Noi/NOTES/2014-6-20: Difference between revisions

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= Preparation of low-input (1ng) RRBS sequencing libraries of cell-free DNA (plasma samples) from 10 pancreastic cancer patients and 10 normal control  =
= Preparation of low-input (1ng) RRBS sequencing libraries of cell-free DNA (plasma samples) from 10 pancreastic cancer patients and 10 normal control  =
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]
[[noi:DMR220k_LabNotes| '''Calendar''']]
==== Sample info ====
==== Sample info ====
* [[Noi/NOTES/2014-6-9#Part_1:_DNA_extraction_from_plasma_samples| Plasma from pancreatic cancer patients (PC-P)]]
* [[Noi/NOTES/2014-6-9#Part_1:_DNA_extraction_from_plasma_samples| Plasma from pancreatic cancer patients (PC-P)]]
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== 1) MspI digestion ==
== 1) MspI digestion ==
* Note that I had condition of MspI digestion of this experiment different from the low-input experiment (5ng) on 06-03-2014 since I used 2ul of MspI in 18ul reaction. I need to increase volume of of Klenow exo- reaction and ligation to 25 and 30ul, respectively to reduce concentration of glycerol in the reaction as there is no purification steps prior to bisulfite conversion.
* Note that I had condition of MspI digestion of this experiment different from the low-input experiment (5ng) on 06-03-2014 since I used 2ul of MspI in 18ul reaction. I need to increase volume of of Klenow exo- reaction and ligation to 25 and 30ul, respectively to reduce concentration of glycerol in the reaction as there is no purification steps prior to bisulfite conversion.
* For the future, I do not need to use 2ul of MspI as 1ul showed similar activity to 2ul based on agarose gel analysis. I can use the same condition as experiment on 06-03-2014
* In the future, I do not need to use 2ul of MspI as 1ul showed similar activity to 2ul based on agarose gel analysis. I can use the same condition as experiment on 06-03-2014
* I spike in 1% of unmeth lambda DNA. This mean that I included 10pg of lambda DNA for 1ng input DNA.
* I spike in 1% of unmeth lambda DNA. This mean that I included 10pg of lambda DNA for 1ng input DNA.
==== <u>MspI digestion set up</u> ====
==== <u>MspI digestion set up</u> ====
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|}
|}
==== MspI Reaction Mix ====
==== MspI Reaction Mix ====
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn'''
| align="center" style="width:80px;background:#f0f0f0;"|'''26x rxn'''
|-
| MspI||2.00||52.00
|-
| 10x Tango Buffer||2.00||52.00
|-
| 12pg/ul unmeth lambda DNA||0.83||21.67
|-
| H2O||3.17||82.33
|-
| Total||8.00||
|}
:- Add H2O to each tube
:- Add 1ng of DNA to each tube
:- Add 8ul of MspI reaction Mix to each tube and pipette few times
:- Spin down the tubes
:- Mix by gentle pulse-vortexing on PCR rack 10x
:- Spin down the tubes
:- 37C, 3h --> 65C, 20min --> hold at 4C (set heat lid)<br>
<br>
== 2) End-repair/dA-tailing ==
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
* I increased reaction volume to 25ul as reason above
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn'''
| align="center" style="width:80px;background:#f0f0f0;"|'''28x rxn'''
|-
| MspI-digested DNA||18.00||0.00
|-
| 10X Tango Buffer||0.50||14.00
|-
| Klenow, exo-||1.00||28.00
|-
| dA:dC:dG||1.00||28.00
|-
| H2O||4.50||126.00
|-
| Total||25.00||196.00
|}
:- Aliquot 24.50 of Klwnow, exo- and dA:dC:dG reaction mix to 8-tube strip
:- Add 7ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of MspI digested DNA. Pipette few times
:- Spin down the tube
:- Mix by gentle pulse-vortexing on PCR rack 10x
:- Spin down the tube
:- 30C, 20min --> 37C, 20min --> hold at 4C  (no heat lid)
:- 75C, 10min --> hold at 4C (heat lid on)
* No AMPure bead purification<br>
<br>
== 3) Methylated adaptor ligation ==
* Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 30ul
==== <u>Ligation reaction set up</u> ====
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Dilute 1:20 TruSeq adaptor by adding 2ul of TruSeq adaptor to 40ul H2O<br>
====<u> Ligation reaction Mix</u> ====
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="width:70px;background:#f0f0f0;"|'''1x rxn'''
| align="center" style="width:70px;background:#f0f0f0;"|'''26x rxn'''
|-
| dA-tailed DNA||25.00||0.00
|-
| 10X Tango buffer||0.50||13.00
|-
| HC T4 DNA ligase (30units/ul)||1.00||26.00
|-
| 10mM ATP ||1.50||39.00
|-
| H2O||1.00||26.00
|-
| Total||29.00||
|}
:- Aliquot 13ul of ligation reaction mix to 8-tube strip
<u>PC-P samples</u>
:{| {{table}} class = wikitable
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Index'''
|-
| PC_P_1||1
|-
| PC_P_2||2
|-
| PC_P_3||4
|-
| PC_P_4||5
|-
| PC_P_5||6
|-
| PC_P_6||7
|-
| PC_P_7||8
|-
| PC_P_8||10
|-
| PC_P_9||11
|-
| PC_P_10||12
|-
