AlanFung:LabNotes/2015/2015-1-15
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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 |
Part I: Pooling WGBS libraries & Normal Control WGBS libraries (01/16/2015)
(The tube should be labeled ~2014-12-13) | (The tube should be labeled ~2014-12-16) | |||||||
Sample | Conc. (ng/ul) | TruSeq Indx | Volume for 50ng (ul) | Sample | Conc. (ng/ul) | TruSeq Indx | Volume for 50ng (ul) | |
PCP-9 | 26.13 | 8 | 1.91 | NC-1 | 13.73 | 1 | 3.64 | |
PCP-4 | 21.73 | 3 | 2.30 | NC-2 | 16 | 2 | 3.13 | |
7P-6 | 26.93 | 22 | 1.86 | NC-3 | 16.93 | 4 | 2.95 | |
PCP-8 | 19.07 | 7 | 2.62 | NC-5 | 13.07 | 5 | 3.83 | |
PCP-6 | 20.13 | 5 | 2.48 | NC-6 | 15.33 | 6 | 3.26 | |
PCP-7 | 22.8 | 6 | 2.19 | NC-7 | 13.73 | 7 | 3.64 | |
PCP-2 | 22.13 | 1 | 2.26 | NC-8 | 15.87 | 8 | 3.15 | |
PCP-3 | 21.47 | 2 | 2.33 | NC-9 | 16.53 | 10 | 3.02 | |
7P-1 | 14.67 | 18 | 3.41 | NC-12 | 14.8 | 13 | 3.38 | |
PCP-5 | 21.47 | 4 | 2.33 | NC-13 | 13.19 | 14 | 3.79 | |
7P-8 | 22.8 | 25 | 2.19 | NC-14 | 14.93 | 15 | 3.35 | |
7P-3 | 19.07 | 20 | 2.62 | NC-15 | 13.6 | 16 | 3.68 | |
6P-1 | 26 | 9 | 1.92 | NC-16 | 13.87 | 27 | 3.60 | |
6P-8 | 24.8 | 14 | 2.02 | NC-17 | 13.47 | 18 | 3.71 | |
6P-10 | 22.27 | 16 | 2.25 | NC-18 | 14.67 | 19 | 3.41 | |
6P-9 | 20.27 | 15 | 2.47 | NC-19 | 13.47 | 20 | 3.71 | |
6P-2 | 19.6 | 10 | 2.55 | NC-20 | 12.79 | 11 | 3.91 | |
6P-3 | 19.87 | 11 | 2.52 | NC-21 | 12.31 | 12 | 4.06 | |
6P-4 | 19.2 | 12 | 2.60 | NC-22 | 16 | 21 | 3.13 | |
7P-10 | 25.33 | 27 | 1.97 | NC-23 | 13.6 | 22 | 3.68 | |
7P-7 | 29.6 | 23 | 1.69 | NC-24 | 11.97 | 3 | 4.18 | |
7P-2 | 19.73 | 19 | 2.53 | NC-27 | 14.67 | 9 | 3.41 | |
7P-5 | 18 | 21 | 2.78 | NC-29 | 12.96 | 23 | 3.86 | |
6P-5 | 24.67 | 13 | 2.03 | NC-30 | 15.47 | 25 | 3.23 | |
Total Volume | 55.83 | Total Volume | 84.70 |
- - Re-purified the two pools with 1X AMPure beads
- - 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. = 34.5 ng/ul --> total amount in 32ul = 1104 ng
- - Cancer patient WGBS library pool: conc. = 26.5 ng/ul --> total amount in 32ul = 848 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/B (0.8ul each). I pool the same amount of the 24 indexes in kit A & B 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 4.5uL SeqCap Epi probe pool on ice.
- Vortex quickly for 3s and at maximum speed for 10s at RT.
- Add 20l mineral oil on top.
- 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 77h and transferred the reaction to 4C to continue the next day.
Part IV: Washing and recovering captured bisulfite-converted DNA samples
Date of experiment 01/20/2015
IMPORTANT NOTE
- It is extremely important to maintain the water bath at 47C. I just have two samples, so I used thermomixer and prepare all washing buffer in 1.5mL or 2mL tube
- Equilibrate buffer at 47C for at least 2h before washing the captured Multiplex DNA sample. This should mean Stringent Wash Buffer and Wash Buffer I as they were used at 47C.
