat step 2.5-2.7 for a total 2 washes
  9. Resuspend the bead with 200ul Bead Wash Buffer and vortex (100ul/sample)
  10. Aliquot 100ul of resuspended beads into new lo-bind 0.2mL tube.
  11. Place the tube on the magnet. Remove the clear liquid.
  12. The capture beads are now READY to bind to captured DNA. PROCEED IMMEDIATELY TO STEP 3. BINDING DNA TO THE CAPTURE BEADS


Step 3. Binding DNA to the Capture Beads

  1. Transfer the hybridization samples to the capture beads prepare in step 2.11
  2. Mix thoroughly by pipetting up and down 10X. NOTE: I have carried a small volume of mineral oil with the hybridization sample as I wanted to transfer as much as possible of the hybridization sample.
  3. Place the tube containing the beads and DNA on a thermocycler set at 47C for 45min (heat lid maintained at 57C). I just keep thermocycler after after hybridization on.
    1. Mix the samples by vortexing 3s every 15min to ensure that hte beads remain in suspension. Should have vortex close to thermocycler.


Step 4. Washing the Capture Beads Plus Bound DNA

  1. After 45-min incubation, add 100ul of 1X Wash Buffer I heated to 47C to the 15ul capture beads plus bound DNA. I added while the tube maintained on thermocycler at 47C.
  2. Mix by vortexing 10X
  3. Transfer the entire contents to Lo-bind 1.5mL tube
  4. Place the tube in the magnet to bind the beads. Remove and discard spnt
  5. Remove the tube from the magnet and add 200ul of 1X Stringent Wash Buffer heated to 47C. Pipette up and down 10X. WORK QUICKLY so that the temperature does not drop down much below 47C.
    1. I place the tube to thermomixter next to Stringent Wash Buffer and add the buffer.
  6. Incubate at 47C for 5min
  7. Repeat step 4.4 - 4.6 for a total 2 washes using 1X stringent Wash Buffer heated to 47C
  8. Place the tube back to the magnet and remove spnt
  9. Add 200ul of RT 1X Wash Buffer I -> mix by vortexing for 2min
  10. Place the tube back to the magnet and remove spnt
  11. Add 200ul of RT 1X Wash Buffer II -> mix by vortexing for 1min
  12. Place the tube back to the magnet and remove spnt
  13. Add 200ul of RT 1X Wash Buffer III -> mix by vortexing for 30s
  14. Place the tube back to the magnet and remove spnt
  15. Remove the tube from the magnet and add 50ul H2O. Mix by vortexing.
  16. Continue to Amplification Step or store the beads plus captured DNA at -15 to -20C.

THERE IS NO NEED TO ELUTE DNA FROM THE BEADS

Part IV: Amplifying Captured DNA Using LM-PCR

Step 1. Resuspend the Post-LM-PCR Oligos

  1. Spin the lyophilized oligos
  2. Add 480ul H2O to the tube label Post-LM-PCR Oligos 1&2
  3. Briefly vortex and spin down


Preparing the LM-PCR

recommendation conditions for 1rxn with 40ul captured DNA

Components                            Volume (ul)
KAPA HiFi HotStart ReadyMix (2X)        50.00
Post-LM-PCR Oligos 1&2 (5uM)            10.00  --> Final conc. of primers after adding template = 0.5uM
Total                                   60.00   

# Pipette 30ul to 2X tube
# Add 20ul of the bead-bound captured DNA to each tube.
# Mix by vortexing
# For NTC, add H2O      

QUCIK RUN PCR to monitor cycle number

  • The User Guide suggest the actual condition for amplification of captured DNA. However, this is my 1st time doing this experiment. I may have lower efficiency of DNA capture. I will do PCR -1 & +1 of the recommendation cycle number (16X).
Components                            Volume (ul)
KAPA HiFi HotStart ReadyMix (2X)        25.00
Post-LM-PCR Oligos 1&2 (5uM)            5.00
Total                                   30.00   