| NTC_1||11
|-
| NTC_2||12
|}
<u>NC-P samples</u>
:{| {{table}} class = wikitable
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Index'''
|-
| NC-1||13
|-
| NC-2||14
|-
| NC-3||15
|-
| NC-5 ||16
|-
| NC-6 ||27
|-
| NC-7 ||18
|-
| NC-8 ||19
|-
| NC-9 ||20
|-
| NC-10 ||21
|-
| NC-11 ||22
|-
| NTC_1||21
|-
| NTC_2||22
|}
:- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
:- Add 4ul of ligation reaction mix
:- Spin down the tube
:- Mix by gentle pulse-vortexing on PCR rack 10x
:- Spin down the tube
:- 16C, 30h (no heat lid) --> I had no time to come back to continue to next step. I incubate longer.
:- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
:- Continue to bisulfite conversion
== 4) Bisulfite conversion ==
* Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer.
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
:- Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
:- Mix well and spin down the tube
:- Put on thermomixer at 37C for 15min, 1000rpm
:- Solution should be clear
:- Spin down the tube
:- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA  --> total volume 150ul
:- Mix by pipetting 10X with multi-channel pipette
:- Spin down the tube
:- Incubate following below program
::- 98°C for 10 minutes (DNA denaturation)
::- 64°C for 2.5 hours (Bisulfite conversion)
::- 4°C storage for up to 20 hours or continue to desulfonation
:- Add 601ul Binding Buffer to each column (with 10ng of tRNA in each 601ul Binding Buffer
:- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
:- Mix by pipetting 5x
:- Spin down column at 14,000 rpm for 30s
:- <u>Discard spnt</u>
:- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
:- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u>
:- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
:- Spin down column at 14,000 rpm for 30s  <u>(do not discard spnt)</u>
:- Add 200ul Wash Buffer
:- Spin down column at 14,000 rpm for 30s
:- <u>Discard spnt</u>
:- Add 200ul Wash Buffer
:- Spin down column at 14,000 rpm for 3min <u>(do not discard spnt)</u>
:- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
:- Add 30ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
:- Wait for 2min
:- Spin down column at 12,000 rpm for 1min
:- Continue to amplification (During elution step, PCR mix should be prepared)
== Amplification ==
=== 1st round PCR (fix 12 cycles) ===
{| {{table}} border = 1
| align="center" style="width:140px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''25 rxn mix'''
|-
| Bis-cvt DNA||||||||||30.00||0.00
|-
| 10X Reaction buffer||10||X||1||X||5.00||125.00
|-
| dNTP mix||10||mM||0.25||mM||1.25||31.25
|-
| TruS_F/R||10||uM||0.3||uM||1.50||37.50
|-
| 50X SYBG||50||X||0.8||X||0.80||20.00
|-
| PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00||25.00
|-
| H2O||||||||||10.45||261.25
|-
| Total ||||||||||50.00||
|}
:- Aliquot 20 + 30ul BIS-DNA template
:- Mix well
:: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 cycles --> 72C for 2min''
:- Purify with 1X AMPure beads (incubate DNA with beads 15min, the rest steps are the same as previous experiment)
:- Elute with 40ul EB Buffer
:- Continue to 2nd round PCR. No PAGE verification.
=== 2nd round PCR  ===
==== OPTIMIZE PCR cycle number ====
* I optimize PCR cycle number between 10-16 cycles by doing PCR in small volume (~10ul) and stopped reaction at 10X, 12X, 14X, and 16X cycles.
* Then analyze in 12% TBE gel
* Select the optimum cycle number to amplify the rest of 1st round PCR
<u>'''PC-P smaple'''</u>
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''13 rxn mix'''
|-
| Purified 1st round DNA||||||||||12.00||0.00
|-
| 5X Phusion HF buffer||5||X||1||X||8.00||104.00
|-
| dNTP mix||10||mM||0.25||mM||1.00||13.00
|-
| TruS_F/R||10||uM||0.3||uM||1.20||15.60
|-
| 50X SYBR||50||X||0.4||X||0.32||4.16
|-
| Phusion HF||2||unit/ul||||||0.80||10.40
|-
| H2O||||||||||16.68||216.84
|-
| Total||||||||||40.00||
|}
* Note: From the condition above, I used Phusion HF 1ul  (2units) in 50ul reaction (normalized to 50ul reaction), which is too high. In the following steps, I reduced it to 0.5ul (1unit) in 50ul reaction.
:- Aliquot 28 + 12ul DNA template
:- Mix well. Split 4X of 8ul
::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X [10X, 12X, 14X, 16X] cycles --> 72C for 2min''<br>
:- Added 10ul 2X loading dye to each strip tube & mix well
:- Load 10ul PCR/dye mix in 12% gel and run at 250 volts for 40min
[[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TT-10X.jpg| 500px]]  [[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TEST-12X.jpg| 480px]]
* Note: I did a wrong labeling for 12 cycles of PC-P samples. They should be P in stead of N
[[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TEST-14X.jpg| 495px]]  [[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TEST-16X.jpg| 490px]]
 
