Noi/NOTES/2011-8-4

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Test BSPP DNA capture with DMR330k probe set with all new reagents[edit]

  • Sample:
  • 3 human iPS lines with single Nonsynonymous mutations and 2 GA samples from Kang Zhang's lab
    • 2HFF 8d-10 (conc. 214.6 ng/ul)
    • 218HFF 2-11 (conc. 411.1 ng/ul)
    • 218 HFF 4-11 (conc. 359.6 ng/ul)
    • GA sample well A5 (conc. of bis-cvt DNA 88 ng/ul)
    • GA sample well ID B5 (conc. of bis-cvt DNA 50.4 ng/ul)

Note: Since S5 probe was lately made and I a little bit concern about the stuffs in the probe like salts or acrylamide contaminations even it never show the effect before (The volume of S5 probe is the largest in the reaction). I will set up the reaction with or without S5 probe.

Sample ID Sample ID in Zhang lab Conc. (ng/ul) volume for 1200ng (ul) H20 (ul) Total amount for bis-cvt (ng) CT conversion reagent (ul) Total volume (ul)
2HFF 8d_10 8d_10 214.60 5.59 14.41 1200.00 130.00 150.00
218HHF211 2_11 411.10 2.92 17.08 1200.00 130.00 150.00
218HHF411 4_11 359.60 3.34 16.66 1200.00 130.00 150.00
  • Note: prepared two reactions for each samples

Qubit ss-DNA assay[edit]

Sample ID Conc. In Qubit (ng/mL) Volume (ul) Final conc (ng/ul) Yields (ng) Volume for 250ng
8d_10 95.60 38.00 19.12 726.56 13.08
2_11 69.80 38.00 13.96 530.48 17.91
4_11 79.30 38.00 15.86 602.68 15.76


Capture set up[edit]

Probe preparation and reaction mix[edit]

Include S5 probe[edit]

  • 5 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 6rxn (ul)
DMR.s1 0.05 2.24 0.11 0.39 0.29 1.72
DMR.s2 0.07 2.24 0.16 0.35 0.47 2.79
DMR.s3 0.23 18.90 4.41 23.20 0.19 1.14
DMR.s4 1.00 18.90 18.90 23.20 0.81 4.89
DMR.s5** 4.83 18.90 91.29 29.20 3.13 18.76
DMR.Exp1 0.23 18.90 4.44 31.88 0.14 0.83
DMR.Exp2 0.19 18.90 3.50 24.11 0.15 0.87
DMR.Exp3 0.27 18.90 5.03 28.90 0.17 1.04
1.05uM Oligo suppressor-set1 0.13 0.75
1.05uM Oligo suppressor-set2 0.13 0.75


Components 1rxn (ul) 6 rxn mix (ul)
10X AmpLigase Buffer 2.50 15.00
DMR330k probe mix (ul) 5.34 32.04
105nM Oligo suppressor 0.25 1.50
Total volume (ul) 8.09 48.54


sample ID Final conc. (ng/ul) Volume for 250ng(ul) H2O (ul) Total amount of bis-cvt gDNA (ng) Probe mix (ul) 10x Amligase buffer (ul) 1.05uM Oligo suppressor set1&2 (ul) Total volume (ul)
8d_10 19.12 13.08 3.83 250.00 5.34 2.50 0.25 25.00
2_11 13.96 17.91 -1.00 250.00 5.34 2.50 0.25 25.00
4_11 15.86 15.76 1.15 250.00 5.34 2.50 0.25 25.00
A5 (GA) 88.00 2.84 14.07 250.00 5.34 2.50 0.25 25.00
B5 (GA) 50.40 4.96 11.95 250.00 5.34 2.50 0.25 25.00


Not include S5 probe[edit]

  • 3 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 4rxn (ul)
DMR.s1 0.05 2.24 0.11 0.39 0.29 1.15
DMR.s2 0.07 2.24 0.16 0.35 0.47 1.86
DMR.s3 0.23 18.90 4.41 23.20 0.19 0.76
DMR.s4 1.00 18.90 18.90 23.20 0.81 3.26
DMR.Exp1 0.23 18.90 4.44 31.88 0.14 0.56
DMR.Exp2 0.19 18.90 3.50 24.11 0.15 0.58
DMR.Exp3 0.27 18.90 5.03 28.90 0.17 0.70
1.05uM Oligo suppressor-set1 0.13 0.50
1.05uM Oligo suppressor-set2 0.13 0.50


