Noi/NOTES/2014-6-2: Difference between revisions
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:- ''Incubate at 16C for 16h (no heat lid)'' | :- ''Incubate at 16C for 16h (no heat lid)'' | ||
:- ''Heat inactivate at 65C for 20min'' | :- ''Heat inactivate at 65C for 20min'' | ||
= Sensitivity test of MONOD V1N3 BSPP capture with amplification = | |||
* There are many things need to concern about BSPP capture of serum/plasma samples for this project. | |||
** DNA amount from plasma is very low in the range of 5-10ng from 1mL plasma. | |||
** Serum samples showed higher amount than plasma, but Dr. Zhang suggest that the high amount is from the lysis of white blood cell. | |||
** If we use serum samples, we need to increase the sensitivity of cancer DNA detection. | |||
** If we use plasma sample, we need to do some pre-amplification to amplify <10ng of cell-free DNA by ~10-fold for padlock capture. | |||
* The amount that Dr. Zhang meant is amount of extracted DNA not after bisulfite conversion. I have done some experiment to test the recovery rate of bisulfite conversion at different input amounts. | |||
==== Bisulfite conversion recovery rate #1 ==== | |||
* Note: The resulting BIS-DNAs were used for sensitivity test experiment | |||
{| {{table}} class = wikitable | |||
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Conc. in the Qubit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''uL used''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Dilution''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Sample conc. (ng/ul)''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Yield in 15ul (ng)''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Input DNA (ng)''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|''% Recovery'' | |||
|- | |||
| JK-1||15.2||ng/mL||1||200||3.03||45.45||bgcolor="Aqua"|100||bgcolor="Aqua"|45.45 | |||
|- | |||
| JK-2||19.1||ng/mL||1||200||3.82||57.30||bgcolor="Aqua"|100||bgcolor="Aqua"|57.30 | |||
|- | |||
| JK-3||13.3||ng/mL||1||200||2.66||39.90||bgcolor="Aqua"|100||bgcolor="Aqua"|39.90 | |||
|- | |||
| JK-4||41||ng/mL||1||200||8.21||123.15||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|61.58 | |||
|- | |||
| JK-5||36.6||ng/mL||1||200||7.31||109.65||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|54.83 | |||
|- | |||
| JK-6||39.4||ng/mL||1||200||7.88||118.20||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|59.10 | |||
|- | |||
| JK-7||133||ng/mL||1||200||26.6||399.00||bgcolor="royalBlue"|600||bgcolor="royalBlue"|66.50 | |||
|- | |||
| JK-8||131||ng/mL||1||200||26.2||393.00||bgcolor="royalBlue"|600||bgcolor="royalBlue"|65.50 | |||
|} | |||
==== Bisulfite conversion recovery rate #2 ==== | |||
{| {{table}} class = wikitable | |||
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Conc. in the Qubit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''uL used''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Dilution''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Sample conc. (ng/ul)''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Yield in 9ul (ng)''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Input DNA (ng)''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''% Recovery''' | |||
|- | |||
| JK-1||3.8||ng/mL||1||200||0.760||6.84||bgcolor="lightcyan"|25||bgcolor="lightcyan"|27.36 | |||