* 12-16 cycles are over-amplified for PC-P samples. Even 10X cycles is still too high intensity compare to some examples of the reference.
*  Did the same way for NC-P samples, but only monitor at 8X and 9X. However, I looked at qPCR curve too late, so I could only stopped at 9X and 10X cycles.
[[File:ZhangLab_2 2014-06-23 16hr 09min_NC_1ngRRBS_2nd_TEST_9X.jpg| 500px]]  [[File:ZhangLab_2 2014-06-23 16hr 09min_NC_1ngRRBS_2nd_TEST_10X.jpg| 500px]]
* N10 & N11 did not work!!
* These steps may spend a lot of time and gels. However, in the future, I do not need to do this optimization as I would know roughly number of cycle to stop at.
* The reason that I need to look at the gel image first is that the adaptor dimers level from ligation reaction is very hight  (no purification before bisulfite conversion). Therefore, the qPCR curve of NTC and the samples with DNA are not different.
* From this optimization, I  know that I should stop  before qPCR curve reaches the plateau as the gel intensity is consistent with the qPCR curve. Letting PCR go after it reaches the plateau causing over-amplification and a smear a long the lane instead of a smear at the range between ~160-360bp
* I would do 8 cycles for the rest of 1st round PCR for both PC-P and NC-P samples but do 9 cycles for sample PC-P-10 as it had very low intensity.
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''18.5 rxn mix'''
|-
| Purified 1st round DNA||||||||||25.00||0.00
|-
| 5X Phusion HF buffer||5||X||1||X||20.00||370.00
|-
| dNTP mix||10||mM||0.25||mM||2.50||46.25
|-
| TruS_F/R||10||uM||0.3||uM||3.00||55.50
|-
| 50X SYBR||50||X||0.4||X||0.80||14.80
|-
| Phusion HF||2||unit/ul||||||1.00||18.50
|-
| H2O||||||||||47.70||882.45
|}
:- Aliquot 75ul, add 25ul 1st round PCR
:- Mix well and split 48ul, 2X.
::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X [8X, 9X for PC-P-10] cycles --> 72C for 2min''<br>
[[File:ZhangLab_2 2014-06-23 20hr 02min_1ngRRBS_PC-P_PQ-3ulof55.jpg| 500px]]  [[File:ZhangLab_2 2014-06-23 20hr 02min_1ngRRBS_NC-P_PQ-3ulof55.jpg| 450px]]
==== PAGE quantification results ====
<u>PC-P samples</u>
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)'''
|-
| PC-P-1||1||7.12||356.17
|-
| PC-P-2||2||8.09||404.41
|-
| PC-P-3||4||10.82||540.90
|-
| PC-P-4||5||11.25||562.37
|-
| PC-P-5||6||7.49||374.73
|-
| PC-P-6||7||8.61||430.26
|-
| PC-P-7||8||10.23||511.64
|-
| PC-P-8||10||7.87||393.59
|-
| PC-P-9||11||3.25||162.28
|-
| PC-P-10||12||1.94||96.97
|}
<u>NC-P samples</u>
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)'''
|-
| NC-P-1||13||4.82||241.12
|-
| NC-P-2||14||7.60||380.06
|-
| NC-P-3||15||9.37||468.59
|-
| NC-P-5||16||3.86||192.81
|-
| NC-P-6||27||6.31||315.29
|-
| NC-P-7||18||2.62||131.19
|-
| NC-P-8||19||3.76||187.99
|-
| NC-P-9||20||4.61||230.44
|}
==== Library pooling ====
* To avoid index duplication, I need to split PC-P and NC-P libraries into 2 pools for each
:- <span style="color:crimson">(10M each) NP-RRBS-CTT-5ng, Jun3: <u>Index</u> 9, 10, 11, 13</span>
:- <span style="color:crimson">(20M each) NP-RRBS-CTT-100ng, Jun13: <u>Index</u> 18, 19, 20, 21</span>
<br>
:- <span style="color:green">(20M each) NP-RRBS-PCT-100ng-Jun16: <u>Index</u> 1, 2, 4, 5, 6, 7, 8, 12, 14, 15</span> --> ''(This pool should take the whole lane. Will not be mixed with other pool)''
<br>
:- <span style="color:crimson">(4.5M each) NP-RRBS-PC-P-1ng-p1-Jun20: <u>Index</u> 1, 2, 4, 5, 6, 7, 8, 12</span>
