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= SeqCap Epi probe pool (UMR v1) liquid hybridization on WGBS libraries generated from normal and cancer patients = | = SeqCap Epi probe pool (UMR v1) liquid hybridization on WGBS libraries generated from normal and cancer patients = | ||
[[noi:DMR220k_LabNotes#2014|'''Back to calendar''']] | [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]<br> | ||
[[Media:Users Guide_SeqCapEpi_0114-2.pdf| '''SeqCap Epi User Guide''']]<br> | [[Media:Users Guide_SeqCapEpi_0114-2.pdf| '''SeqCap Epi User Guide''']]<br> | ||
* 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 | * 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 |
Revision as of 15:30, 8 November 2014
SeqCap Epi probe pool (UMR v1) liquid hybridization on WGBS libraries generated from normal and cancer patients
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 |