AlanFung:LabNotes/2015/2015-1-15: Difference between revisions
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# Have ~1ug of the WGBS library pool in a Lo-bind 1.5 mL tube prepare above, including normal control WGBS library pool and cancer patient WGBS library pool | # Have ~1ug of the WGBS library pool in a Lo-bind 1.5 mL tube prepare above, including normal control WGBS library pool and cancer patient WGBS library pool | ||
# Thaw the resuspended SeqCap HE Universal Oligos (1000uM) and the resuspended SeqCap HE Index Oligo (1000uM) that matches the DNA adaptor index in the WGBS library preparation | # Thaw the resuspended SeqCap HE Universal Oligos (1000uM) and the resuspended SeqCap HE Index Oligo (1000uM) that matches the DNA adaptor index in the WGBS library preparation | ||
::- In this experiment, I pooled all libraries using barcode adaptor match with all | ::- In this experiment, I pooled all libraries using barcode adaptor match with all 24 indexes in HE-Oligo KitA (0.8ul each). I pool the same amount of the 12 indexes in kit A and use 1ul of the pooled oligos in step 4.3 | ||
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=== Step 4. Prepare the hybridization samples === | === Step 4. Prepare the hybridization samples === | ||
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## 3ul of Hybridization Component A (vial 6) | ## 3ul of Hybridization Component A (vial 6) | ||
# Vortext the mixture for 10s and centrifuge at maximum speed for 10s. I transfer the contents in the tube to 0.2mL Lo-bind PCR tube. Make sure that you resuspend the mixture very well. | # Vortext the mixture for 10s and centrifuge at maximum speed for 10s. I transfer the contents in the tube to 0.2mL Lo-bind PCR tube. Make sure that you resuspend the mixture very well. | ||
# Heat the mixture at 95C for 10min with a heat lid on thermocycler. During this time, I thaw a 4.5ul aliquot of SeqCap Epi probe pool in a 0.2mL PCR tube on ice and | # Heat the mixture at 95C for 10min with a heat lid on thermocycler. During this time, I thaw a 4.5ul aliquot of SeqCap Epi probe pool in a 0.2mL PCR tube on ice and spin down | ||
# Centrifuge the heated tubes at maximum speed for 10s at RT. | # Centrifuge the heated tubes at maximum speed for 10s at RT. | ||
# Transfer the WGBS library pool/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail to the 4.5mL SeqCap Epi probe pool on ice. | # Transfer the WGBS library pool/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail to the 4.5mL SeqCap Epi probe pool on ice. | ||
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* Composed of 50% (1,000pmol) SeqCap HE Universal Oligo & 50% (1,000pmol) of the appropriate SeqCap HE Index Oligo. | * Composed of 50% (1,000pmol) SeqCap HE Universal Oligo & 50% (1,000pmol) of the appropriate SeqCap HE Index Oligo. | ||
* For this experiment, I incubate the hybridization sample for | * For this experiment, I incubate the hybridization sample for 72h. | ||
<br> | <br> | ||
<br> | <br> |
Revision as of 21:53, 16 January 2015
SeqCap Epi probe pool (UMR v1) hybridization capture on normal and cancer patient WGBS libraries
- Protocol: Dropbox/ZhangLab/Capture/Noi SeqCap Epi probe pool protocol
Background
- There were a number of differences on the experimental protocols between the two batches of data.
- 141112_HiSeq: Noi used the Tang et al RRBS method for end-repair and polishing Noi/NOTES/2014-10-29; capture was done on Noi/NOTES/2014-11-4;
- 141216_HiSeq: Noi used the Kapa Hyper Prep kit Noi/NOTES/2014-11-30; capture was done on Noi/NOTES/2014-12-25
- Noi did the capture experiment: Noi/NOTES/2014-12-25.
- The libraries were sequenced in one HiSeq PE100bp rapid run 141214_SN1001.
- Dinh mapped the reads: Dinh/Dinh_2015/NOTES/2015-1-7.
- Dr. Zhang analyzed the Hiseq Rapid run data, mapping rate is good. But specificity and enrichment factors are much lower than the first experiment
- Kun did the comparison: Kun:LabNotes/MONOD/2015-1-9.
- Kun did additional comparison for the two sets of data, he is sure the first batch of data has good on-target rates but low library complexity, and the second batch was the opposite.
- Kun:LabNotes/MONOD/2015-1-9#Further_comparison_of_the_two_batches_of_library_preparation_and_capturing_experiments.
- Conclusion 1 Plasma DNA needs to be end-polished using the standard library prep protocol such as the one implemented in the Kapa Hyper Prep kit.