:- <span style="color:blue">(4.5M each) NP-RRBS-PC-P-1ng-p2-Jun20: <u>Index</u> 10, 11</span> --> MiSeq
<br>
:- <span style="color:crimson">(4.5M each) NP-RRBS-NC-P-1ng-p1-Jun20: <u>Index</u> 14, 15, 16, 27</span>
:- <span style="color:blue">(4.5M each) NP-RRBS-NC-P-1ng-p2-Jun20: <u>Index</u> 13, 18, 19, 20</span> --> MiSeq
NP-RRBS-PC-P-1ng-p1-Jun20
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 120ng (ul)'''
|-
| PC-P-1||1||7.12||16.85
|-
| PC-P-2||2||8.09||14.84
|-
| PC-P-3||4||10.82||11.09
|-
| PC-P-4||5||11.25||10.67
|-
| PC-P-5||6||7.49||16.01
|-
| PC-P-6||7||8.61||13.94
|-
| PC-P-7||8||10.23||11.73
|-
| PC-P-10*||12||1.94||50.00
|}
NP-RRBS-PC-P-1ng-p2-Jun20
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 90ng (ul)'''
|-
| PC-P-8||10||7.87||11.43
|-
| PC-P-9||11||3.25||27.73
|}
NP-RRBS-NC-P-1ng-p1-Jun20
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)'''
|-
| NC-P-2||14||7.60||13.16
|-
| NC-P-3||15||9.37||10.67
|-
| NC-P-5||16||3.86||25.93
|-
| NC-P-6||27||6.31||15.86
|}
NP-RRBS-NC-P-1ng-p2-Jun20
{| {{table}} border = 1
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Index'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)'''
|-
| NC-P-1||13||4.82||20.74
|-
| NC-P-7||18||2.62||38.11
|-
| NC-P-8||19||3.76||26.60
|-
| NC-P-9||20||4.61||21.70
|}
==== PAGE size selection ====
[[File:ZhangLab_2 2014-06-24 11hr 14min_PAGE-SS_PC-P_p1-p2.jpg| 400px]]
PC-P pool1: Resuspend with 10ul H2O x 3tubes --> volume 30ul, 4ul for PAGE verification
PC-P pool2: Resuspend with 15ul H2O x 1tubes --> volume 15ul, 4ul for PAGE verification
[[File:ZhangLab_2 2014-06-24 11hr 14min_PAGE-SS_NC-P_pool1.jpg| 300px]]
NC-P pool1: Resuspend with 12ul H2O x 2tubes --> volume 24ul, 4ul for PAGE verification
[[File:ZhangLab_2 2014-06-24 11hr 14min_PAGE-SS_NC-P_pool2.jpg| 400px]]
NC-P pool2: Resuspend with 10ul H2O x 3tubes --> volume 30ul, 4ul for PAGE verification
==== PAGE verification ====
[[File:ZhangLab_2 2014-06-24 17hr 06min_RRBS_PAGE-verify.jpg| 500px]]
=== 2014-06-25 ===
==== Preparing sequencing library pool for IGM ====
* We will do HiSeq Rapid run, PE 100+7bp
* Spike in with 5% PhiX in each lane
:1). NP-RRBS-PCT_100ng-Jun16 --> rename to '''NP_RRBS_PCT_20140625'''
:- This library requires 200M reads, so it'll take the whole lane. I assigned it in lane 1.
:- I aliquoted 20ul for sequencing. Conc. by qPCR = 19.29nM
:2). '''NP_RRBS_CTT_P_20140625'''
:{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Seq. Library'''
| align="center" style="width:60px;background:#f0f0f0;"|'''# of ind. Library'''
| align="center" style="width:80px;background:#f0f0f0;"|'''# of read for each (M)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Total # of reads (M)'''
| align="center" style="width:60px;background:#f0f0f0;"|'''Loading % '''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. by qPCR (nM)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Ratio of loading % to CTT-5ng'''
| align="center" style="width:80px;background:#f0f0f0;"|'''# of mole to pool (nmole)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume to pool (ul)'''
|-
| NP-RRBS-CTT_5ng-Jun3||4||10||40||23.0||15.28||1||50.88||3.33
|-
| NP-RRBS-CTT_100ng-Jun13||4||20||80||46.0||7.00||2||101.76||14.54
|-
| NP-RRBS-PC_P_1ng_p1-Jun20||8||4.5||36||20.7||28.25||0.9||45.79||1.62
|-
| NP-RRBS-NC_P_1ng_p1-Jun20||4||4.5||18||10.3||18.95||0.45||22.90||1.21
|-
| ||||||||||||Total||221.33||20.70
|}
:- Final conc. of NP_RRBS_CTT_P_20140625 = 221.33nmole/20.70ul = 10.69nM
:- It should be loaded in lane 2

