Revision as of 09:04, 4 December 2014 by >RuiLiu
Purpose
- repeat selector procedure:
- selector : gDNA = 200:1; linker : selector = 3:1
- direct gDNA input: 12, 24, 48ng
- compare selector vs padlock probe
- premade
Samples
- HapMap gDNA from Coriell GM18506 (diluted to 50ng/ul)
|
gDNA input
|
Selector set
|
Probe ratio
|
Indx
|
Test
|
#1
|
48ng
|
linkerV2_UMI
|
200:1; 3:1
|
Indx01
|
selector_UMI
|
#2
|
24ng
|
linkerV2_UMI
|
200:1; 3:1
|
Indx02
|
selector_UMI
|
#3
|
12ng
|
linkerV2_UMI
|
200:1; 3:1
|
Indx03
|
selector_UMI
|
#4
|
NTC
|
linkerV2_UMI
|
200:1; 3:1
|
Indx04
|
selector_UMI
|
#5
|
48ng
|
linkerV2
|
200:1; 3:1
|
Indx05
|
selector
|
#6
|
24ng
|
linkerV2
|
200:1; 3:1
|
Indx06
|
selector
|
#7
|
12ng
|
linkerV2
|
200:1; 3:1
|
Indx07
|
selector
|
#8
|
NTC
|
linkerV2
|
200:1; 3:1
|
Indx08
|
selector
|
#9
|
48ng
|
linkerV2
|
200:1
|
Indx09
|
padlock probe
|
#10
|
24ng
|
linkerV2
|
200:1
|
Indx10
|
padlock probe
|
#11
|
12ng
|
linkerV2
|
200:1
|
Indx11
|
padlock probe
|
#12
|
NTC
|
linkerV2
|
200:1
|
Indx12
|
padlock probe
|
#13
|
50ng_digested (Kun's, asPC)
|
linkerV2_UMI
|
200:1; 3:1
|
Indx13
|
selector_UMI
|
#14
|
20ng_digested (Kun's, asPC)
|
linkerV2_UMI
|
200:1; 3:1
|
Indx14
|
selector_UMI
|
Procedure
1a. R.E digestion
Components
|
48ng
|
24ng
|
12ng
|
NTC
|
gDNA (12ng/ul)
|
4.00
|
2.00
|
1.00
|
0.00
|
10x cutsmart buffer
|
1.00
|
1.00
|
1.00
|
1.00
|
DpnII (50U/ul) -> dilute to 5U/ul
|
2.50
|
1.25
|
0.63
|
0.63
|
BfaI (10U/ul) -> dilute to 2U/ul
|
2.50
|
1.25
|
0.63
|
0.63
|
H2O
|
0.00
|
4.50
|
6.75
|
7.75
|
Total
|
10.00
|
10.00
|
10.00
|
10.00
|
Temperature
|
Duration
|
Digestion
|
37C, 1hr
|
hold
|
4C
|
Components
|
Volume (ul)
|
Final conc.
|
10X NEBuffer 3.1
|
1
|
1x
|
Temperature
|
Duration
|
Digestion
|
37C, 30min
|
heat inactivation
|
80C, 20min
|
hold
|
4C
|
1b. Assembly of selecotr and linker
Components Vol. (ul) Final
LMS_selector V2 (1ng/ul: 33nM) 20.00 0.4ng/ul ratio 200:1
linkerV2 or linkerV2_UMI (100nM) 20.00 3:1 linker:selector
10X AmpLigase buffer 5.00 1x
H2O 5.00
Total 50.00
2. Hybridization: Ampligase buffer needs to be aliquot and stored individually
Components
|
Final conc.
|
48ng
|
24ng
|
12ng
|
NTC
|
48ng
|
24ng
|
12ng
|
NTC
|
digested gDNA
|
0/12/24/48ng
|
10.00
|
10.00
|
10.00
|
10.00
|
4.00
|
2.00
|
1.00
|
0.00
|
10x Ampligase buffer
|
1x
|
1.36
|
1.68
|
1.84
|
1.84
|
2.1
|
2.1
|
2.1
|
2.1
|
MgCl2 (25mM)
|
2mM
|
1.60
|
1.60
|
1.60
|
1.60
|
0.00
|
0.00
|
0.00
|
0.00
|
NAD (50mM)