Step 1. Preparing Sequence Capture and Bead Wash Buffer
Concentrated Buffer | Volume/sample (ul) | H2O (ul) | Total (ul) | Volume for 2.1X | H2O for 2.1X |
10X Stringent Wash Buffer (vial 4) | 40 | 360 | 400 | 84 | 756 |
10X Wash Buffer I (vial 1) | 30 | 270 | 300 | 63 | 567 |
10X Wash Buffer II (vial 2) | 20 | 180 | 200 | 42 | 378 |
10X Wash Buffer III (vial 3) | 20 | 180 | 200 | 42 | 378 |
2.5X Bead Wash Buffer (vial 7) | 200 | 300 | 500 | 420 | 630 |
- Dilute 10X Wash Buffer (I, II, III and Stringent) and 2.5X Bead Wash Buffer to 1X working solutions as table above.
- Warm 1X Wash Buffer I and 1X Stringent Wash Buffer at 47C on thermocycler at least 47C as describe above. I have a very short time to prepare, so I incubated only ~1h.
- NOTE: All of Stringent Wash Buffer is used at 47C. Wash Buffer I will be used at 47C and RT (100ul at 47C and 200 at RT per rxn). I May need to aliquot accordingly.
Step 2. Preparing the Capture Beads
- Step below for 2 captures
- Allow the capture beads to warm to RT for 30min prior to use.
- Mix the beads thoroughly by vortexing to 15s.
- Aliquot 100ul of beads for each capture into a 1.5mL tube. For 2 captures, aliquot 200ul. of beads.
- Place the tube with beads on magnet device for 1.5mL tube for 3-5min until the liquid is clear. Remove and discard the liquid.
- Add 400ul of Bead Wash Buffer (200ul/sample)
- Vortex the beads for 10s
- Place the tube back to the magnet. Remove the liquid once the liquid is clear.
- Repeat step 2.5-2.7 for a total 2 washes
- Resuspend the bead with 200ul Bead Wash Buffer and vortex (100ul/sample)
- Aliquot 100ul of resuspended beads into new lo-bind 0.2mL tube.
- Place the tube on the magnet. Remove the clear liquid.
- The capture beads are now READY to bind to captured DNA. PROCEED IMMEDIATELY TO STEP 3. BINDING DNA TO THE CAPTURE BEADS
Step 3. Binding DNA to the Capture Beads
- Transfer the hybridization samples to the capture beads prepare in step 2.11
- Mix thoroughly by pipetting up and down 10X. NOTE: I have carried a small volume of mineral oil with the hybridization sample as I wanted to transfer as much as possible of the hybridization sample.
- Place the tube containing the beads and DNA on a thermocycler set at 47C for 45min (heat lid maintained at 57C). I just keep thermocycler after after hybridization on.
- Mix the samples by vortexing 3s every 15min to ensure that hte beads remain in suspension. Should have vortex close to thermocycler.
Step 4. Washing the Capture Beads Plus Bound DNA
- After 45-min incubation, add 100ul of 1X Wash Buffer I heated to 47C to the 15ul capture beads plus bound DNA. I added while the tube maintained on thermocycler at 47C.
- Mix by vortexing 10X
- Transfer the entire contents to Lo-bind 1.5mL tube
- Place the tube in the magnet to bind the beads. Remove and discard spnt
- Remove the tube from the magnet and add 200ul of 1X Stringent Wash Buffer heated to 47C. Pipette up and down 10X. WORK QUICKLY so that the temperature does not drop down much below 47C.
- I place the tube to thermomixter next to Stringent Wash Buffer and add the buffer.
- Incubate at 47C for 5min
- Repeat step 4.4 - 4.6 for a total 2 washes using 1X stringent Wash Buffer heated to 47C
- Place the tube back to the magnet and remove spnt
- Add 200ul of RT 1X Wash Buffer I -> mix by vortexing for 2min
- Place the tube back to the magnet and remove spnt
- Add 200ul of RT 1X Wash Buffer II -> mix by vortexing for 1min
- Place the tube back to the magnet and remove spnt
- Add 200ul of RT 1X Wash Buffer III -> mix by vortexing for 30s
- Place the tube back to the magnet and remove spnt
- Remove the tube from the magnet and add 50ul H2O. Mix by vortexing.
- Continue to Amplification Step or store the beads plus captured DNA at -15 to -20C.
- I stored the captured DNA in -20C
THERE IS NO NEED TO ELUTE DNA FROM THE BEADS
Part V: Amplifying Captured DNA Using LM-PCR
Date of experiment 01/21/2015
Preparing the LM-PCR
QUICK RUN PCR to monitor cycle number
- The user guide has suggestions on the pcr condition for amplification of captured DNA
- But since this is my first time doing the capture experiment, my capture efficiency might not be as high as expected.