Latest revision as of 20:46, 28 July 2014

Preparation of low-input (1ng) RRBS sequencing libraries of cell-free DNA (plasma samples) from 10 pancreastic cancer patients and 10 normal control[edit]

Calendar

Sample info[edit]

1) MspI digestion[edit]

  • Note that I had condition of MspI digestion of this experiment different from the low-input experiment (5ng) on 06-03-2014 since I used 2ul of MspI in 18ul reaction. I need to increase volume of of Klenow exo- reaction and ligation to 25 and 30ul, respectively to reduce concentration of glycerol in the reaction as there is no purification steps prior to bisulfite conversion.
  • In the future, I do not need to use 2ul of MspI as 1ul showed similar activity to 2ul based on agarose gel analysis. I can use the same condition as experiment on 06-03-2014
  • I spike in 1% of unmeth lambda DNA. This mean that I included 10pg of lambda DNA for 1ng input DNA.

MspI digestion set up[edit]

1.1) PC-P samples

# Sample Original conc. (ng/ul) Volume for 1ng (ul) H2O to 10ul MspI Mix Total Code
1 PC-P_1 0.473 2.11 7.89 8.00 18.00 P_1
2 PC-P_2 0.852 1.17 8.83 8.00 18.00 P_2
3 PC-P_3 1.360 0.74 9.26 8.00 18.00 P_3
4 PC-P_4 0.340 2.94 7.06 8.00 18.00 P_4
5 PC-P_5 0.813 1.23 8.77 8.00 18.00 P_5
6 PC-P_6 0.767 1.30 8.70 8.00 18.00 P_6
7 PC-P_7 0.644 1.55 8.45 8.00 18.00 P_7
8 PC-P_8 0.524 1.91 8.09 8.00 18.00 P_8
9 PC-P_9 0.238 4.20 5.80 8.00 18.00 P_9
10 PC-P_10 0.370 2.70 7.30 8.00 18.00 P_10
11 NTC_1 0.000 0.00 10.00 8.00 18.00 P_11
12 NTC_2 0.000 0.00 10.00 8.00 18.00 P_12

1.2) NC-P samples

# Sample Original conc. (ng/ul) Volume for 1ng (ul) H2O to 10ul MspI Mix Total Code
1 NC-1 0.293 3.41 6.59 8.00 18.00 N_1
2 NC-2 0.325 3.08 6.92 8.00 18.00 N_2
3 NC-3 0.538 1.86 8.14 8.00 18.00 N_3
4 NC-5 0.156 6.41 3.59 8.00 18.00 N_5
5 NC-6 0.148 6.76 3.24 8.00 18.00 N_6
6 NC-7 0.245 4.08 5.92 8.00 18.00 N_7
7 NC-8 0.141 7.09 2.91 8.00 18.00 N_8
8 NC-9 0.171 5.85 4.15 8.00 18.00 N_9
9 NC-10 0.134 7.46 2.54 8.00 18.00 N_10
10 NC-11 0.152 6.58 3.42 8.00 18.00 N_11
11 NTC_1 0.000 0.00 10.00 8.00 18.00 N_12
12 NTC_2 0.000 0.00 10.00 8.00 18.00 N_13

MspI Reaction Mix[edit]

