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[[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]<br>
= WGBS sequencing library preparation using KAPA protocol (Eppendorf) and epMotion (Repeat!) =
= WGBS sequencing library preparation using KAPA protocol (Eppendorf) and epMotion (Repeat!) =
* The step of library sequencing prep will be filled soon.
* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-27
== Amplification of bisulfite sequencing llibraries with N2 barcoded primers ==
* I have sheared gDNA from the previous batch left ~22ul (conc. ~40ng/ul based on conc. from the HAPMAP plate). I added 25ul H2O to each tube -> total volume 47ul, and will prepare end repair reaction mix in 23ul
* Note: Since I start with sheared 880ng, I reduce the amount of enzymes in each step, but keep total volume the same (except ligation 50ul -> 40ul total)
== End repair ==
{| {{table}}
| width ="150pt" align="center" style="background:#f0f0f0;"|'''Total reaction'''
| align="center" style="background:#f0f0f0;"|'''70.00'''
| align="center" style="background:#f0f0f0;"|'''ul'''
|-
| DNA ||47.00||ul
|-
| End repair reaction mix ||23.00||ul
|}<br>
{| {{table}}
| width ="150pt" align="center" style="background:#f0f0f0;"|'''KAPA End repair mix'''
| width ="80pt" align="center" style="background:#f0f0f0;"|'''1x rxn'''
| width ="80pt" align="center" style="background:#f0f0f0;"|'''8x rxn mix'''
|-
| 10X End repai buffer||7.00||56.00
|-
| KAPA End repair enzyme||3.00||24.00
|-
| H2O||13.00||104.00
|-
| Total||23.00||184.00
|}
* Aliquot to column #1: 23ul
== A-tailing ==
A-tailing reaction mix: Total 30ul/reaction<br>
{| {{table}}
| width ="150pt" align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix'''
| width ="80pt" align="center" style="background:#f0f0f0;"|'''1x rxn'''
| width ="80pt" align="center" style="background:#f0f0f0;"|'''8x rxn mix'''
|-
| KAPA 10X A-tailing buffer||3.00||24.00
|-
| KAPA A-tailing enzyme||2.00||16.00
|-
| H2O||25.00||200.00
|-
| Total||30.00||240.00
|}
* Aliquot to column #2: 30ul
== Adapter ligation ==
Adapter ligation mix: Total 40ul/reaction -> 10ul is methylation adapters
{| {{table}}
| width ="150pt"  align="center" style="background:#f0f0f0;"|'''Ligation reaction mix'''
| width ="80pt" align="center" style="background:#f0f0f0;"|'''1x rxn'''
| width ="80pt" align="center" style="background:#f0f0f0;"|'''8x rxn mix'''
|-
| KAPA 5X Ligation buffer||8.00||64.00
|-
| KAPA DNA ligase||3.00||24.00
|-
| H2O||19.00||152.00
|-
| Total||30.00||240.00
|}
* Aliquot to column #3: 30ul
== Methylation adapter preparation ==
'''Alan's note:''' <br>
# 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
# 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)
Prepare adapter 2ulx 8rxn --> 16.00ul<br>
H2O 8ulx8rxn --> 64.00ul<br>
Total 80.00ul<br>
== Reagent preparation ==
{| {{table}}
| width ="150pt" align="center" style="background:#f0f0f0;"|'''Reagents'''
| width ="80pt" align="center" style="background:#f0f0f0;"|'''1rxn'''
| width ="120pt" align="center" style="background:#f0f0f0;"|'''Min volume in 30mL reservior'''
|-
| AMPure beads||120ul||~1.1mL
|-
| 20% PEG/2.5M NaCl||90+50 = 140ul||~1.4mL
|-
| 80% EtOH||140x6times||~10mL
|-
| H2O for elution||22ul||~1mL
|}
 
