Noi/NOTES/2014-11-25
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Comparison of the efficiency between TruSeq adaptor and a customized methylated Y-adaptor with UMI[edit]
- 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[edit]
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[edit]
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[edit]
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[edit]
Adaptor ligation reaction mix[edit]
- 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[edit]
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[edit]
- 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[edit]
- 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[edit]
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[edit]
End repair & dA-tailing reaction mix[edit]
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[edit]
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[edit]
Adaptor ligation reaction mix[edit]
- 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[edit]
- 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[edit]
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[edit]
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.