|- | |||
| JK-2||4.9||ng/mL||1||200||0.980||8.82||bgcolor="lightcyan"|25||bgcolor="lightcyan"|35.28 | |||
|- | |||
| JK-3||2.5||ng/mL||1||200||0.500||4.50||bgcolor="lightcyan"|25||bgcolor="lightcyan"|18.00 | |||
|- | |||
| JK-4||5.11||ng/mL||1||200||1.022||9.20||bgcolor="lightcyan"|25||bgcolor="lightcyan"|36.79 | |||
|- | |||
| JK-5||10.8||ng/mL||1||200||2.160||19.44||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|38.88 | |||
|- | |||
| JK-6||9.06||ng/mL||1||200||1.812||16.31||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|32.62 | |||
|- | |||
| JK-7||12.3||ng/mL||1||200||2.460||22.14||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|44.28 | |||
|- | |||
| JK-8||11.2||ng/mL||1||200||2.240||20.16||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|40.32 | |||
|- | |||
| JK-9||25||ng/mL||1||200||5.000||45.00||bgcolor="aqua"|100||bgcolor="aqua"|45.00 | |||
|- | |||
| JK-10||26.7||ng/mL||1||200||5.340||48.06||bgcolor="aqua"|100||bgcolor="aqua"|48.06 | |||
|- | |||
| JK-11||28.4||ng/mL||1||200||5.680||51.12||bgcolor="aqua"|100||bgcolor="aqua"|51.12 | |||
|- | |||
| JK-12||24.3||ng/mL||1||200||4.860||43.74||bgcolor="aqua"|100||bgcolor="aqua"|43.74 | |||
|- | |||
| JK-13||90.1||ng/mL||1||200||18.020||162.18||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|81.09 | |||
|- | |||
| JK-14||89||ng/mL||1||200||17.800||160.20||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|80.10 | |||
|- | |||
| JK-15||79.7||ng/mL||1||200||15.940||143.46||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|71.73 | |||
|- | |||
| JK-16||85.4||ng/mL||1||200||17.080||153.72||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|76.86 | |||
|} | |||
* Input 200ng had too high recovery rate compare to previous experiment. |
Revision as of 15:23, 3 June 2014
RRBS library preparation of colon tumor tissue samples from Biochain
- [Link to calendar]
- References
Sample list and details
- Sample type: genomic DNA from primary tumor tissue
- Colon tumor tissue, frozen (CTT-frozen): re-quantified concentration with Qubit dsDNA HS assay
- Colon tumor tissue, FFPE (CTT-FFPE)
- 2014-06-01, I added TE buffer to make conc. to 50ng/ul (total volume 40ul) based on original conc. and re-measured conc. with Qubit dsDNA HS assay again (1ul for the assay).
- Dr. Zhang suggested to do two replicates for each level
- I will spike in 0.5% of unmethylated lambda DNA to check bisulfite conversion rate.
Sample Original conc. (ng/ul) Estimate volume (ul) Dilute to 50ng/ul (total volume) Add TE buffer (ul) Qubit conc. (ng/ul) CTT-Frozen 580.00 3.45 40.00 36.55 52.3 CTT-FFPE 76.00 26.32 40.00 13.68 64.8
Library preparation
- Low-input RRBS (5ng)
- Standard RRBS (100ng)
Experimental procedures
- I firstly dilute CTT gDNA to 2ng/ul volume 50ul
Sample Conc. (ng/ul) Stock volume (ul) TE (ul) CTT-frozen 52.30 1.91 48.09 CTT-FFPE 64.80 1.54 48.46
- For 5ng input, I will add 25pg of unmethylated lambda DNA (add 2.08ul of stock 12pg/ul)
- For 100ng input, I will add 0.5ng of unmethylated lambda DNA (add 0.5ul of stock 1ng/ul)
1) DNA fragmentation with MspI
1.1) Low-input RRBS MspI digestion set up
CTT-5ng_1 CTT-5ng_2 CTT-F-5ng_1 CTT-F-5ng_2 5ng NTC