Components 1rxn (ul) 4 rxn mix (ul)
10X AmpLigase Buffer 2.50 10.00
DMR330k probe mix (ul) 2.22 8.88
105nM Oligo suppressor 0.25 1.00
Total volume (ul) 4.97 19.88


sample ID Final conc. (ng/ul) Volume for 250ng(ul) H2O (ul) Total amount of bis-cvt gDNA (ng) Probe mix (ul) 10x Amligase buffer (ul) 1.05uM Oligo suppressor set1&2 (ul) Total volume (ul)
8d_10 19.12 13.08 6.95 250.00 2.22 2.50 0.25 25.00
2_11 13.96 17.91 2.12 250.00 2.22 2.50 0.25 25.00
4_11 15.86 15.76 4.27 250.00 2.22 2.50 0.25 25.00


Program

   -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
   -> add 2.5ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (2011_08_05)
   -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_08_06)

SLN mix[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume 23 ul
AmpliTaq Stoffel 10.00 U/ul 2.00 U/ul 4.60
AmpLigase 5.00 U/ul 0.50 U/ul 2.30
dNTP 1000.00 uM 100.00 uM 2.30
10x AmpLigase Buffer 10.00 x 1.00 x 2.30
H2O 11.50


Amplification[edit]

  • Performed PCR to test if the capture work well in 50ul reaction
Reagents volume (ul) 8.5 rxn mix
captured template 4.00 0.00
100uM AmpFV6.3NH2 0.10 0.85
100uM AmpRV6.3NH2 0.10 0.85
50x SYBG 0.40 3.40
2x PhusionHF MM 25.00 212.50
H2O 20.40 173.40
Total volume (ul) 50.00 aliquot 46ul add 4ul template


Program 98C 30sec
(98C 10sec -> 58C 20sec-> 72C 20sec) x 20 cycles
72C 2min
15C hold. 2011_08_10

  • Perform PCR to amplify the three human iPS line samples
Reagents volume (ul) 6.5 rxn mix
captured template 8.00 0.00
100uM AmpFV6.3NH2 0.20 1.30
100uM AmpRV6.3NH2 0.20 1.30
50x SYBG 0.80 5.20
2x PhusionHF MM 50.00 325.00
H2O 40.80 265.20
Total volume (ul) 100.00 aliquot 92ul add 8ul template


Program 98C 30sec
(98C 10sec -> 58C 20sec-> 72C 20sec) x 20 cycles
72C 2min
15C hold.

  • Purify with 0.8 vol. Ampure beads, elute with 25ul EB/tube, so total volume = 50ul
  • PAGE quantify DNA concentration (loaded sample 0.5 ul)

File:ZhangLab 2 2011-08-10 19hr 02min purified amplicon.jpg

Ct values[edit]

Sample Ave Ct value
8d_10 15.08
2_11 14.59
4_11 15.77


PAGE quantification and MmeI digestion set up[edit]

Sample ID Conc.(ng/ul) Total volume(ul) Total amount (ng)
8d_10 10.88 50.00 543.78
2_11 11.75 50.00 587.57
4_11 9.44 50.00 471.83


Sample ID Conc.(ng/ul) Total volume(ul) Total amount (ng) H2O 10X NEB buffer4 5mM SAM MmeI Total volume(ul)
8d_10 10.88 22.99 250.00 7.21 4.00 0.80 5.00 40.00
2_11 11.75 21.27 250.00 8.93 4.00 0.80 5.00 40.00
4_11 9.44 26.49 250.00 3.71 4.00 0.80 5.00 40.00


  • Prepare reaction mix
Components 1 rxn 3.2 rxn
10x NEB buffer4 4.00 12.80
5mM SAM 0.80 2.56
MmeI 5.00 16.00
Total 9.80 Aliquot 9.8 ul


