Noi/NOTES/2014-6-10

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MONOD V1N3 BSPP capture of serum and solid tumor DNA[edit]

serum DNA[edit]

  • Some samples had volume >20ul, I evaporated to reduce the volume (~from 30ul --> 13/14ul, 30C for 10min)
Sample Original conc. (ng/ul) Original vol. before evap. Volume for 50ng (ul) H2O (ul) Lightning Conv. Reagent (ul) Total volume (ul) Total DNA amount (ng)
PC-S_2 (evap) 1.66 27.00 14.00 6.00 130.00 150.00 44.82
PC-S_3 21.40 NA 2.34 17.66 130.00 150.00 50.00
PC-S_5 (evap) 1.58 27.00 14.00 6.00 130.00 150.00 42.66
PC-S_6 17.50 NA 2.86 17.14 130.00 150.00 50.00
PC-S_7 (evap) 1.80 25.00 14.00 6.00 130.00 150.00 45.00
PC-S_9 5.72 NA 8.74 11.26 130.00 150.00 50.00
PC-S_10 (evap) 1.71 27.00 13.00 7.00 130.00 150.00 46.17

Solid tumor tissue DNA[edit]

Sample Conc. (ng/ul) Volume for 50ng (ul) H2O (ul) Lightning Conv. Reagent (ul) Total volume (ul) Total DNA amount (ng)
PC-T-1 82.70 0.60 19.40 130.00 150.00 50.00
PC-T-2 35.80 1.40 18.60 130.00 150.00 50.00
PC-T-4 42.80 1.17 18.83 130.00 150.00 50.00
PC-T-6 15.20 3.29 16.71 130.00 150.00 50.00
PC-C-7 16.30 3.07 16.93 130.00 150.00 50.00
  • I did the 2nd set for those samples with high amount of DNA. Just in case there is something wrong with the result of some samples.
Sample Conc. (ng/ul) Volume for 50ng (ul) H2O (ul) Lightning Conv. Reagent (ul) Total volume (ul) Total DNA amount (ng)
PC-S_3 21.40 2.34 17.66 130.00 150.00 50.00
PC-S_6 17.50 2.86 17.14 130.00 150.00 50.00
PC-S_9 5.72 8.74 11.26 130.00 150.00 50.00
PC-T-1 82.70 0.60 19.40 130.00 150.00 50.00
PC-T-2 35.80 1.40 18.60 130.00 150.00 50.00
PC-T-4 42.80 1.17 18.83 130.00 150.00 50.00
PC-T-6 15.20 3.29 16.71 130.00 150.00 50.00
PC-C-7 16.30 3.07 16.93 130.00 150.00 50.00
  • Elute BIS-DNA with 9.2ul Elution buffer and expected DNA volume left ~8.4ul for capture, but I only have DNA left 7ul. Next time, I will elute with ~10ul Elution buffer.

BSPP capture set up[edit]

  • Since the input DNA for BIS-CVT was quite low, I did not measure BIS-DNA concentration since I did not want to lose DNA. I assumed that there was DNA after BIS-CVT ~10ng from 20% recovery rate.
  • I will use probe to target ration 1000:1 --> requires V1N3 amount 0.44ug from 10X diluted V1N3 (0.73ng/ul)
strip 1 PC-S_2 PC-S_3 PC-S_5 PC-S_6 PC-S_7 PC-S_9 PC-S_10
Volume (ul) 7.00 7.00 7.00 7.00 7.00 7.00 7.00
V1N3 amount (ng) 0.44 0.44 0.44 0.44 0.44 0.44 0.44
V1N3 conc. (ng/ul) 0.73 0.73 0.73 0.73 0.73 0.73 0.73
V1N3 volume (ul) 0.60 0.60 0.60 0.60 0.60 0.60 0.60
H2O in buffer mix 1.40 1.40 1.40 1.40 1.40 1.40 1.40
10X Ampligase Buffer 1.00 1.00 1.00 1.00 1.00 1.00 1.00
Total 10.00 10.00 10.00 10.00 10.00 10.00 10.00
strip 2 PC-T-1 PC-T-2 PC-T-4 PC-T-6 PC-C-7 NTC
Volume (ul) 7.00 7.00 7.00 7.00 7.00 7.00
V1N3 amount (ng) 0.44 0.44 0.44 0.44 0.44 0.44
V1N3 conc. (ng/ul) 0.73 0.73 0.73 0.73 0.73 0.73
V1N3 volume (ul) 0.60 0.60 0.60 0.60 0.60 0.60
10X Ampligase Buffer 1.00 1.00 1.00 1.00 1.00 1.00
H2O in buffer mix 1.40 1.40 1.40 1.40 1.40 1.40
Total 10.00 10.00 10.00 10.00 10.00 10.00
extra set[edit]
  • With the same condition as samples above
PC-S_3 PC-S_6 PC-S_9 PC-T-1 PC-T-2 PC-T-4 PC-T-6 PC-C-7

V1N3 & 10X Ampligase Buffer Mix[edit]

Components 1x rxn 25x rxn
V1N3 (0.73ng/ul) 0.60 15.00
10X Ampligase Buffer Mix 1.00 25.00
H2O 1.40 35.00
Total 3.00

Program

-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 1ul KLN mix (2U/ul Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 6h-> 94C 2min -> add 1.5ul Exo I/III mix (1ul of 20U/ul ExoI & 0.5ul of 200U/ul of ExoIII)-> 37C 2h -> 94C 5min -> 4C hold.