# Aliquot 7.5ul + 5ul bead-bound captured DNA (total 12.5ul).
# I include NTC of PCR by adding 5ul of H2O
# Split 4ul 3X

LM-PCR program

98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 15X, 16X, 17X --> 72C, 1min --> Hold @8C

  • I used thermocycler as including the beads in PCR will interfere fluorescent reading.
  • After taking PCR tube from thermocycler, I notice evaporation. This might be because, I used too small volume and did not close the lid tight enough. I never had evaporation problem when I used Realplex qPCR even I set up reaction as small as 5ul.
  • I verify all PCR product in 6% TBE gel. I was not very sure that evaporation will cause a big problem.
File:ZhangLab 2 2014-11-07 15hr 22min TEST 15-17X-SeqCapNor-Canc.jpg

2nd QUCIK RUN PCR to monitor cycle number

  • PCR fail in the 1st run. I suspect it should be evaporation problem. However, I still saw primer dimer bands and the unexpected band ~125bp, which is not what I want. This meant PCR was actually100% fail, but why I did not see the expected 300bp band?
  • I a little doubt about the primers. It should be STD primers used to amplify the libraries prepared by Illumina adaptor. I decide to use the amplification primers I used to amplify the libraries (TruS F/R). I will use the same primer concentration as I used to amplify the library at 0.3uM instead of 5uM.
  • I will increase total reaction to be larger (12.5ul) and close the lid very tight.
Components                            Volume (ul)
KAPA HiFi HotStart ReadyMix (2X)        50.00
TruS F/R (10uM)                          3.00  --> Final conc. of primers after adding template = 0.3uM
H2O                                      7.00
Total                                   60.00

# Aliquot 7.5ul + 5ul bead-bound captured DNA (total 12.5ul) 2X
# Ran PCR at 16 and 17 cycles on different thermocycler.

 LM-PCR program

98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 16X, 17X --> 72C, 1min --> Hold @8C
  • I still observe evaporation on one of the reaction ran at 16cycles, but not the one at 17cycles.
  • It's possible that the lid was losen by itself.
  • I MAY NEED TO BE VERY CAREFUL WHEN I INCUBATE SMALL VOLUME REACTION ON THERMOCYCLER
  • I continue to verify PCR product in 6% TBE gel by loading 5ul PCR product
File:ZhangLab 2 2014-11-07 16hr 34min TEST 17X-16X-SeqCapNor-Canc.jpg
  • This time, I observe the expected band at 300bp. YES!!!. This meant that the capture worked well. The signal of 16cycles as recommended by the User Guide looked a little faint while the signal of 17 cycles was very saturated. I think that evaporation for the 16-cycle reaction was not very efficient because of evaporation more than 50% of total volume.
  • I continue to amplify the rest of the bead-bound captured DNA (~42ul left)

Amplification in a large volume

Components                            Volume (ul)  2.2rxn (ul) 
KAPA HiFi HotStart ReadyMix (2X)        50.00          110.00
TruS F/R (10uM)                          3.00            6.60
H2O                                      7.00           15.40
Total                                   60.00

# Aliquot 63ul + 42ul bead-bound captured DNA (total ul)
# Split 52ul 2X
 
  LM-PCR program

98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 16X --> 72C, 1min --> Hold @8C
- Purified amplified captured DNA with 1X AMPure beads
- Elute with total volume 50ul of EB buffer
- Verify purified sequencing library in 6% TBE gel by loading 3ul.
File:ZhangLab 2 2014-11-07 18hr 48min beadpurified UMRv1-SeqCap-Normal-cancer-3ul.jpg
 
Library ID:
1 = NP-DNASMRT-Dec_SeqCapUMRv1-Oct29 
2 = NP-SeqCap-pNormalSetA-Nov4
3 = NP-SeqCap-pCancerSetA-Nov4
  • I am not sure if the upper background will interfere sequencing. I might overloaded DNA. Anyway,next time, I might reduce PCR cycle number to 15 cycles.