==epMotion run ==
'''Report file of epMotion run:''' [[Media:MasterReport_LibPrep_HAPMAPAug1.txt]]<br>
[[File:LibPreP_HAPMAP_Aug1.png| 500px]]
* Note: I used all adapter ligated DNA for bisulfite conversion, so I don't have the image showing DNA before and after end repair, A-tailing and adapter ligation. However I did most of the step the same as I did on July 27.
'''Image of DNA before end repair and after adapter ligation performed on 2012.07.27
[[File:ZhangLab_2 2012-07-26 12hr 55min_PAGE-Quan-shearedDNA.png| 350px]] [[File:ZhangLab_2 2012-07-27 20hr 33min_KAPA-adapter.jpg| 400px]]
 
= Bisulfite conversion using EZ-96 DNA Methylation-Lightning™ MagPrep =
* Performed bisulfite conversion manually and followed the product's protocol: [[Media:EZ-96DNAMethylation-LightninMagPrep.pdf| EZ-96DNAMethylation-LightninMagPrep.pdf]]
* Elute with elution buffer 40ul
== ssDNA Qubit quantification ==
* Used 3ul of bis-cvt DNA for the assay (dilution 200/3)
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''uL used'''
| align="center" style="background:#f0f0f0;"|'''Dilution'''
| align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Yields in 40ul'''
|-
| #1||17.2||ng/mL||3||66.67||1.15||45.87
|-
| #2||4.4||ng/mL||3||66.67||0.29||11.73**
|-
| #3||12.3||ng/mL||3||66.67||0.82||32.80
|-
| #4||65.6||ng/mL||3||66.67||4.37||174.93
|-
| #5||32.9||ng/mL||3||66.67||2.19||87.73
|-
| #6||7.16||ng/mL||3||66.67||0.48||19.09**
|-
| #7||48.3||ng/mL||3||66.67||3.22||128.80
|-
| #8||30.6||ng/mL||3||66.67||2.04||81.60
|}
* The yield after bisulfite conversion is relatively low when compare to initial amount of DNA. However, the DNA amount would be reduced in each step. From the image, bead purification did'n recover the fragments < ~200bp, so I don't expect to get very high after bisulfite conversion. The total yields I got after bisulfite conversion are varied as well, and the yield of some sample didn't correlated with the intensity before bisulfite conversion. However, I could not rely on to this image because it is the image of previous experiment. Next time I should collect sample for verification.
* **= samples have very low yields --> If compare to the intensity and total yield after amplification with barcode primers, these concentrations by ssDNA Qubit quantification seemed to agree to the results below.
== Amplification of bisulfite sequencing llibraries (Test) ==
* Test if the adapter ligation reaction worked before amplify the libraries with barcoded primers
* Test if the adapter ligation reaction worked before amplify the libraries with barcoded primers
 
{| {{table}}
* Loaded 2.5 ul of bis-cvt DNA and 2.5ul of amplified amplicons
| width="160pt" align="center" style="background:#f0f0f0;"|'''Components'''
| width="60pt" align="center" style="background:#f0f0f0;"|'''1rxn'''
| align="center" style="background:#f0f0f0;"|'''10 rxn mix'''
|-
| DNA template ||2.50||0.00
|-
| 10uM PCR_F||0.50||5.00
|-
| 10uM PCR_R.Ind1||0.50||5.00
|-
| 2x iQ MM||12.50||125.00
|-
| H2O||9.00||90.00
|-
| Total||25.00||250.00
|}
* Aliquot 22.5ul, add 2.5ul of bis-cvt DNA template (2.5 out of 40ul)
'''Program'''
98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x15cycles -> 72C 3min -> 15CHold
'''qPCR results'''<br>
[[File:20120801_qPCR_adapterligated.png| 600px]]
<br>
* Loaded 2.5 ul of bis-cvt DNA (#1 & #4) and 2.5ul of amplified amplicons
[[File:ZhangLab_2 2012-08-02 11hr 31min-HAPMAP-bis-test.jpg| 400px]] <br>
[[File:ZhangLab_2 2012-08-02 11hr 31min-HAPMAP-bis-test.jpg| 400px]] <br>