KLN Solution Mix[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume 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]

  • Used the same condition for PCR as experiment on 2014-06-02 with 10ul captured DNA in total 75ul
Components 1x rxn 22.5 rxn mix
Captured template 10.00 0.00
10uM AmpF6.4Sol 1.50 33.75
10uM AmpR6.3.IndX 1.50 0.00
2X KAPA SYBR MM 37.50 843.75
H2O 24.50 551.25
Total 75.00
Program (Eppendorf Realplex)

95C 30s -> (95C 10s -> 58C 20s -> 72C 20s)x8 -> (95C 10s -> 72C 20s)x19 -> 72C 3min

quantification of individual sequencing libraries[edit]

V1N3-S
File:ZhangLab 2 2014-06-12 15hr 10min PC-S V1N3.jpg
I did not include index43 in the library pools as the concentration is too low.
Sample Index Conc. (ng/ul) Yield in 35ul (ng) Volume for 20ng (ul)
PC-S_2 Ind_41 2.26 79.04 8.86
PC-S_3 Ind_42 1.11 38.78 18.05
PC-S_6 Ind_44 0.71 24.86 28.16
PC-S_7 Ind_45 1.39 48.75 14.36
PC-S_9 Ind_46 0.75 26.14 26.78
PC-S_10 Ind_47 0.78 27.47 25.48
  • Purified in 2 well of 5-well 6% TBE PAGE
V1N3-T
File:ZhangLab 2 2014-06-12 15hr 10min PC-T V1N3.jpg
Sample Index Conc. (ng/ul) Yield in 35ul (ng) Volume for 30ng (ul)
PC-T-1 Ind_51 2.99 104.81 10.02
PC-T-2 Ind_52 2.50 87.33 12.02
PC-T-4 Ind_53 2.96 103.45 10.15
PC-T-6 Ind_54 1.13 39.70 26.45
PC-T-7 Ind_55 2.99 104.62 10.04
  • Purified in 2 well of 5-well 6% TBE PAGE
NP-BSPP-V1N3-S-Jun10, 2014                 NP-BSPP-V1N3-T-Jun10, 2014     
File:ZhangLab 2 2014-06-13 10hr 19min V1N3 serum PAGE-SS.jpg File:ZhangLab 2 2014-06-13 10hr 19min V1N3 tumor PAGE-SS.jpg
  • Since we are going to get sequencing at Illumina, I pooled three library pools to one tube with the same molar ratio
Library pool # of libraries Ratio toV1N3-P Conc.by Qubit dsDNA HS (ng/ul) Pool 4ng of V1N3-P (ul)
V1N3-S 6 1.500 0.590 10.169
V1N3-T 5 1.250 0.610 8.197
V1N3-P 4 1.000 0.488 8.197
  • Library ID: NP-BSPP-V1N3-TSP-Jun10 --> was changed to NP-BSPP-V1N3-TSP-Jun13
  • Conc. by Qubit dsDNA HS quantification: 0.58ng/ul
  • Handed sequencing libraries to Alan

Index for serum samples[edit]

  • I used barcoded primers in column #6
strip 1 PC-S_2 PC-S_3 PC-S_5 PC-S_6 PC-S_7 PC-S_9 PC-S_10
Index Ind_41 Ind_42 Ind_43 Ind_44 Ind_45 Ind_46 Ind_47
  • I used barcoded primers in column #7 and start at well C1 (= Ind_51) to avoid bad barcodes
Project ID Sample type Zhang lab ID Index
UCSD-004-05 Serum PC-S_2 Ind_41
UCSD-004-06 Serum PC-S_3 Ind_42
UCSD-004-08 Serum PC-S_5 Ind_43
UCSD-004-09 Serum PC-S_6 Ind_44
UCSD-004-10 Serum PC-S_7 Ind_45
UCSD-004-12 Serum PC-S_9 Ind_46
UCSD-004-13 Serum PC-S_10 Ind_47

Index for solid tumor tissue samples[edit]

strip 2 PC-T-1 PC-T-2 PC-T-4 PC-T-6 PC-T-7 NTC
Index Ind_51 Ind_52 Ind_53 Ind_54 Ind_55 Ind_56
Project ID Sample type Zhang lab ID Index
UCSD-004-04 Solid tumor tissue PC-T-1 Ind_51
UCSD-004-05 Solid tumor tissue PC-T-2 Ind_52
UCSD-004-07 Solid tumor tissue PC-T-4 Ind_53
UCSD-004-09 Solid tumor tissue PC-T-6 Ind_54
UCSD-004-10 Solid tumor tissue PC-C-7 Ind_55

Index for plasma sample from Kang Zhang's lab with pre-amplification[edit]

  • Lab note on 2014-06-07: [[1]]
Index Sample Conc. (ng/ul) Yield in 70ul (ng) Volume for 40ng (ul)
Ind_33 NC-P-5ng_1 1.00 69.68 40.19
Ind_34 NC-P-5ng_2 0.62 43.70 50.00**
Ind_35 NC-P-10ng_1 0.88 61.38 45.62
Ind_36 NC-P-10ng_2 1.26 88.54 31.63
  • MiSeq data: /home/kunzhang/seqStore/20140613_MiSeq