Noi/NOTES/2014-6-2

From ZhangLabWiki
Revision as of 15:58, 7 June 2014 by >Noi (→‎BSPP capture set up)
Jump to navigation Jump to search

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 18h (no heat lid)
- Heat inactivate at 65C for 20min

2014-06-03, continued

Low-input RRBS

- Heat inactivate at 65C, 20min
- Wait for STD RRBS sample for AMPure bead purification

STD RRBS

- AMPure bead purification with 2X volume of the bead

- Mix 50ul of AMPure beads with 25ul ligated DNA. Mix by pipetting 10x
- Sit for 30min
- Transfer to sit on magnet for 5min
- Wash twice with 160ul freshly prepared 75% EtOH
- Dry the bead for 3-5min
- Resuspend with 27 H2O
- Transfer to new tube for bisulfite conversion. 25ul will be used for bisulfite conversion.

5) Bisulfite conversion

  • I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 31ul elution buffer.

Prep
- Prepare 1 tubes of CT Conversion Reagent, by adding 850ul H2O, 50ul of Resuspension Buffer, and 300ul of Dilution Buffer to CT Conversion Reagent (for 25ul DNA sample --> reduce H2O from 900 to 850)

- Add125ul of complete CT Conversion Reagent to adaptor ligated DNA (no sample transfer to the new tube)
- Mix by pipetting 10X with multi-channel pipette
- Spin down the plate at 2,000rpm for 1min
- 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

Prep
- Mix 600:1 ratio of Binding Buffer and 10ng/ul tRNA
- For 10.2 rxn, I mixed 6.12mL of Binding Buffer with 10.2ul of 10ng/ul tRNA (this actually for low-input, but I think it should be fine)

- Add 601ul of Binding Buffer/tRNA mix to the column
- Bind DNA to column by transfer bisulfite-treated DNA to the column and mixing by pipetting up and down for 5X. I rinse the well with small amount of Binding Buffer to transfer DNA to the column as much as possible
- Spin down 14,000rpm for 30sec. Discard spnt
- Wash with 100ul Wash buffer
- Spin down 14,000rpm for 30sec
- Incubate with 200ul of Desulphonation Buffer for 18min
- Spin down 14,000rpm for 30sec
- Wash column with 200ul Wash Buffer.
- Spin down 14,000rpm for 30sec. Discard spnt
- Wash the column with 200ul Wash Buffer.
- Spin down 14,000rpm for 2min
- Elute converted DNA with warm (~60C) 31ul Elution Buffer. Incubate column with Elution Buffer at room temp for 2min.
- Spin down at 14,000rmp for 1min. This should have ~30ul DNA left for PCR

Sensitivity test of MONOD V1N3 BSPP capture with pre-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 (400uM dNTP, 2.5U of Klenow,exo---> 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
-The numbers showed input DNA for amplification.
  • From the gel image, it showed that amplification in all input DNA worked well as no amplification in NTC and un-amplified BIS-DNA. I was wondering where is the top band of BIS-DNA. I am concern if it could be purified with Zymo column (DNA Clean & Concentrator™-5 - Capped Columns).
  • The pattern of amplified DNA looked similar to Illumina's amplified DNA.

BSPP capture set up

  • Since I expected that the amount of template would increase after amplification, I roughly estimate it to be ~10X.
  • I therefor increase the amount of BSPP to 5x based on 1000:1 probe to target ratio.
  • Perform BSPP capture the same volume and incubation time as experiment on 2014-06-01: [[4]]
    • I will add all experimental detail later.

Amplification

  • I used 10ul of captured DNA in total 75ul PCR reaction.
Components 1x rxn 13.5 rxn mix
Captured template 10.00 0.00
10uM AmpF6.4Sol 1.50 20.25
10uM AmpR6.3.IndX (X=73-93) 1.50 0.00
2X KAPA SYBR MM 37.50 506.25
H2O 24.50 330.75
Total 75.00
- Aliquot 63.5ul + 1.5ul AmpR6.3 IndX + 10ul template
- Set cycle number maximum at 27X
Program (Eppendorf Realplex)

98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x19 -> 72C 3min
Sample Index
1000:1_5ng-preamp_1 Ind_1
1000:1_5ng-preamp_2 Ind_2
1000:1_10ng-preamp_1 Ind_3
1000:1_10ng-preamp_2 Ind_4
1000:1_25ng-preamp_1 Ind_5
1000:1_25ng-preamp_2 Ind_6
1000:1_50ng-preamp_1 Ind_7
1000:1_50ng-preamp_2 Ind_8
  • Loaded 4ul of PCR products in 6% TBE gel (the same volume as 2014-06-01)

File:ZhangLab 2 2014-06-06 14hr 13min V1N3 CANC2-5-50ng-pream.jpg

Compare the qPCR results with experiment on 2014-06-01 with no pre-amplification before BSPP capture
  • Note that, I only focus on 5 and 10ng input
no pre-amplification
File:2014-06-03 5ng-noamp.png  File:2014-06-03 10ng-noamp.png

Pre-amplification
File:2014-06-06 5ng-preamp.png  File:2014-06-06 10ng-preamp.png

* The qPCR curves between no-amp and pre-amp may not be able to use for comparison as there is more background in pre-amp capture.
Compare the gel image
 no pre-amplification
File:ZhangLab 2 2014-06-03 16hr 20min V1N3 1000to1.jpg

Pre-amplification

File:ZhangLab 2 2014-06-06 14hr 13min V1N3 CANC2-5-50ng-pream.jpg
  • From the gel images, it looked like the intensity of BSPP capture with pre-amplification is higher than no pre-amplification, especially 10ng input. For 5ng, the reactions with pre-amp seemed to be a little brighter than no-amp.
  • However, there is more background in pre-amp capture. I guess this might be decrease with 1-2 less cycle numbers.
  • I should have done these experiments side by side to confirm that a smear band did not come from the original BIS-DNA template as without amplification, the capture worked.

Gel image of BSPP capture of pre-amplified DNA without column purification before ecapture

experiment on 2014-04-10
File:ZhangLab 2 2014-04-10 18hr 50min GP1-V4-gel2 ee.png