- Conclusion 2 The Tang et al scRRBS protocol is based on some assumptions of the DNA fragment ends, which are not applicable to all plasma DNA fragments.
- I just need to repeat the capture experiments on those libraries made in the second batch, then we should be fine.
Experiment | End Repair | Capture | Hiseq Run | Specificity | Enrichment Factors | Complexity | Clonal Rates | Conclusion |
#1 | Tang et al RRBS | 141104 | 141112 | higher | higher | lower | higher | lower complexity and higher clonal rates due to Tang et assumption the DNA fragment ends |
#2 | Kapa Hyper Prep kit | 141225 | 141216 | lower | lower | higher | lower | lower specificity and enrichment due to capture condition |
Task
Locate remaining WGBS libraries, and the NimbleGen capturing reagents
Noi recently re-amplified WGBS libraries of cancer patients and normal control The tubes of the most recent batches should be labeled in December 2014 They all should be in PCR rack (3 of 8-tube strip for cancer patient and 3 of 8-tube strip for normal control) with the cover. Please do not use the sequencing libraries in the yellow PCR rack as those libraries are sequencing libraries from BSPP capture. Library location -20C WGBS from ctDNA of cancer patients were generated using KAPA Hyper Prep Kit They were generated on 2014-12-13 and should be located in the PCR rack with cover
Part I: Pooling WGBS libraries & Normal Control WGBS libraries
(The tube should be labeled ~2014-12-13) | (The tube should be labeled ~2014-12-16) | ||||||
Sample | Conc. (ng/ul) | TruSeq Indx | Volume for 100ng (ul) | Sample | Conc. (ng/ul) | TruSeq Indx | Volume for 100ng (ul) |
PCP-9 | 26.13 | 8 | 3.83 | NC-1 | 13.73 | 1 | 7.28 |
PCP-4 | 21.73 | 3 | 4.60 | NC-2 | 16 | 2 | 6.25 |
PCP-8 | 19.07 | 7 | 5.24 | NC-3 | 16.93 | 4 | 5.91 |
PCP-6 | 20.13 | 5 | 4.97 | NC-5 | 13.07 | 5 | 7.65 |
PCP-7 | 22.8 | 6 | 4.39 | NC-6 | 15.33 | 6 | 6.52 |
PCP-2 | 22.13 | 1 | 4.52 | NC-7 | 13.73 | 7 | 7.28 |
PCP-3 | 21.47 | 2 | 4.66 | NC-8 | 15.87 | 8 | 6.30 |
PCP-5 | 21.47 | 4 | 4.66 | NC-9 | 16.53 | 10 | 6.05 |
6P-1 | 26 | 9 | 3.85 | NC-12 | 14.8 | 13 | 6.76 |
6P-8 | 24.8 | 14 | 4.03 | NC-13 | 13.19 | 14 | 7.58 |
6P-10 | 22.27 | 16 | 4.49 | NC-14 | 14.93 | 15 | 6.70 |
6P-9 | 20.27 | 15 | 4.93 | NC-15 | 13.6 | 16 | 7.35 |
6P-5 | 24.67 | 13 | 4.05 | NC-16 | 13.87 | 27 | 7.21 |
6P-2 | 19.6 | 10 | 5.10 | NC-17 | 13.47 | 18 | 7.42 |
6P-3 | 19.87 | 11 | 5.03 | NC-18 | 14.67 | 19 | 6.82 |
6P-4 | 19.2 | 12 | 5.21 | NC-19 | 13.47 | 20 | 7.42 |
7P-8 | 22.8 | 25 | 4.39 | NC-20 | 12.79 | 11 | 7.82 |
7P-3 | 19.07 | 20 | 5.24 | NC-21 | 12.31 | 12 | 8.12 |
7P-10 | 25.33 | 27 | 3.95 | NC-22 | 16 | 21 | 6.25 |
7P-7 | 29.6 | 23 | 3.38 | NC-23 | 13.6 | 22 | 7.35 |
7P-6 | 26.93 | 22 | 3.71 | NC-24 | 11.97 | 3 | 8.35 |
7P-1 | 14.67 | 18 | 6.82 | NC-27 | 14.67 | 9 | 6.82 |
7P-2 | 19.73 | 19 | 5.07 | NC-29 | 12.96 | 23 | 7.72 |
7P-5 | 18 | 21 | 5.56 | NC-30 | 15.47 | 25 | 6.46 |
Total volume: | 111.67 | Total Volume: | 169.41 |
- - Re-purified the two pools with 1X AMOPure bead
- - Resuspended each library with 33ul H2O
- - Measure conc. with Qubit dsDNA HS assay using 1ul of purified library pool. Total volume left ~32ul
- - Normal WGBS library pool: conc. = ng/ul --> total amount in 32ul = ng
- - Cancer patient WGBS library pool: conc. = ng/ul --> total amount in 32ul = ng
- - The total amount of the two library pools is close to 1ug as recommended by User guide.
