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]

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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[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.