Noi/NOTES/2012-7-27

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WGBS sequencing library preparation using KAPA protocol (Eppendorf) and epMotion[edit]

  • Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-6
  • Note that I used the same total volume of reaction similar to the protocol from Eppendorf. This might be slightly different from the original protocol from KAPA kit, but the total amount of enzyme is the same. I also adjust the volume of adapters in ligation step, but total volume and ratio of adapter to A-tailed DNA are the same.

Image of sheared DNA (loaded 0.5ul)

File:ZhangLab 2 2012-07-26 12hr 55min PAGE-Quan-shearedDNA.png

Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters[edit]

  • First of all, need to check if I have enough reagents for 8 rxn

Sample list[edit]

Labeled Sample IDs
1	GM12891
2	GM12892
3	GM10861
4	GM11994
5	GM11995
6	GM18506
7	GM18507
8	GM18505
End repair 
Total reaction in        70ul		
DNA 	                 50ul
End repair reaction mix 20ul

KAPA End repair mix	       1x rxn	8x rxn mix
10X End repai buffer	       7.00	56.00
KAPA End repair enzyme	       5.00	40.00
H2O	                       8.00	64.00
==================================================
Total	                      20.00   160.00
Aliquot to column #1: 20ul

20C for 30min
A-tailing
Total reaction in 30ul

A-tailing reaction mix	      1x rxn	8x rxn mix
KAPA 10X A-tailing buffer	3.00	24.00
KAPA A-tailing enzyme	        3.00	24.00
H2O	                       24.00   192.00
==================================================
Total	                       30.00   240.00
Aliquot to column #2: 30ul

30C for 30min
Adapter ligation
Total reaction in 50ul 
Ligation reaction mix 40ul
Methylation adapter   10ul

Ligation reaction mix	      1x rxn	8x rxn mix
KAPA 5X Ligation buffer	        10.00	80.00
KAPA DNA ligase	                 5.00	40.00
H2O	                        25.00	200.00
==================================================
Total	                        40.00	320.00
Aliquot to column #3: 40ul

Methylation adapter preparation
Alan's note: 
1. Use the methylated adapter with paired-end pcr primer pairs(PCR primer PE1.0 and PCR primer PE 2.0) provided in illumina's paired end genomic DNA sample prep kit
2. Use 10ul of methylated adapter to 5ug gDNA insert, if starting DNA amount is less than 5ug, titrate the volume of adapter reagent accordingly to maintain 10:1 ratio of DNA
Since I used 1ug of DNA, used 2ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same)

Methylation adapter	2ulx 8rxn	16.00
H2O	                 8ulx8rxn	64.00
==================================================
Total		                        80.00

20C for 15min
Reagent preparation
Reagents	                     1rxn	Min volume in 30mL reservior
AMPure beads	                    120ul	~1.1mL
20% PEG/2.5M NaCl	    90+50 = 140ul	~1.4mL
80% EtOH	            140x6 = 840ul      ~10.0mL
H2O for elution	                        23ul 	~1.0mL

epMotion run[edit]

Report file of epMotion run:

epMotion Started:5:00pm, finished: 7:30pm (~3hr for 8 samples)
  • Very important, when I added AMPure bead to end-repaired DNA and mixed for ten times, I have changed liquid type to water. This should be changed back to glycerol as before since the last step, the liquid was flushed out really quick, and it might cause contamination if bubbles are formed.
  • Note:
    • After adapter ligation I eluted DNA with 23ul H2O, and transfered eluted DNA to 96-well plate (on the same plate, column #3)
    • I checked adapter ligated DNA in 6% TBE gel (loaded 0.5 ul each, also load sheared DNA of sample #1 and #2 as control)
File:ZhangLab 2 2012-07-27 20hr 33min KAPA-adapter.jpg
 Labeled Sample IDs
#1B = #1 before end repair
#2B = #2 before end repair
#1	GM12891
#2	GM12892
#3	GM10861
#4	GM11994
#5	GM11995
#6	GM18506
#7	GM18507
#8	GM18505

2012.07.30

  1. Took 1ul of Adapter ligated DNA and added to 8ul EB buffer -> for some validation, like qPCR or PAGE quantification to compare to the bisulfite conversion products

qPCR to check if the end repair, A-tailing, and adapter ligation works

Components 1rxn 10 rxn mix
DNA template (1:9 diluted) 2.00 0.00
10uM PCR_F 1.00 10.00
10uM PCR_R.Ind1 1.00 10.00
2x iQ MM 25.00 250.00
H2O 21.00 210.00
Total 50.00 500.00
Program		
95C 	2min	 -> (95C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles
72C	3min
15C	Hold
# Run the gel check (the same labeled as above picture, but #1B, #1, #2B, #2, #3,....,#8) 
## 2ul of 10x diluted adapter ligated DNA (the same amount that added to 50ul qPCR)
## 3ul of PCR product (out of 50ul)
File:ZhangLab 2 2012-07-30 16hr 17min adapterligated qPCR.jpg
# From the image above, I can see the amplification of all samples. This suggests that the reaction in the previous steps performed by epMotion worked very well. Even this result is not the  final after bisulfite conversion, but it showed that the library prep before bisulfite conversion was success.

PROBLEM[edit]

  • When I performed bisulfite conversion of these 8 adapter ligated DNA, the reaction leaked out from the plate at the end, and they leaked to another well beside. I need to discard all of these reactions and start over the library prep again. I still have 1ug of sheared DNA left.

CAUSE OF PROBLEM[edit]

  • I never used this eppendorf plate for bisulfite conversion before. It should be the sealing problem because the well beside without DNA were sealed with plastic seal whereas the well with reaction sealed with a smalled strip of aluminum seal. The partial overlapping between the aluminum seal and plastic seal (on bottom) cause the reaction leaking. In the future, I should pay attention on sealing the plate seriously, since this problem never happen when I used the plate came with the kit and I did the bisulfite conversion on the whole plate and seal very well.
  • After I tested the plated by incubating few well of reagent at the same thermalcycler profile for Lightning MagPrep, I found that it's possible that I forgot to set the machine to heat lid. This because i observed the water drop on the top of the well even it didn't leak out like last time, but I didn't observed this when I heated the lid.
  • Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-8-1