- - Continue to SeqCap hybridization capture
Part II: SeqCap hybridization capture with optimized conditions
- Followed exactly the instruction describe in the User Guide page 22-24
Step 1. Preparing for hybridization
- Turn on a heat block to 95C. I use thermocycler to heat DNA mix at step 4.9 as I transfer the DNA mix from 1.5mL tube to 0.2ul PCR tube.
Step 2. Resuspend the SeqCap HE Universal and SeqCap HE Index Oligos
- Noi prepared this step the first time she opened the kit
- We have HE Oligo Index set A
- Spin the lyophilized oligo tubes, contained in the SeqCap HE-Oligo KitA briefly to allow the contents to pellet to the bottom of the tube.
- Add 120ul PCR-grade H2O to SeqCap HE Universal Oligo tube (1000uM final concentration).
- Add 10ul PCR-grade H2O to SeqCap HE Index Oligo tube (1000uM final concentration).
- Vortex the oligos plus H2O for 5s and spin down the resuspended oligo tube.
- After use, they should be stored at -15 to -25C
Step 3. Prepare the bisulfite-converted DNA sample library and HE oligos for hybridization
- Have ~1ug of the WGBS library pool in a Lo-bind 1.5 mL tube prepare above, including normal control WGBS library pool and cancer patient WGBS library pool
- Thaw the resuspended SeqCap HE Universal Oligos (1000uM) and the resuspended SeqCap HE Index Oligo (1000uM) that matches the DNA adaptor index in the WGBS library preparation
- - In this experiment, I pooled all libraries using barcode adaptor match with all 24 indexes in HE-Oligo KitA (0.8ul each). I pool the same amount of the 12 indexes in kit A and use 1ul of the pooled oligos in step 4.3
Step 4. Prepare the hybridization samples
- Add 10ul of Bisulfite Capture Enhancer (found in the SeqCap Epi Accessary Kit) to a new Lo-bind 1.5 mL tube)
- Add 1ug of WGBS library pool
- Add 1,000pmol SeqCap HE Universal Oligo 1 and 1,000pmol of the 1000uM SeqCap HE Index Oligo SetA as described above.
- This equal to 1ul of 1,000uM of oligos
- Close the tube and make a hole in the top of the tube's cap with an 20 gauge needle (I poked many holes)
- Dry the mixture of WGBS library pool/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos in a vacuum concentrator on high heat 60C for ~15-20min until it dry up. A little liquid left in the tube between 1-2ul should be fine to prevent over dry.
- Cover the holes in the tube's cap with a sticker
- To each tube, add
- 7.5ul of 2X Hybridization Buffer (vial 5)
- 3ul of Hybridization Component A (vial 6)
- Vortext the mixture for 10s and centrifuge at maximum speed for 10s. I transfer the contents in the tube to 0.2mL Lo-bind PCR tube. Make sure that you resuspend the mixture very well.
- Heat the mixture at 95C for 10min with a heat lid on thermocycler. During this time, I thaw a 4.5ul aliquot of SeqCap Epi probe pool in a 0.2mL PCR tube on ice and spin down
- Centrifuge the heated tubes at maximum speed for 10s at RT.
- Transfer the WGBS library pool/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail to the 4.5mL SeqCap Epi probe pool on ice.
- Vortext quickly for 3s and at maximum speed for 10s at RT.
- Incubate on a thermo cycler at 47C for 64-72h. Turn on the thermocycler 's heat lid and set to maintain at 57C (10C above the hybridization temperature).
- NOTE: I overlaid the hybridization sample with 20ul mineral oil as Alice did before to prevent evaporation
The hybridization sample contains the following components: Component Solution Capture Bisulfite Capture Enhancer 10ug WGBS libraries 1ug Hybridization Enhancing Oligos 2,000pmol* ------------------------------------------------------- (All component above were heated up to dry) 2X Hybridization Buffer (vial 5) 7.50ul Hybridization Component A (vial 6) 3.00ul SeqCap Epi probe pool 4.50ul Total 15.00ul * Composed of 50% (1,000pmol) SeqCap HE Universal Oligo & 50% (1,000pmol) of the appropriate SeqCap HE Index Oligo.
- For this experiment, I incubate the hybridization sample for 72h.