Sample Conc. (ng/ul) Volume for 5ng (ul) 10X Tango Buffer MspI (10U/ul) 12pg/ul unmeth-lambda DNA H2O (ul) Total (ul) 1.1 CTT-frozen 2.00 2.5 2.00 1.00 2.08 10.42 18.00 1.2 CTT-FFPE 2.00 2.5 2.00 1.00 2.08 10.42 18.00 1.3 NTC 0.00 2.5 2.00 1.00 2.08 10.42 18.00
- MspI reaction mix I
Components 1 rxn 5.5 rxn mix MspI (10U/ul) 1.00 5.50 10X Tango Buffer 2.00 11.00 12pg/ul unmeth-lambda DNA 2.08 11.44 H2O 10.42 57.31 Total 15.50
- - Aliquot 15.50ul to each tube
- - Add 2.5ul of diluted CTT DNA to each tube
- - Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down
1.2) STD RRBS MspI digestion set up
CCT-100ng_1 CTT-100ng_2 CTT-F-100ng_1 CTT-F-100ng_2 100ng NTC
Sample Conc. (ng/ul) Volume for 100ng (ul) 10X Tango Fuffer MspI (10U/ul) H2O to adjust volume (ul) 1ng/ul unmeth-lambda DNA H2O in MspI mix(ul) Total (ul) 1.1 CTT-frozen 52.30 1.91 2.00 1.00 5.09 0.50 7.50 18.00 1.2 CTT-FFPE 64.80 1.54 2.00 1.00 5.46 0.50 7.50 18.00 1.3 NTC 0.00 0.00 2.00 1.00 7.00 0.50 7.50 18.00
- MspI reaction mix II
Components 1 rxn 5.5 rxn mix MspI (10U/ul) 1.00 5.50 10X Tango Buffer 2.00 11.00 1ng/ul unmeth-lambda DNA 0.5 2.75 H2O 7.50 41.25 Total 11.00
- - Add H2O to adjust volume as table above
- - Aliquot 11ul to each tube
- - Add CTT DNA to each tube following the number in table above
- - Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down
- - Incubate at 37C for 3hr
- - Heat inactivate at 65C for 20min
3) End-repair/dA-tailing
Prep
- To prevent contamination to enzyme tube by multiple time pipetting, I aliquot 11ul each of Klenow fragment, exo- and dA:dC:dG mix into PCR tube.
- dA:dC:dG solution was prepared in the concentration (20mM:2mM:2mM). Final concentration in 20ul reaction is 1mM:0.1mM:0.1mM.
- - Add 2ul of dA:dC:dG/Klenow fragment exo- to each tube
- - Spin down the tube
- - Mix by gentle pulse-vortexting for 10x
- - Spin down the tube
- - Incubate at 30C for 20min (for gap-filling) --> 37C for 20min (for extra dA-tailing) no heat lid will help to protect Klenow fragment, exo
- - Heat inactivate enzyme at 75C for 10min
- - Set program to hold at 4C
- - Spin down the tube before continuing to next step
Methylated adaptor ligation
- To avoid the overlapping of indexes to some scRRBS libraries that may need to sequence in the same run (HiSeq Rapid run has only 2 lanes), I will not use index 2, 4, 5, and 12
- I will use 1ul of 1:10 diluted TruSeq adaptors for both low-input RRBS and STD RRBS.
Index list
CCT-5ng_1 | CTT-5ng_2 | CTT-F-5ng_1 | CTT-F-5ng_2 | 5ng NTC |
Ind_9 | Ind_10 | Ind_11 | Ind_13 | Ind_14 |
CCT-100ng_1 | CTT-100ng_2 | CTT-F-100ng_1 | CTT-F-100ng_2 | 100ng NTC |
Ind_15 | Ind_16 | Ind_18 | Ind_19 | Ind_20 |
Prep
- Diluted 1:10 of TrueSeq methylated adaptors by mixing 1ul of adaptor with 10ul H2O and mix
- Prepare ligation reaction mix
Components Volume (ul) 11x rxn mix dA-tailed reaction 20.00 0.00 10X Tango buffer 0.50 5.50 HC T4 DNA ligase (30units/ul) 1.00 11.00 10mM ATP 1.25 13.75 H2O 1.25 13.75 Total 24.00 44.00
- - Add 1ul of diluted methylated adapter
- - Add 4ul of ligation reaction mix
- - Spin down the tube
- - Mix by gentle pulse-vortexting for 10x
- - Spin down the tube
- - Incubate at 16C for 16h (no heat lid)
- - Heat inactivate at 65C for 20min
Sensitivity test of MONOD V1N3 BSPP capture with amplification
- There are many things need to concern about BSPP capture of serum/plasma samples for this project.