Components 1x rxn 26x rxn
MspI 2.00 52.00
10x Tango Buffer 2.00 52.00
12pg/ul unmeth lambda DNA 0.83 21.67
H2O 3.17 82.33
Total 8.00
- Add H2O to each tube
- Add 1ng of DNA to each tube
- Add 8ul of MspI reaction Mix to each tube and pipette few times
- Spin down the tubes
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tubes
- 37C, 3h --> 65C, 20min --> hold at 4C (set heat lid)


2) End-repair/dA-tailing[edit]

Prep

  • I increased reaction volume to 25ul as reason above
Components 1x rxn 28x rxn
MspI-digested DNA 18.00 0.00
10X Tango Buffer 0.50 14.00
Klenow, exo- 1.00 28.00
dA:dC:dG 1.00 28.00
H2O 4.50 126.00
Total 25.00 196.00
- Aliquot 24.50 of Klwnow, exo- and dA:dC:dG reaction mix to 8-tube strip
- Add 7ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of MspI digested DNA. Pipette few times
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- 75C, 10min --> hold at 4C (heat lid on)
  • No AMPure bead purification


3) Methylated adaptor ligation[edit]

  • Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 30ul

Ligation reaction set up[edit]

Prep
- Dilute 1:20 TruSeq adaptor by adding 2ul of TruSeq adaptor to 40ul H2O

Ligation reaction Mix[edit]

Components 1x rxn 26x rxn
dA-tailed DNA 25.00 0.00
10X Tango buffer 0.50 13.00
HC T4 DNA ligase (30units/ul) 1.00 26.00
10mM ATP 1.50 39.00
H2O 1.00 26.00
Total 29.00
- Aliquot 13ul of ligation reaction mix to 8-tube strip

PC-P samples

Sample Index
PC_P_1 1
PC_P_2 2
PC_P_3 4
PC_P_4 5
PC_P_5 6
PC_P_6 7
PC_P_7 8
PC_P_8 10
PC_P_9 11
PC_P_10 12
NTC_1 11
NTC_2 12

NC-P samples

Sample Index
NC-1 13
NC-2 14
NC-3 15
NC-5 16
NC-6 27
NC-7 18
NC-8 19
NC-9 20
NC-10 21
NC-11 22
NTC_1 21
NTC_2 22
- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- Add 4ul of ligation reaction mix
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 30h (no heat lid) --> I had no time to come back to continue to next step. I incubate longer.
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- Continue to bisulfite conversion

4) Bisulfite conversion[edit]

  • Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer.

Prep

- Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1000rpm
- Solution should be clear
- Spin down the tube
- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul
- Mix by pipetting 10X with multi-channel pipette
- Spin down the tube
- Incubate following below program
- 98°C for 10 minutes (DNA denaturation)
- 64°C for 2.5 hours (Bisulfite conversion)
- 4°C storage for up to 20 hours or continue to desulfonation
- Add 601ul Binding Buffer to each column (with 10ng of tRNA in each 601ul Binding Buffer
- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
- Mix by pipetting 5x
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 3min (do not discard spnt)
- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
- Add 30ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- Wait for 2min
- Spin down column at 12,000 rpm for 1min
- Continue to amplification (During elution step, PCR mix should be prepared)

Amplification[edit]

1st round PCR (fix 12 cycles)[edit]

Components Conc unit Final conc./amount unit Volume (ul) 25 rxn mix
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 5.00 125.00
dNTP mix 10 mM 0.25 mM 1.25 31.25
TruS_F/R 10 uM 0.3 uM 1.50 37.50
50X SYBG 50 X 0.8 X 0.80 20.00
PfuTurbo Cx 2.5 Unit/ul 1 unit 1.00 25.00
H2O 10.45 261.25
Total 50.00
- Aliquot 20 + 30ul BIS-DNA template
- Mix well
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 cycles --> 72C for 2min
- Purify with 1X AMPure beads (incubate DNA with beads 15min, the rest steps are the same as previous experiment)
- Elute with 40ul EB Buffer
- Continue to 2nd round PCR. No PAGE verification.

2nd round PCR[edit]

OPTIMIZE PCR cycle number[edit]

  • I optimize PCR cycle number between 10-16 cycles by doing PCR in small volume (~10ul) and stopped reaction at 10X, 12X, 14X, and 16X cycles.
  • Then analyze in 12% TBE gel
  • Select the optimum cycle number to amplify the rest of 1st round PCR

