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

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==== End repair & dA-tailing reaction mix ====
==== End repair & dA-tailing reaction mix ====
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="width:200px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:240px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume'''
| align="center" style="width:80px;background:#f0f0f0;"|'''half rxn'''
| align="center" style="width:80px;background:#f0f0f0;"|'''half rxn'''
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| Total||10.00||5.00||
| Total||10.00||5.00||
|}
|}
==== Reaction set up ====
==== Reaction set up ====
{| {{table}} border = 1
{| {{table}} border = 1

Revision as of 02:34, 28 November 2014

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
- 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