== Amplification of bisulfite sequencing libraries with N2 barcoded primers ==
{| {{table}} border = 1
{| {{table}} border = 1
| width="160pt" align="center" style="background:#f0f0f0;"|'''Components'''
| width="160pt" align="center" style="background:#f0f0f0;"|'''Components'''
Line 23: Line 166:
|}
|}
* Aliquot 88ul, add 2ul of 10uM PCR_R Ind (Ind1-8), 10ul of bis-cvt DNA template
* Aliquot 88ul, add 2ul of 10uM PCR_R Ind (Ind1-8), 10ul of bis-cvt DNA template
  '''Program'''<br>
  '''Program'''
  98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x10cycles -> 72C 3min -> 15CHold
  98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x10cycles -> 72C 3min -> 15CHold
{| {{table}}  
{| {{table}}  
| width="80pt" align="center" style="background:#f0f0f0;"|'''Sample ID'''
| width="80pt" align="center" style="background:#f0f0f0;"|'''Sample ID'''
Line 46: Line 189:
| WGBS.GM18505||#8||PCR_R.N2Indx8
| WGBS.GM18505||#8||PCR_R.N2Indx8
|}
|}
'''qPCR results'''<br>
[[File:20120801_qPCRWGBS1-8-1.png| 600px]]
<br>
* 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
* 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]]<br>
[[File:BeadPure_Aug2.png| 500px]]
[[File:ZhangLab_2 2012-08-02 16hr 05min_bead-HAPMAP-WGBS.jpg| 400px]] <br>
[[File:ZhangLab_2 2012-08-02 16hr 05min_bead-HAPMAP-WGBS.jpg| 400px]] <br>


== PAGE quantification of 400-600bp fragments ==
== PAGE quantification of 400-600bp fragments ==
* Dr. Zhang suggested to cut the band around 400-600bp because 250bp is too short 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.  
* 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| 400px]]<br>
[[File:ZhangLab_2 2012-08-02 16hr 05min_bead-HAPMAP-WGBS-400-600bp_PQ.jpg| 400px]]<br>
Line 74: Line 223:
|-
|-
| #8||11.29||790.44||5.31
| #8||11.29||790.44||5.31
|}
|}<br>
 
== PAGE size-selection ==
[[File:ZhangLab_2 2012-08-02 18hr 23min-PAGE-SS.jpg| 350px]]
* 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 dsDNA Qubit HS  -> conc. 1.47ng/ul or 4.83nM
[[File:ZhangLab_2 2012-08-28 14hr 52min_PQ_NP_WGBS_HAPMAP_Aug01.png| 200px]]
- Concentration by PAGE quantification is 4.42ng/ul
* '''Library ID: NP_WGBS_HAPMAP1-8_July27''', sequenced on GAII '''HL128''' run. The eight libraries were spiked 1/10 in lane 1.

Latest revision as of 23:32, 29 August 2012

[Link to calendar]

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

  • Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-27
  • I have sheared gDNA from the previous batch left ~22ul (conc. ~40ng/ul based on conc. from the HAPMAP plate). I added 25ul H2O to each tube -> total volume 47ul, and will prepare end repair reaction mix in 23ul
  • Note: Since I start with sheared 880ng, I reduce the amount of enzymes in each step, but keep total volume the same (except ligation 50ul -> 40ul total)

End repair[edit]

Total reaction 70.00 ul
DNA 47.00 ul
End repair reaction mix 23.00 ul


KAPA End repair mix 1x rxn 8x rxn mix
10X End repai buffer 7.00 56.00
KAPA End repair enzyme 3.00 24.00
H2O 13.00 104.00
Total 23.00 184.00
  • Aliquot to column #1: 23ul

A-tailing[edit]

A-tailing reaction mix: Total 30ul/reaction

A-tailing reaction mix 1x rxn 8x rxn mix
KAPA 10X A-tailing buffer 3.00 24.00
KAPA A-tailing enzyme 2.00 16.00
H2O 25.00 200.00
Total 30.00 240.00
  • Aliquot to column #2: 30ul

Adapter ligation[edit]

Adapter ligation mix: Total 40ul/reaction -> 10ul is methylation adapters

Ligation reaction mix 1x rxn 8x rxn mix
KAPA 5X Ligation buffer 8.00 64.00
KAPA DNA ligase 3.00 24.00
H2O 19.00 152.00
Total 30.00 240.00
  • Aliquot to column #3: 30ul

