Noi/NOTES/2014-11-25: Difference between revisions

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= Comparison of the efficiency between TruSeq adaptor and an unmethylated customized Y-adaptor with UMI =
= Comparison of the efficiency between TruSeq adaptor and a customized unmethylated Y-adaptor with UMI =
[[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]
[[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]
* 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.  
* 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 oligo --> final conc. 15uM to be equivalent to the concentration of TruSeq adaptor
* I received top and bottom oligos to generated Y-adaptor oligo --> final conc. 15uM to be equivalent to the concentration of TruSeq adaptor
<br>
<br>
  Conditions
  '''<u>Annealing of Y-adaptor</u>'''
'''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'''<br>
'''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 6P-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 #1 and #9 will be compared the effect of total volume.
==== End repair & dA-tailing reaction mix ====

Revision as of 01:00, 28 November 2014

Comparison of the efficiency between TruSeq adaptor and a customized unmethylated Y-adaptor with UMI

Back to calendar

  • 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 oligo --> 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 6P-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 #1 and #9 will be compared the effect of total volume.

End repair & dA-tailing reaction mix