  • Prepare 6.4ul of 5mM SAM by adding 1ul of 32mM SAM to 5.4ul H2O
  • Incubate 37C 2hr
  • purify with 1 vol. Ampure beads, elute with 25 ul EB
  • PAGE quantify DNA concentration (loaded sample 2ul)

File:ZhangLab 2 2011-08-11 08hr 41min MmeI.jpg

N2 adaptor ligation set up[edit]

Adaptor preparation[edit]

Components Volume (ul) Final conc. (uM)
100uM PE_N2_adaptor 2.00 20.00
100uM PE_b_A 2.00 20.00
10x Stoffel buffer 1.00 1x
H2O 5.00
Total 10.00


Program

94C 2min -> 0.2C/sec to 20C -> 4C hold

Adapters to ligated product ratio: 10:1
Est length of digested products: 212bp (after MmeI)
MW digested products = (212bp*607.4 D/bp +157.9 D) = 128.927kD = 128,927 g/mole

For 100 ng digested product = 100ng / 128,927g/mole = 7.76E-4 nmole * 10:1 = 0.008 nmole adapters required.
20uM Adapters (ul) = 0.008nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.008nmoles/ (20E-3 nmoles/ul) = 0.40 ul

  • 0.40 ul of 20uM adapters per 100ng digested products.
  • so 0.24ul of 20uM adapters per 60ng digested products.
Sample ID conc.(ng/ul) Volume (ul) Total amount (ng) N2 adapters conc (uM) N2 adaptors volume (ul) 5xQuickLigase buffer QuickLigase (ul) H2O (ul) Total (ul)
8d_10 3.19 18.80 60.00 20uM 0.24 8.00 1.00 11.96 40.00
2_11 3.99 15.02 60.00 20uM 0.24 8.00 1.00 15.74 40.00
4_11 2.78 21.57 60.00 20uM 0.24 8.00 1.00 9.19 40.00


Reaction mix

Reagents 1x 4.5 rxn mix
5xQuickLigase buffer 8.00 36.00
QuickLigase 1.00 4.50
20uM N2 adapters 0.24 1.08
Total 9.24 Aliquot 9.24ul

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  • Incubate at RT for 15 min
  • Purify with Ampure beads, elute with 30ul EB

Amplification with N2 barcoded primers[edit]

  • From the previous experiment and optimization, Use 2% N2 ligated DNA template in PCR
Reagents 1 rxn 7.2 rxn mix
N2 adapter ligated template 2.00 0.00
10uM PCR_F 1.00 7.20
10uM PCR_R.N2 IndX(X= 21,2 2 & 23) 1.00 0.00
50x SYBG 0.40 2.88
2x PhusionHF MM 50.00 360.00
H2O 45.60 328.32
Total volume (ul) 100.00 aliquot 97ul


  • Add 1ul of PCR_R.N2 IndX and 2ul DNA template
Sample ID IndX
8d_10 PCR_R.N2Ind21
2_11 PCR_R.N2Ind22
4_11 PCR_R.N2Ind23


Program 98C 30sec

  (98C 10sec -> 63C 20sec-> 72C 30sec) x 12 cycles
  72C 2min  
  15C hold
  • Purify amplicon with 0.8 volume Ampure beads, elute with 50ul EB each tube (total = 100ul)
  • PAGE quantify DNA concentration (loaded sample 1ul)

File:ZhangLab 2 2011-08-11 11hr 57min N2Amp.jpg

Sample Conc. (ng/ul) Volume (ul) Yields Volume for 200ng (ul)
8d_10 2.75 100.00 275.23 72.67
2_11 3.05 100.00 305.29 65.51
4_11 2.75 100.00 274.65 72.82


  • Perform PAGE size selection with 6% TBE 5-well gel (1gel)
  • Total volume of of multiplexed libraries: 24ul

File:ZhangLab 2 2011-08-11 15hr 21min NP3smuiPSSeqLib.jpg

  • 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 10.58ng/ul, 51.8nM (336bp), yields = 10.58 ng/ul * 24ul = 253.9ng
  • Qubit quantification: ng/ul
  • Sequencing: Sample ID:NP-3SMUips-Ind21_23-Aug42011, HL100 run 110811 SE 80+7bp, s1-s3