Methylation adapter preparation[edit]

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)

Prepare adapter 2ulx 8rxn --> 16.00ul
H2O 8ulx8rxn --> 64.00ul
Total 80.00ul

Reagent preparation[edit]

Reagents 1rxn Min volume in 30mL reservior
AMPure beads 120ul ~1.1mL
20% PEG/2.5M NaCl 90+50 = 140ul ~1.4mL
80% EtOH 140x6times ~10mL
H2O for elution 22ul ~1mL

epMotion run[edit]

Report file of epMotion run: Media:MasterReport_LibPrep_HAPMAPAug1.txt

File:LibPreP HAPMAP Aug1.png
  • Note: I used all adapter ligated DNA for bisulfite conversion, so I don't have the image showing DNA before and after end repair, A-tailing and adapter ligation. However I did most of the step the same as I did on July 27.

Image of DNA before end repair and after adapter ligation performed on 2012.07.27

File:ZhangLab 2 2012-07-26 12hr 55min PAGE-Quan-shearedDNA.png File:ZhangLab 2 2012-07-27 20hr 33min KAPA-adapter.jpg

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

ssDNA Qubit quantification[edit]

  • Used 3ul of bis-cvt DNA for the assay (dilution 200/3)
Sample Concentration in the Qubit Unit uL used Dilution Final conc. (ng/ul) Yields in 40ul
#1 17.2 ng/mL 3 66.67 1.15 45.87
#2 4.4 ng/mL 3 66.67 0.29 11.73**
#3 12.3 ng/mL 3 66.67 0.82 32.80
#4 65.6 ng/mL 3 66.67 4.37 174.93
#5 32.9 ng/mL 3 66.67 2.19 87.73
#6 7.16 ng/mL 3 66.67 0.48 19.09**
#7 48.3 ng/mL 3 66.67 3.22 128.80
#8 30.6 ng/mL 3 66.67 2.04 81.60
  • The yield after bisulfite conversion is relatively low when compare to initial amount of DNA. However, the DNA amount would be reduced in each step. From the image, bead purification did'n recover the fragments < ~200bp, so I don't expect to get very high after bisulfite conversion. The total yields I got after bisulfite conversion are varied as well, and the yield of some sample didn't correlated with the intensity before bisulfite conversion. However, I could not rely on to this image because it is the image of previous experiment. Next time I should collect sample for verification.
  • **= samples have very low yields --> If compare to the intensity and total yield after amplification with barcode primers, these concentrations by ssDNA Qubit quantification seemed to agree to the results below.

Amplification of bisulfite sequencing llibraries (Test)[edit]

  • Test if the adapter ligation reaction worked before amplify the libraries with barcoded primers
Components 1rxn 10 rxn mix
DNA template 2.50 0.00
10uM PCR_F 0.50 5.00
10uM PCR_R.Ind1 0.50 5.00
2x iQ MM 12.50 125.00
H2O 9.00 90.00
Total 25.00 250.00
  • Aliquot 22.5ul, add 2.5ul of bis-cvt DNA template (2.5 out of 40ul)
Program
98C 2min (98C 15sec ->	64C 20sec -> 72C 30sec) x15cycles -> 72C 3min -> 15CHold

qPCR results

File:20120801 qPCR adapterligated.png


  • Loaded 2.5 ul of bis-cvt DNA (#1 & #4) and 2.5ul of amplified amplicons

File:ZhangLab 2 2012-08-02 11hr 31min-HAPMAP-bis-test.jpg

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

PAGE quantification of 400-600bp fragments[edit]

  • Dr. Zhang suggested to cut the band around 400-600bp because 250bp is too short 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 dsDNA Qubit HS -> conc. 1.47ng/ul or 4.83nM
File:ZhangLab 2 2012-08-28 14hr 52min PQ NP WGBS HAPMAP Aug01.png

- Concentration by PAGE quantification is 4.42ng/ul
  • Library ID: NP_WGBS_HAPMAP1-8_July27, sequenced on GAII HL128 run. The eight libraries were spiked 1/10 in lane 1.