- DNA amount from plasma is very low in the range of 5-10ng from 1mL plasma.
- Serum samples showed higher amount than plasma, but Dr. Zhang suggest that the high amount is from the lysis of white blood cell.
- If we use serum samples, we need to increase the sensitivity of cancer DNA detection.
- If we use plasma sample, we need to do some pre-amplification to amplify <10ng of cell-free DNA by ~10-fold for padlock capture.
- The amount that Dr. Zhang meant is amount of extracted DNA not after bisulfite conversion. I have done some experiment to test the recovery rate of bisulfite conversion at different input amounts.
Bisulfite conversion recovery rate #1
- Note: The resulting BIS-DNAs were used for sensitivity test experiment
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Sample conc. (ng/ul) | Yield in 15ul (ng) | Input DNA (ng) | % Recovery |
JK-1 | 15.2 | ng/mL | 1 | 200 | 3.03 | 45.45 | 100 | 45.45 |
JK-2 | 19.1 | ng/mL | 1 | 200 | 3.82 | 57.30 | 100 | 57.30 |
JK-3 | 13.3 | ng/mL | 1 | 200 | 2.66 | 39.90 | 100 | 39.90 |
JK-4 | 41 | ng/mL | 1 | 200 | 8.21 | 123.15 | 200 | 61.58 |
JK-5 | 36.6 | ng/mL | 1 | 200 | 7.31 | 109.65 | 200 | 54.83 |
JK-6 | 39.4 | ng/mL | 1 | 200 | 7.88 | 118.20 | 200 | 59.10 |
JK-7 | 133 | ng/mL | 1 | 200 | 26.6 | 399.00 | 600 | 66.50 |
JK-8 | 131 | ng/mL | 1 | 200 | 26.2 | 393.00 | 600 | 65.50 |
Bisulfite conversion recovery rate #2
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Sample conc. (ng/ul) | Yield in 9ul (ng) | Input DNA (ng) | % Recovery |
JK-1 | 3.8 | ng/mL | 1 | 200 | 0.760 | 6.84 | 25 | 27.36 |
JK-2 | 4.9 | ng/mL | 1 | 200 | 0.980 | 8.82 | 25 | 35.28 |
JK-3 | 2.5 | ng/mL | 1 | 200 | 0.500 | 4.50 | 25 | 18.00 |
JK-4 | 5.11 | ng/mL | 1 | 200 | 1.022 | 9.20 | 25 | 36.79 |
JK-5 | 10.8 | ng/mL | 1 | 200 | 2.160 | 19.44 | 50 | 38.88 |
JK-6 | 9.06 | ng/mL | 1 | 200 | 1.812 | 16.31 | 50 | 32.62 |
JK-7 | 12.3 | ng/mL | 1 | 200 | 2.460 | 22.14 | 50 | 44.28 |
JK-8 | 11.2 | ng/mL | 1 | 200 | 2.240 | 20.16 | 50 | 40.32 |
JK-9 | 25 | ng/mL | 1 | 200 | 5.000 | 45.00 | 100 | 45.00 |
JK-10 | 26.7 | ng/mL | 1 | 200 | 5.340 | 48.06 | 100 | 48.06 |
JK-11 | 28.4 | ng/mL | 1 | 200 | 5.680 | 51.12 | 100 | 51.12 |
JK-12 | 24.3 | ng/mL | 1 | 200 | 4.860 | 43.74 | 100 | 43.74 |
JK-13 | 90.1 | ng/mL | 1 | 200 | 18.020 | 162.18 | 200 | 81.09 |
JK-14 | 89 | ng/mL | 1 | 200 | 17.800 | 160.20 | 200 | 80.10 |
JK-15 | 79.7 | ng/mL | 1 | 200 | 15.940 | 143.46 | 200 | 71.73 |
JK-16 | 85.4 | ng/mL | 1 | 200 | 17.080 | 153.72 | 200 | 76.86 |
- Input 200ng had too high recovery rate compare to previous experiment.