PC-P smaple

Components Conc unit Final conc. unit Volume (ul) 13 rxn mix
Purified 1st round DNA 12.00 0.00
5X Phusion HF buffer 5 X 1 X 8.00 104.00
dNTP mix 10 mM 0.25 mM 1.00 13.00
TruS_F/R 10 uM 0.3 uM 1.20 15.60
50X SYBR 50 X 0.4 X 0.32 4.16
Phusion HF 2 unit/ul 0.80 10.40
H2O 16.68 216.84
Total 40.00
  • Note: From the condition above, I used Phusion HF 1ul (2units) in 50ul reaction (normalized to 50ul reaction), which is too high. In the following steps, I reduced it to 0.5ul (1unit) in 50ul reaction.
- Aliquot 28 + 12ul DNA template
- Mix well. Split 4X of 8ul
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [10X, 12X, 14X, 16X] cycles --> 72C for 2min
- Added 10ul 2X loading dye to each strip tube & mix well
- Load 10ul PCR/dye mix in 12% gel and run at 250 volts for 40min
File:ZhangLab 2 2014-06-23 11hr 45min 1ngRRBS 2nd TT-10X.jpg  File:ZhangLab 2 2014-06-23 11hr 45min 1ngRRBS 2nd TEST-12X.jpg
* Note: I did a wrong labeling for 12 cycles of PC-P samples. They should be P in stead of N
File:ZhangLab 2 2014-06-23 11hr 45min 1ngRRBS 2nd TEST-14X.jpg  File:ZhangLab 2 2014-06-23 11hr 45min 1ngRRBS 2nd TEST-16X.jpg

 
* 12-16 cycles are over-amplified for PC-P samples. Even 10X cycles is still too high intensity compare to some examples of the reference. 
  • Did the same way for NC-P samples, but only monitor at 8X and 9X. However, I looked at qPCR curve too late, so I could only stopped at 9X and 10X cycles.
File:ZhangLab 2 2014-06-23 16hr 09min NC 1ngRRBS 2nd TEST 9X.jpg  File:ZhangLab 2 2014-06-23 16hr 09min NC 1ngRRBS 2nd TEST 10X.jpg

* N10 & N11 did not work!!
  • These steps may spend a lot of time and gels. However, in the future, I do not need to do this optimization as I would know roughly number of cycle to stop at.
  • The reason that I need to look at the gel image first is that the adaptor dimers level from ligation reaction is very hight (no purification before bisulfite conversion). Therefore, the qPCR curve of NTC and the samples with DNA are not different.
  • From this optimization, I know that I should stop before qPCR curve reaches the plateau as the gel intensity is consistent with the qPCR curve. Letting PCR go after it reaches the plateau causing over-amplification and a smear a long the lane instead of a smear at the range between ~160-360bp
  • I would do 8 cycles for the rest of 1st round PCR for both PC-P and NC-P samples but do 9 cycles for sample PC-P-10 as it had very low intensity.
Components Conc unit Final conc. unit Volume (ul) 18.5 rxn mix
Purified 1st round DNA 25.00 0.00
5X Phusion HF buffer 5 X 1 X 20.00 370.00
dNTP mix 10 mM 0.25 mM 2.50 46.25
TruS_F/R 10 uM 0.3 uM 3.00 55.50
50X SYBR 50 X 0.4 X 0.80 14.80
Phusion HF 2 unit/ul 1.00 18.50
H2O 47.70 882.45
- Aliquot 75ul, add 25ul 1st round PCR
- Mix well and split 48ul, 2X.
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [8X, 9X for PC-P-10] cycles --> 72C for 2min
File:ZhangLab 2 2014-06-23 20hr 02min 1ngRRBS PC-P PQ-3ulof55.jpg  File:ZhangLab 2 2014-06-23 20hr 02min 1ngRRBS NC-P PQ-3ulof55.jpg

PAGE quantification results[edit]

PC-P samples

Sample Index Conc. (ng/ul) Amount in 50ul (ng)
PC-P-1 1 7.12 356.17
PC-P-2 2 8.09 404.41
PC-P-3 4 10.82 540.90
PC-P-4 5 11.25 562.37
PC-P-5 6 7.49 374.73
PC-P-6 7 8.61 430.26
PC-P-7 8 10.23 511.64
PC-P-8 10 7.87 393.59
PC-P-9 11 3.25 162.28
PC-P-10 12 1.94 96.97

NC-P samples

Sample Index Conc. (ng/ul) Amount in 50ul (ng)
NC-P-1 13 4.82 241.12
NC-P-2 14 7.60 380.06
NC-P-3 15 9.37 468.59
NC-P-5 16 3.86 192.81
NC-P-6 27 6.31 315.29
NC-P-7 18 2.62 131.19
NC-P-8 19 3.76 187.99
NC-P-9 20 4.61 230.44

Library pooling[edit]

