Revision as of 12:33, 29 November 2014 by >Noi
Comparison of the efficiency between TruSeq adaptor and a customized unmethylated Y-adaptor with UMI
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- Dr. Zhang wanted to compare the efficiency of standard adaptor and a customized Y-adaptor with UMI for generating sequencing libraries using KAPA Hyper Prep kit. In this experiment, I used TruSeq adaptor to represent standard adaptor.
- In addition to compare between the two versions of adaptor, I also compare between a half reaction volume (30ul of end-repair/A-tailing and 55ul of ligation reaction) and a full reaction (60ul of end-repair/A-tailing and 110ul of ligation reaction) following the reaction set up of the instruction from KAPA. The reason is that the reaction is optimum for DNA input between 1ng-1ug. My experiment uses 1ng of input, so I think that the enzymes/and reagents should be more than enough. I therefore set up a half reaction volume to save the reagents and the concentrations of all components are still the same.
- I received top and bottom oligos to generated Y-adaptor --> final conc. 15uM to be equivalent to the concentration of TruSeq adaptor
Annealing of Y-adaptor
Component Volume (ul)
SG_Adt.top_v1 (100uM) 30.00
SG_Adt.bottom_v3 (100uM) 30.00
10X Stoffel Buffer 20.00
H2O 120.00
Total 200.00
I split 100ul to 2X of 0.2mL PCR tube and incubate at the following templarature
95C, 5min --> -0.1C/s to 20C --> hols at 20C, 5min and put on ice
Sample list
No Sample IDs Conc. (ng/ul)
#1 6P-2 1.10
#2 6P-3 1.57
#3 7P-2 1.14
- Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally.
- I set up one reaction of sample 7P-2 in a full reaction volume for comparison
- KAPA Hyper Prep reaction set up (exactly in the instruction):
- - 50ul of fragmented DNA
- - 7ul of End repair & dA-tailing buffer
- - 3ul of End repair & dA-tailing enzyme mix
- - Total reaction: 60ul
End repair & dA-tailing
Sample IDs Adaptor Reaction volume Tube #
6P-2 TruS half rxn #1
6P-3 TruS half rxn #2
7P-2 TruS half rxn #3
NTC TruS half rxn #4
6P-2 UMI half rxn #5
6P-3 UMI half rxn #6
7P-2 UMI half rxn #7
NTC UMI half rxn #8
7P-2 TruS full rxn #9
TruS = TruSeq adaptor
UMI = customized unmethylated Y-adaptor with UMI
Tube #3 and #9 will be compared the effect the reaction volume.
End repair & dA-tailing reaction mix
Components
|
Volume
|
half rxn
|
10.2x rxn
|
End repair/dA-tailing buffer |
7.00 |
3.50 |
35.70
|
End repair/dA-tailing enzyme mix |
3.00 |
1.50 |
15.30
|
Total |
10.00 |
5.00 |
|
Reaction set up
Sample IDs
|
Conc. (ng/ul)
|
Volume for 1ng (ul)
|
End repair/dA-tailing rxn mix
|
H2O (ul)
|
Total (ul)
|
6P-2 |
1.10 |
0.91 |
5.00 |
24.09 |
30.00
|
6P-3 |
1.57 |
0.64 |
5.00 |
24.36 |
30.00
|
7P-2 |
1.14 |
0.88 |
5.00 |
24.12 |
30.00
|
NTC |
0.00 |
0.00 |
5.00 |
25.00 |
30.00
|
6P-2 |
1.10 |
0.91 |
5.00 |
24.09 |
30.00
|
6P-3 |
1.57 |
0.64 |
5.00 |
24.36 |
30.00
|
7P-2 |
1.14 |
0.88 |
5.00 |
24.12 |
30.00
|
NTC |
0.00 |
0.00 |
5.00 |
25.00 |
30.00
|
7P-2 |
1.14 |
0.88 |
10.00 |
49.12 |
60.00
|
- - Mix well
- - 20C, 30min
- - 65C, 30min,
- - Hold 4C
- - Proceed to Adaptor ligation immediately
Adaptor ligation
Adaptor ligation reaction mix
- I prepare the new tube of 1:20 diluted TruSeq adaptor (conc. after dilution = 714nM) Index3. Since Dr. Zhang does not want to sequence the library, I just used the same barcoded adaptor for all samples.