- Noi did the optimization before and I will modify her protocol.
- I will do PCR -1 & +1 of the recommendation cycle number (16X).
- I will use the amplification primers Noi used to amplify the libraries (TruS F/R).
- I will use the same primer concentration as I used to amplify the library at 0.3uM instead of 0.5uM.
- Noi had issue with evaporation when doing pcr in small volume, I will add 20ul mineral oil for the pcr reaction.
Components | Volume (ul) |
KAPA HiFi HotStart ReadyMix (2X) | 20.83 |
TruS F/R (10uM) > 0.3uM after adding template | 1.25 |
H2O | 2.92 |
Total | 25.00 |
# Aliquot 7.5ul MM + 5ul bead-bound captured DNA (total 12.5ul) 2X # Aliquot 7.5ul MM + 5ul ddH2O for NTC (total 12.5ul) 2X # Aliquot 4ul pcr section mix to pcr tubes # Ran PCR at 15,16 and 17 cycles on different thermocycler. LM-PCR program 98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 16X, 17X --> 72C, 1min --> Hold @8C
- TBE Gel
File:ZhangLab 2 2015-01-21 15hr 31min.jpg
Amplification in a large volume
Components Volume (ul) 2.2rxn (ul) KAPA HiFi HotStart ReadyMix (2X) 50.00 110.00 TruS F/R (10uM) 3.00 6.60 H2O 7.00 15.40 Total 60.00 # Aliquot 63ul + 42ul bead-bound captured DNA (total ul) # Split 52ul 2X LM-PCR program 98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 15X --> 72C, 1min --> Hold @8C
- - Purified amplified captured DNA with Qiaquick PCR columns
- - Elute with total volume 50ul of EB buffer, total 100ul for each set.
- - Verify purified sequencing library in 6% TBE gel by loading 3ul.
Library ID: 1 = HL-Seqcap-pNormalSetA-Jan15 2 = HL-Seqcap-pCancerSetA-Jan15
Information for sequencing and sample tracking
HL-Seqcap-pNormalSetA-Jan15
- Average size: 300bp
- Primer system: Illumina TruSeq
Sample | TruSeq Indx | Seq (RC) |
NC-1 | 1 | ATCACG |
NC-2 | 2 | CGATGT |
NC-3 | 4 | TGACCA |
NC-5 | 5 | ACAGTG |
NC-6 | 6 | GCCAAT |
NC-7 | 7 | CAGATC |
NC-8 | 8 | ACTTGA |
NC-9 | 10 | TAGCTT |
NC-12 | 13 | AGTCAA |
NC-13 | 14 | AGTTCC |
NC-14 | 15 | ATGTCA |
NC-15 | 16 | CCGTCC |
NC-16 | 27 | ATTCCT |
NC-17 | 18 | GTCCGC |
NC-18 | 19 | GTGAAA |
NC-19 | 20 | GTGGCC |
NC-20 | 11 | GGCTAC |
NC-21 | 12 | CTTGTA |
NC-22 | 21 | GTTTCG |
NC-23 | 22 | CGTACG |
NC-24 | 3 | TTAGGC |
NC-27 | 9 | GATCAG |
NC-29 | 23 | GAGTGG |
NC-30 | 25 | ACTGAT |
HL-Seqcap-pCancerSetA-Jan15
- Average size: 300bp
- Primer system: Illumina TruSeq
Sample | TruSeq Indx | Seq (RC) |
PCP-9 | 8 | ACTTGA |
PCP-4 | 3 | TTAGGC |
7P-6 | 22 | CGTACG |
PCP-8 | 7 | CAGATC |
PCP-6 | 5 | ACAGTG |
PCP-7 | 6 | GCCAAT |
PCP-2 | 1 | ATCACG |
PCP-3 | 2 | CGATGT |
7P-1 | 18 | GTCCGC |
PCP-5 | 4 | TGACCA |
7P-8 | 25 | ACTGAT |
7P-3 | 20 | GTGGCC |
6P-1 | 9 | GATCAG |
6P-8 | 14 | AGTTCC |
6P-10 | 16 | CCGTCC |
6P-9 | 15 | ATGTCA |
6P-2 | 10 | TAGCTT |
6P-3 | 11 | GGCTAC |
6P-4 | 12 | CTTGTA |
7P-10 | 27 | ATTCCT |
7P-7 | 23 | GAGTGG |
7P-2 | 19 | GTGAAA |
7P-5 | 21 | GTTTCG |
6P-5 | 13 | AGTCAA |