Noi/NOTES/2012-8-3

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  • Note: This page was the summary of WGBS prep to share with David Lin from Eppendorf

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

  • 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 gDNA (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]

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
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)
  • 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.

Bisulfite conversion using EZ-96 DNA Methylation-Lightning™ MagPrep[edit]

  • Performed bisulfite conversion manually and followed the product's protocol: EZ-96DNAMethylation-LightninMagPrep.pdf
    • For bisulfite conversion in the deep well plate, I did it manually since we don't have TM 1000-8 pipette and discarding spnt multiple rounds with TM 300-8 pipette might be prone to contamination
  • Elute with elution buffer 40ul

Amplification of bisulfite sequencing libraries with N2 barcoded primers[edit]

Components 1 rxn 26rxn mix
DNA template 10.00 0.00
10uM PCR_F 2.00 52.00
10uM PCR_R.Indx (1-8) 2.00 0.00
2x iQ Supermix 50.00 1,300.00
H2O 36.00 936.00
  • Aliquot 88ul, add 2ul of 10uM PCR_R Ind (Ind1-8), 10ul of bis-cvt DNA template
Program	
98C 2min (98C 15sec ->	64C 20sec -> 72C 30sec) x10cycles -> 72C 3min -> 15CHold
Sample ID Labeling Indx
WGBS.GM12891 #1 PCR_R.N2Indx1
WGBS.GM12892 #2 PCR_R.N2Indx2
WGBS.GM10861 #3 PCR_R.N2Indx3
WGBS.GM11994 #4 PCR_R.N2Indx4
WGBS.GM11995 #5 PCR_R.N2Indx5
WGBS.GM18506 #6 PCR_R.N2Indx6
WGBS.GM18507 #7 PCR_R.N2Indx7
WGBS.GM18505 #8 PCR_R.N2Indx8

qPCR results
File:20120801 qPCRWGBS1-8-1.png

  • Purified sequencing libraries with 0.8x vol AMPure bead with epMotion (3 strips), eluted with 25ul EB for each well -> total volume at the end ~70ul

Report file of epMotion run:Media:MasterReport_20120802_beadPure.txt

File:BeadPure Aug2.png

File:ZhangLab 2 2012-08-02 16hr 05min bead-HAPMAP-WGBS.jpg

  • Note: To validate the quality of sequencing library, Dr. Zhang suggested to do small scale sequencing before doing deep sequencing, so I pooled small amount of each sequencing library and performed PAGE-size selection.

PAGE quantification of 400-600bp fragments[edit]

  • Cut the band around 400-600bp for PE sequencing.
  • Perform PAGE size-selection of the size range 400-600bp for a small scale sequencing. Then pooled 60ng of each library and performed PAGE size-selection.

File:ZhangLab 2 2012-08-02 16hr 05min bead-HAPMAP-WGBS-400-600bp PQ.jpg

Lab IDs Final conc.

of 400-600bp (ng/ul)

Total yields in 70ul (ng) Volume for 60ng (ul)
#1 13.50 945.28 4.44
#2 9.66 675.94 6.21
#3 11.24 786.94 5.34
#4 13.64 955.09 4.40
#5 13.85 969.55 4.33
#6 8.95 626.85 6.70
#7 12.40 867.78 4.84
#8 11.29 790.44 5.31


PAGE size-selection[edit]

File:ZhangLab 2 2012-08-02 18hr 23min-PAGE-SS.jpg

  • Note: From the image, it seemed like I cut the gel a little smaller than 600bp
  • Resuspend with 12ul H2O each tube -> total volume 24ul.
  • Quantify concentration by PAGE quantification (also verify the size of sequencing libraries) and by dsDNA Qubit -> conc. 1.47ng/ul or 4.83nM
  • Library ID: NP_WGBS_HAPMAP1-8_July27, sequenced on GAII HL128 run. The eight libraries were spiked 1/10 in lane 1.

Mapping result of NP_WGBS_HAPMAP1-8 sequencing libraries[edit]

  • Mapping 1st read to hg19
Sample IDs Total # of reads Mappable reads Mapping rate (%) Bisulfite conversion rate (%)
GM10861** 7,419,387 583,598 7.87 98.70
GM11994 687,322 604,611 87.97 99.30
GM11995 774,373 689,816 89.08 99.10
GM12891 715,640 631,161 88.20 98.50
GM12892 638,358 553,167 86.65 99.30
GM18505 810,806 717,095 88.44 99.40
GM18506 586,667 513,178 87.47 99.30
GM18507 686,850 606,360 88.28 99.10
  • Note: Sample ID GM10861 has about 10times more sequencing read than other libraries. I think this was because it was loaded on the same lane with bacterial sequencing library, which used the same barcode.