- I also diluted a customized Y-adaptore 1:20 with 1X Stoffel buffer to be easier for ligation reaction mix prep.
- I prepared two tube of ligation reaction mix containing TruSeq adaptor and customized adaptore with UMI
Components
|
Volume
|
half rxn
|
4.1x rxn
|
Ligation buffer |
30.00 |
15.00 |
61.50
|
DNA ligase |
10.00 |
5.00 |
20.50
|
H2O |
8.00 |
4.00 |
16.40
|
Total |
48.00 |
24.00 |
98.40
|
1:20 diluted adaptor (714nM) |
2.00 |
1.00 |
4.10
|
Total |
50.00 |
25.00 |
|
- Final conc. of adaptor in ligation reaction is 13.64nM (the kit recommends 14nM)
Adaptor ligation reaction set up
Components
|
Volume (ul)
|
End repair & dA-tailing rxn product |
30.00
|
Adaptor ligation rxn mix |
25.00
|
Total |
55.00
|
- For a full reaction, I add reagent directly to the tube woth 2ul of 1:20 diluted adaptor
- - Add 25ul of adaptor ligation reaction mix
- - Mix well
- - 20C, 15min
- - Continue to SPRI clean up immediately
1X AMPure bead purification
- The instruction suggested to use 0.8X of AMPure bead for clean up, but I use 1X of AMPure bead
- Aliquot AMPure bead in 1.5mL tube and leave at RT
- - Add 55ul AMPure bead for a half reaction and 110ul for a full reaction. Mix by pipetting 10x
- - Sit for 15min
- - Transfer to sit on magnet for 5min
- - Wash twice with 200ul freshly prepared 80% EtOH
- - Dry the bead for 3-5min
- - Resuspend with 25ul EB buffer
Amplification
- I prepared 2 tubes of PCR mix for primer pairs of TruSeq adaptor and a customized adaptor with UMI
Components
|
1X 50ul rxn
|
10ul rxn
|
6 rxn
|
Adaptor ligated DNA |
20.00 |
2.00 |
0.00
|
2X KAPA HiFi Hotstart ReadyMix |
25.00 |
5.00 |
30.00
|
10uM TruS F/R or 10uM SG_PCR F/RInd1 |
1.50 |
0.30 |
1.80
|
50X SYBR |
0.40 |
0.08 |
0.48
|
H2O |
3.10 |
2.62 |
15.72
|
Total |
50.00 |
10.00 |
|
- I used less of DNA template than the instruction
- - Aliquot 8 + 2ul adaptor ligated DNA
- - Mix well
- 98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 10 --> 72C for 1min
File:2014-11-27 qPCR-TruSeqAdaptor-10x.PNG File:2014-11-27 qPCR-CustomUMIAdaptor-10x.PNG
- - Verify 4ul of PCR product in 6% TBE gel
File:ZhangLab 2 2014-11-27 14hr 44min KAPAHyper TruS-CustomUMI-10x v2.jpg
- Dr. Zhang thought that there should be something about the number of annealed adaptor molecules even I used the same concentration for annealing. The present of 125bp adaptor dimers in TruSeq adaptor but very faint in customized adaptor could also partially contribute to qPCR curve.
- He suggested me to repeat experiment using one plasma DNA sample and use1X, 2X and 4X of customized adaptor.
Repeat WGS library prep by varying concentration of customized adaptor
2014-11-28
- I used sample PCP-3 for this experiment as it has total amount more than 30ng.
- Conc. o fPCP-3 = 1.36ng/ul
Sample IDs Adaptor Adaptor conc. Tube #
PCP-3 TruS 1X 1
PCP-3 UMI 1X 2
PCP-3 UMI 2X 3
PCP-3 UMI 4X 4
End repair & dA-tailing
End repair & dA-tailing reaction mix
Components
|
Volume
|
half rxn
|
4.2x rxn
|
End repair/dA-tailing buffer |
7.00 |
3.50 |
14.70
|
End repair/dA-tailing enzyme mix |
3.00 |
1.50 |
6.30
|
Total |
10.00 |
5.00 |
|
Reaction set up
Sample IDs
|
Conc. (ng/ul)
|
Volume for 1ng (ul)
|
End repair/dA-tailing rxn mix
|
H2O (ul)
|
Total (ul)
|
PCP-3_TruS |
1.36 |
0.74 |
5.00 |
24.26 |
30
|
PCP-3_1x |
1.36 |
0.74 |
5.00 |
24.26 |
30
|
PCP-3_2X |
1.36 |
0.74 |
5.00 |
24.26 |
30
|
PCP-3_4X |
1.36 |
0.74 |
5.00 |
24.26 |
30
|
- - Mix well
- - 20C, 30min
- - 65C, 30min,
- - Hold 4C
- - Proceed to Adaptor ligation immediately
Adaptor ligation
Adaptor ligation reaction mix
- I used TruSeq adaptor prepare on 2014-11-25 (diluted 1:20, conc. 714nM)
- I diluted customized adaptor 1:20 by mixing 2ul of 15uM annealed adaptor (prepared on 2014-11-25) with 40ul of H2O.
