Noi/NOTES/2014-6-2

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RRBS library preparation of colon tumor tissue samples from Biochain[edit]

Sample list and details[edit]

  • 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[edit]

  • Low-input RRBS (5ng)
  • Standard RRBS (100ng)

Experimental procedures[edit]

  • 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[edit]

1.1) Low-input RRBS MspI digestion set up[edit]

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[edit]

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[edit]

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[edit]

- 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[edit]

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

STD RRBS[edit]

- 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

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

Pre-amplification set up[edit]

bis-CANC_2 Conc. (ng/ul) Amount required (ng) Volume for XX ng (ul) H2O 50uM N9 10X reaction buffer Total
5a 5.00 5.00 1.00 4.80 0.40 1.00 7.20
5a 5.00 5.00 1.00 4.80 0.40 1.00 7.20
10a 5.00 10.00 2.00 3.80 0.40 1.00 7.20
10a 5.00 10.00 2.00 3.80 0.40 1.00 7.20
25a 21.70 25.00 1.15 4.65 0.40 1.00 7.20
25a 21.70 25.00 1.15 4.65 0.40 1.00 7.20
50a 21.70 50.00 2.30 3.50 0.40 1.00 7.20
50a 21.70 50.00 2.30 3.50 0.40 1.00 7.20
NTC-a 0.00 0.00 0.00 5.80 0.40 1.00 7.20

N9 and reaction buffer for Klenow, exo- mix

N9 & Buffer Mix 1x rxn 15X rxn
10X Reaction Buffer 1.00 15.00
50uM N9 0.40 6.00
Total 1.40

Klenow, exo- & dNTP mix

Klenow, exo- & dNTP mix 1x rxn 12x rxn
10mM dNTP Mix 0.40 4.80
Klenow Fragment, exo - (5U/ul) 0.50 6.00
H2O 1.90 22.80
Total 2.80
  • Final conc. of dNTP in the reaction = 0.4mM or 400uM
  • Final conc. of N9 primers in the reaction = 2uM
  • Total amount of Klenow, exo- in 10ul = 2.5 units
- Mix DNA with H2O and 1.4ul of N9/reaction buffer mix
- Heat at 94C, 3min --> 30C (ramp 0.2C/sec) 3min --> add 2.8ul of dNTP & Klenow, exo- mix (400uM dNTP, 2.5U of Klenow,exo- in the hood)---> 30C, 1h --> heat inactivate at 75C, 20min
- Purified with Zymo DNA Clean & Concentrator-5 column (I added 60ul of binding buffer, 6x volume) --> elute 8ul


- I used 5ul for MONOD V1N3 capture. The left over DNA in the tube ~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 for 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 BIS-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[edit]

  • Since I expected that the amount of template would increase after amplification, I roughly estimate it to be ~10X--> after column purification, I assume 5X left over.
  • 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]]
Input BIS-DNA (ng) in pre-amp Assume 5X amount after pre-amp (ng) Probe required (ng) V1N3 conc. (ng/ul) V1N3 volume (ul)
5 25 1.10 0.73 1.51
10 50 2.20 0.73 3.01
25 125 5.50 6.75 0.81
50 250 11.00 6.75 1.63
strip 1 5a 5a 10a 10a 25a 25a 50a 50a
Volume (ul) 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00
H2O (ul) 2.49 2.49 0.99 0.99 3.19 3.19 2.37 2.37
V1N3 amount (ng) 1.10 1.10 2.20 2.20 5.50 5.50 11.00 11.00
V1N3 conc. (ng/ul) 0.73 0.73 0.73 0.73 6.75 6.75 6.75 6.75
V1N3 volume (ul) 1.51 1.51 3.01 3.01 0.81 0.81 1.63 1.63
10X Ampligase Buffer 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
Total 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 1ul SLN mix (2U/ul Hemo Klentaql fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 5h-> 94C 2min -> add 1.5ul Exo I/III mix (1ul of 20U/ul ExoI & 0.5ul of 200U/ul of ExoIII)-> 37C 2h -> 94C 2min -> 4C hold.

Amplification[edit]

  • 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 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[edit]
  • 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[edit]
 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