  • To avoid index duplication, I need to split PC-P and NC-P libraries into 2 pools for each
- (10M each) NP-RRBS-CTT-5ng, Jun3: Index 9, 10, 11, 13
- (20M each) NP-RRBS-CTT-100ng, Jun13: Index 18, 19, 20, 21


- (20M each) NP-RRBS-PCT-100ng-Jun16: Index 1, 2, 4, 5, 6, 7, 8, 12, 14, 15 --> (This pool should take the whole lane. Will not be mixed with other pool)


- (4.5M each) NP-RRBS-PC-P-1ng-p1-Jun20: Index 1, 2, 4, 5, 6, 7, 8, 12
- (4.5M each) NP-RRBS-PC-P-1ng-p2-Jun20: Index 10, 11 --> MiSeq


- (4.5M each) NP-RRBS-NC-P-1ng-p1-Jun20: Index 14, 15, 16, 27
- (4.5M each) NP-RRBS-NC-P-1ng-p2-Jun20: Index 13, 18, 19, 20 --> MiSeq

NP-RRBS-PC-P-1ng-p1-Jun20

Sample Index Conc. (ng/ul) Volume for 120ng (ul)
PC-P-1 1 7.12 16.85
PC-P-2 2 8.09 14.84
PC-P-3 4 10.82 11.09
PC-P-4 5 11.25 10.67
PC-P-5 6 7.49 16.01
PC-P-6 7 8.61 13.94
PC-P-7 8 10.23 11.73
PC-P-10* 12 1.94 50.00

NP-RRBS-PC-P-1ng-p2-Jun20

Sample Index Conc. (ng/ul) Volume for 90ng (ul)
PC-P-8 10 7.87 11.43
PC-P-9 11 3.25 27.73

NP-RRBS-NC-P-1ng-p1-Jun20

Sample Index Conc. (ng/ul) Volume for 100ng (ul)
NC-P-2 14 7.60 13.16
NC-P-3 15 9.37 10.67
NC-P-5 16 3.86 25.93
NC-P-6 27 6.31 15.86

NP-RRBS-NC-P-1ng-p2-Jun20

Sample Index Conc. (ng/ul) Volume for 100ng (ul)
NC-P-1 13 4.82 20.74
NC-P-7 18 2.62 38.11
NC-P-8 19 3.76 26.60
NC-P-9 20 4.61 21.70

PAGE size selection[edit]

File:ZhangLab 2 2014-06-24 11hr 14min PAGE-SS PC-P p1-p2.jpg 
PC-P pool1: Resuspend with 10ul H2O x 3tubes --> volume 30ul, 4ul for PAGE verification
PC-P pool2: Resuspend with 15ul H2O x 1tubes --> volume 15ul, 4ul for PAGE verification
File:ZhangLab 2 2014-06-24 11hr 14min PAGE-SS NC-P pool1.jpg
NC-P pool1: Resuspend with 12ul H2O x 2tubes --> volume 24ul, 4ul for PAGE verification
File:ZhangLab 2 2014-06-24 11hr 14min PAGE-SS NC-P pool2.jpg
NC-P pool2: Resuspend with 10ul H2O x 3tubes --> volume 30ul, 4ul for PAGE verification

PAGE verification[edit]

File:ZhangLab 2 2014-06-24 17hr 06min RRBS PAGE-verify.jpg

2014-06-25[edit]

Preparing sequencing library pool for IGM[edit]

  • We will do HiSeq Rapid run, PE 100+7bp
  • Spike in with 5% PhiX in each lane
1). NP-RRBS-PCT_100ng-Jun16 --> rename to NP_RRBS_PCT_20140625
- This library requires 200M reads, so it'll take the whole lane. I assigned it in lane 1.
- I aliquoted 20ul for sequencing. Conc. by qPCR = 19.29nM
2). NP_RRBS_CTT_P_20140625
Seq. Library # of ind. Library # of read for each (M) Total # of reads (M) Loading % Conc. by qPCR (nM) Ratio of loading % to CTT-5ng # of mole to pool (nmole) Volume to pool (ul)
NP-RRBS-CTT_5ng-Jun3 4 10 40 23.0 15.28 1 50.88 3.33
NP-RRBS-CTT_100ng-Jun13 4 20 80 46.0 7.00 2 101.76 14.54
NP-RRBS-PC_P_1ng_p1-Jun20 8 4.5 36 20.7 28.25 0.9 45.79 1.62
NP-RRBS-NC_P_1ng_p1-Jun20 4 4.5 18 10.3 18.95 0.45 22.90 1.21
Total 221.33 20.70
- Final conc. of NP_RRBS_CTT_P_20140625 = 221.33nmole/20.70ul = 10.69nM
- It should be loaded in lane 2