Components
|
Volume
|
half rxn
|
4.1x rxn
|
Ligation buffer |
30.00 |
15.00 |
61.50
|
DNA ligase |
10.00 |
5.00 |
20.50
|
H2O |
2.00 |
1.00 |
4.10
|
Total |
42.00 |
21.00 |
|
1:20 diluted adaptor (714nM) |
Varied |
Varied |
|
Total |
110.00 |
55.00 |
|
Sample IDs
|
End repair/dA-tailed DNA
|
H2O
|
1:20 diluted adaptor (714nM)
|
Adaptor
|
Ligation rxn mix
|
Total
|
PCP-3_TruS_1X |
30.00 |
3.00 |
1.00 |
TruS |
21.00 |
55.00
|
PCP-3_1x |
30.00 |
3.00 |
1.00 |
UMI |
21.00 |
55.00
|
PCP-3_2X |
30.00 |
2.00 |
2.00 |
UMI |
21.00 |
55.00
|
PCP-3_4X |
30.00 |
0.00 |
4.00 |
UMI |
21.00 |
55.00
|
- - Add adaptor according to table above and 21ul of adaptor ligation reaction mix
- - Mix well
- - 20C, 15min
- - Continue to SPRI clean up immediately
1X AMPure bead purification
- The instruction suggested to use 0.8X of AMPure bead for clean up, but I use 1X of AMPure bead
- Aliquot AMPure bead in 1.5mL tube and leave at RT
- - Add 55ul AMPure bead for a half reaction and 110ul for a full reaction. Mix by pipetting 10x
- - Sit for 15min
- - Transfer to sit on magnet for 5min
- - Wash twice with 200ul freshly prepared 80% EtOH
- - Dry the bead for 3-5min
- - Resuspend with 25ul EB buffer
Amplification
Components
|
1X 50ul rxn
|
10ul rxn
|
6 rxn
|
Adaptor ligated DNA |
10.00 |
2.00 |
0.00
|
2X KAPA HiFi Hotstart ReadyMix |
25.00 |
5.00 |
30.00
|
10uM TruS F/R or 10uM SG_PCR F/RInd1 |
1.50 |
0.30 |
0.00
|
50X SYBR |
0.40 |
0.08 |
0.48
|
H2O |
13.10 |
2.62 |
15.72
|
Total |
50.00 |
10.00 |
|
- I split PCR mix into two tube and add primers for TruSeq adaptor (add 0.6ul of 10uM TruS F/R to 15.4ul of PCR mix and add 1.2ul of 10uM SG_PCR F/RInd1 to the left over PCR mix in the tube)
- - Aliquot 8 + 2ul adaptor ligated DNA
- - Mix well
- 98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 9 --> 72C for 1min
- I stopped at 9 cycles to avoid amplification saturation for a precise comparison.
File:2014-11-28 qPCR-vary-customAdapt-9X 300nMprimer.PNG
- This time, the qPCR curves of 1X TruSeq and 1X customized adaptor were pretty much the same which is different from previous experiment on 2014-11-25. I will look at gel image for comparison with qPCR curves.
- - Loaded 3ul of PCR product in 6% TBE gel
File:ZhangLab 2 2014-11-28 19hr 20min vary-custAdaptor 9X.jpg
- - I also did PAGE quantification to compare the yield of each condition
PAGE quantification result
Adaptor Relative intensity to TruSeq 1X (14nM)
TruS_1X 1.00
UMI_1X 0.71
UMI_2X 0.87
UMI_4X 1.05
- from this result, Dr. Zhang suggested that it should be fine to use 2X of adaptor (28nM) in ligation reaction.