Noi/NOTES/2014-11-4

From ZhangLabWiki
Revision as of 17:54, 8 November 2014 by >Noi
Jump to navigation Jump to search

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
- Mormal 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 CapSeq hybridization capture


Part III: SeaCap hybridization capture