|
1mM
|
0.40
|
0.40
|
0.40
|
0.40
|
0.00
|
0.00
|
0.00
|
0.00
|
Selector/linker
|
200:1
|
6.38
|
3.20
|
1.60
|
1.60
|
2.55
|
1.28
|
0.64
|
0.64
|
H2O
|
|
0.26
|
3.12
|
4.56
|
4.56
|
11.35
|
14.62
|
16.26
|
17.26
|
Total
|
|
20.00
|
20.00
|
20.00
|
20.00
|
20.00
|
20.00
|
20.00
|
20.00
|
Thermocycler steps
|
Duration
|
Denature
|
95C, 5min
|
Cool down to 60C slowely
|
at -0.2C/sec
|
Hold
|
60C, 16hrs
|
3. Synthesis: Ampligase plus Taq pol.
Components
|
Selector
|
Probe
|
Ampligase (2.5U/ul diluted in water)
|
1
|
0.4
|
HemoKlen Taq (10U/ul)
|
0
|
0.4
|
dNTP mix (1mM)
|
0
|
0.2
|
Total
|
1
|
1
|
Thermocycler steps
|
Duration
|
Synthesis
|
60C, 2hrs
|
Denature
|
94C, 2min
|
hold
|
8C
|
4. ExoM: made mixture of ExoI/III before adding into samples
Components
|
Volume (ul)
|
Final conc.
|
ExoI
|
1
|
|
ExoIII
|
1
|
|
Total
|
2
|
|
Thermocycler steps
|
Duration
|
Digestion
|
37C, 1hr
|
Denature
|
80C, 20min
|
hold
|
8C
|
5. PCR amplification: quick run with 1/10 of sample for an estimation of capture efficiency
Components
|
Volumn (ul)
|
Final conc.
|
cirDNA template
|
22
|
all
|
Q5 MM
|
30
|
1x
|
10 uM CP-2-RA
|
1.2
|
0.2uM
|
10 uM AmpR6.3.Indx
|
1.2
|
0.2uM
|
20x SYBR
|
0.5
|
0.2x
|
H2O
|
5.09999999999999
|
|
Total
|
60
|
|
Realtime-PCR profile
|
QPCR cycles
|
98C 5min
|
1
|
98C 15sec -> 58C 30sec -> 72C 15sec
|
8
|
98C 15sec -> 72C 15sec
|
16
|
72C 3min
|
1
|
Hold 10C
|
|
6. Beads purfication on PCR amplicons
Components
|
Volume (ul)
|
Final conc.
|
PCR amplicon
|
55
|
5ul for gel
|
AMPure beads
|
50
|
0.8X
|
* Clearly remove majority of bands/smear below 200bp
|
|
|
Results
File:Nv4 1-4 QPCR.jpgFile:Nv4 5-12 QPCR.jpg
File:Nv4 1-4 gel.jpgFile:Nv4 5-12 gel.jpg
Mixed lib.
- Following libraries were combined and size selected for sequencing
- Tube label: RL-Nv4_UMI-Dec3
|
gDNA input
|
Selector set
|
Probe ratio
|
Indx
|
Test
|
Mixed V (ul)
|
#1
|
48ng
|
linkerV2_UMI
|
200:1; 3:1
|
Indx01
|
selector_UMI
|
2
|
#2
|
24ng
|
linkerV2_UMI
|
200:1; 3:1
|
Indx02
|
selector_UMI
|
4
|
#3
|
12ng
|
linkerV2_UMI
|
200:1; 3:1
|
Indx03
|
selector_UMI
|
8
|
#13
|
50ng_digested (Kun's, asPC)
|
linkerV2_UMI
|
200:1; 3:1
|
Indx13
|
selector_UMI
|
2
|
#14
|
20ng_digested (Kun's, asPC)
|
linkerV2_UMI
|
200:1; 3:1
|
Indx14
|
selector_UMI
|
4
|
* size selection can be skipped, pretty clean below 200bp
|
|
|
|
|
|
|
|
gDNA input
|
Selector set
|
Probe ratio
|
Indx
|
Test
|
Mixed V (ul)
|
#5
|
48ng
|
linkerV2
|
200:1; 3:1
|
Indx05
|
selector
|
2
|
#6
|
24ng
|
linkerV2
|
200:1; 3:1
|
Indx06
|
selector
|
4
|
#7
|
12ng
|
linkerV2
|
200:1; 3:1
|
Indx07
|
selector
|
8
|
#9
|
48ng
|
linkerV2
|
200:1
|
Indx09
|
padlock probe
|
2
|
#10
|
24ng
|
linkerV2
|
200:1
|
Indx10
|
padlock probe
|
4
|
#11
|
12ng
|
linkerV2
|
200:1
|
Indx11
|
padlock probe
|
8
|
* need size selection, still have a few bands below 200bp
|
|
|
|
|
|
|