Noi/NOTES/2014-6-2
RRBS library preparation of colon tumor tissue samples from Biochain
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- 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 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
Pre-amplification set up
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
- 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
- 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