Noi/NOTES/2014-6-2

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RRBS library preparation of colon tumor tissue samples from Biochain

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.
  • As Dr. Zhang mentioned about pre-amplification before BSPP capture, I wanted to do experiment to test if I can get the capture work on pre-amplified DNa again.
  • I used BIS-DNA of cancer sample #2 (CANC-2) because I had no time to prepare more BIS-DNA.
  • I set up experiment by including 5ng, 10ng, 25ng and 50ng of BIS-DNA in amplification in total 10ul reaction with the following condition (will be added in very details)
    • Mix BIS-DNA with 2uM N9 random primers and 1X Reaction buffer for Klenow, exo-
    • Heat at 94C, 3min --> 30C (ramp 0.2C/sec) 3min --> add dNTP & Klenow, exo- mix --> 30C, 1h --> heat inactivate at 75C, 20min
    • Purified with Zymo column --> elute 8ul
    • I used 5ul for MONOD V1N3 capture. The left over DNA in th etube ~1-1.5ul were loaded in TBE gel to verify if the amplification work well.
    • I loaded 50ng of BIS-DNA before amplification in one lane or comparison
    • I also load 1ul of Illumina amplified DNA as control.

File:ZhangLab 2 2014-06-02 23hr 09min low-amp TBE-verify.jpg