Noi/NOTES/2014-9-10

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MONOD V5 set A + CRC BSPP capture of tumor DNA samples and ctDNA from Illumina[edit]

  • Conditions:
    • Probe set: MONOPD V5 set A + CRC from Illumina
    • Probe:target ratio: 200:1
    • Sample: bisDNA and WGBS libraires amount 100ng
    • Sequencing libraries by BSPP capture will be included with some BSPP libraries generated on 2014-09-05.
      • Set 1: BSPP capture on 4 WGBS libraries generated from cancer ctDNA from Illumina and 4 WGBS libraries generated from normal control cfDNA from Kang Zhang's lab.
      • Set 2: BSPP capture on 4 bis-gDNA from colon primary tumor tissue and 4 WGBS libraries generated the matched pair colon cancer ctDNA.
  • Related notes: 2014-09-05:, 2014-09-08
  • I have made new batch of MONOD V5 set A probe: concentration = 20.9ng/ul.
  • There are some samples that have a little low concentration. I need to take 100ul and evaporate to fit the capture volume of 20ul.

BSPP capture set up[edit]

Components 1x rxn 13.5x rxn
MONOD V5 setA (20.9 ng/ul) 1.29 17.44
CRC (1ng/ul) 0.34 4.62
10X Ampligase Buffer 2.00 27.00
Total 3.63
  • The maximum volume of DNA template is 20 - 3.63 = 16.37ul
Sample Conc. (ng/ul) Volume for 100ng Need to evap?
wNC-6 5.15 19.42 YES
wNC-9 3.95 25.32 YES
wNC-27 6.53 15.31
wNC-30 7.48 13.37
w6P-2 14.30 6.99
w6P-3 5.77 17.33 YES
w6P-4 11.80 8.47
w6P-5 6.45 15.50
wcf1 8.33 12.00
wcf2 5.07 19.72 YES
wcf3 9.43 10.60
wcf4 9.60 10.42
NTC 0.00 0.00
  • After evaporation for ~10min, each sample has different volume. I adjust volume to 16.37ul with H2O during setting up capture reaction.
  • w = WGBS libraries
Sample Conc. (ng/ul) Volume for 100ng H2O Probe/Buffer Mix Total
wNC-6 evap 11.80 4.57 3.63 20.00
wNC-9 evap 16.37 0.00 3.63 20.00
wNC-27 6.53 15.31 1.06 3.63 20.00
wNC-30 7.48 13.37 3.00 3.63 20.00
w6P-2 14.30 6.99 9.38 3.63 20.00
w6P-3 evap 9.00 7.37 3.63 20.00
w6P-4 11.80 8.47 7.90 3.63 20.00
w6P-5 6.45 15.50 0.87 3.63 20.00
wcf1 8.33 12.00 4.37 3.63 20.00
wcf2 evap 11.30 5.07 3.63 20.00
wcf3 9.43 10.60 5.77 3.63 20.00
wcf4 9.60 10.42 5.95 3.63 20.00
NTC 0.00 0.00 16.37 3.63 20.00
  • I have no time to follow standard capture, so I reduce incubation time after adding KLN to 4h.
Program
-> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2ul KLN mix (2U/ul Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 4h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 10C hold.

KLN mix solution Mix[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume (ul), total 30ul
Hemo KlenTaq 100 % 20 % 6.00
AmpLigase 5 U/ul 0.5 U/ul 3.00
dNTP 1 mM 100 uM 3.00
10x AmpLigase Buffer 10 x 1 x 3.00
H2O 15.00

Amplification[edit]

  • Did quick test PCR in 10l reaction
Components 1x rxn 14 rxn mix
Captured template 1.50 0.00
10uM AmpF6.4Sol 0.20 2.80
10uM AmpR6.3.IndX 0.20 2.80
2X KAPA MM 5.00 70.00
H2O 3.10 43.40
Total 10.00
Program (Eppendorf Realplex)

95C 30s -> (95C 10s -> 58C 20s -> 72C 20s)x8 -> (95C 10s -> 72C 20s)xXX -> 72C 3min