Revision as of 18:42, 13 January 2015 by >Noi
LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient[edit]
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Continued from 2014-12-05
- Conditions:
- Probe set: MONOD V5 set A (LMS)
- Probe:target ratio: 150:1 for LMS probe set.
- Please note that I used very high amount of WGBS libraries for BSPP capture up to 600ng. This resulted in a very high amount of BSPP required for capture. I therefor reduced probe to target ratio from 200:1 to 125:1. The main reason is I do not have enough LMS BSPP to capture 50 samples with 600ng DNA each. For experiment I did for Illumina (Li Liu), I used 400ng of WGBS libraries, so I had enough probes to capture that amount. In order to be able to capture all unique molecule of ctDNA, I tried to increase WGBS libraries as much as possible. Ideally, Dr. Zhang wanted 1ug for capture.
- Sample:
- WGBS libraries of ctDNA from cancer patients prepared by KAPA Hyper Prep Kit 600ng.
- WGBS libraries of cfDNA from normal control (re-amplified) prepare by using RRBS protocol but skipping DNA digestion/fragmentation 600ng
Probe calculation[edit]
Probe set Probe size Probe:target ratio
LMS 73,574 150:1
From 2014-12-08 exp., 400ng required 108ng (200:1).
So 600ng (150:1) requires ~100ng of LMS probes
- Since the probe concentration a little low (8.25ng/ul), I will set up capture reaction in 300ul.
NOTE: WHAT ARE DIFFERENT FROM EXP. on 2014-12-08 (ILLUMINA, for Li Liu)[edit]
- Template 600ng instead of 400ng
- Probe: target ratio is 125:1 instead of 200:1
- Capture reaction 30ul instead of 25ul
Precedures[edit]
- I took 600ng of WGBS libraries in 1.5mL LoBind tube. Rationale: total volume of 600ng of WGBS libraries is too large to fit in 30ul capture reaction.
- Speedvac at 60C for ~20-30min to dry up DNA in the tube.
- Resuspend each tube with 14.88ul H2O which is the maximum volume of WGBS libraries to fit in 30ul capture reaction.
- Make sure that we resuspend DNA on the bottom of the tube very well.
- Transfer 14.88ul of DNA to 95-well plate
- I re-organized the location of each sample on the well based on sample ID (small - large)
- Aliquot 15.12ul of Probe & Buffer Mix in below table to each well
- Total volume is 30ul.
- Mix well, seal with plastic seal and spin down the plate
- Layer with 30ul mineral oil
- Seal with Aluminum seal and spin down the plate again.
- Incubate the plate on thermocycler following program below
Sample
|
Conc. (ng/ul)
|
Volume for 600ng (ul)
|
NC-1 |
13.73 |
43.69
|
NC-2 |
16.00 |
37.50
|
NC-3 |
16.93 |
35.43
|
NC-5 |
13.07 |
45.92
|
NC-6 |
15.33 |
39.13
|
NC-7 |
13.73 |
43.69
|
NC-8 |
15.87 |
37.82
|
NC-9 |
16.53 |
36.29
|
NC-12 |
14.80 |
40.54
|
NC-13 |
13.19 |
45.50
|
NC-14 |
14.93 |
40.18
|
NC-15 |
13.60 |
44.12
|
NC-16 |
13.87 |
43.27
|
NC-17 |
13.47 |
44.55
|
NC-18 |
14.67 |
40.91
|
NC-19 |
13.47 |
44.55
|
NC-20 |
12.79 |
46.92
|
NC-21 |
12.31 |
48.75
|
NC-22 |
16.00 |
37.50
|
NC-23 |
13.60 |
44.12
|
NC-25 |
11.97 |
50.11
|
NC-27 |
14.67 |
40.91
|
NC-29 |
12.96 |
46.30
|
NC-30 |
15.47 |
38.79
|
Sample
|
Conc. (ng/ul)
|
Volume for 600ng (ul)
|
PCP-9 |
26.13 |
22.96
|
PCP-4 |
21.73 |
27.61
|
PCP-8 |
19.07 |
31.47
|
PCP-6 |
20.13 |
29.80
|
PCP-7 |
22.80 |
26.32
|
PCP-2 |
22.13 |
27.11
|
PCP-3 |
21.47 |
27.95
|
PCP-5 |
21.47 |
27.95
|
6P-1 |
26.00 |
23.08
|
6P-8 |
24.80 |
24.19
|
6P-10 |
22.27 |
26.95
|
6P-9 |
20.27 |
29.61
|
6P-5 |
24.67 |
24.32
|
6P-2 |
19.60 |
30.61
|
6P-3 |
19.87 |
30.20
|
6P-4 |
19.20 |
31.25
|
7P-8 |
22.80 |
26.32
|
7P-3 |
19.07 |
31.47
|
7P-10 |
25.33 |
23.68
|
7P-7 |
29.60 |
20.27
|
7P-6 |
26.93 |
22.28
|
7P-1 |
14.67 |
40.91
|
7P-2 |
19.73 |
30.41
|
7P-5 |
18.00 |
33.33
|
- In 96-well plate, I re-organized the sample ID from small to large like from PCP2, PCP-3, PCP4,....
Probe & Buffer Mix[edit]
Components
|
1X
|
51X
|
MONOD V5A (LMS) 8.25ng/ul |
12.12 |
618.18
|
10X Ampligase Buffer |
3.00 |
153.00
|
WGBS library (600ng) |
14.88 |
|
Total |
30.00 |
|
Program
-> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 23h
-> add 2.5ul PLN mix (20%v/v Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP)
--> No need to change program while adding PLN mix (keep the tube at 55C all the time)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 5min -> 10C hold.
--> Press proceed once before adding ExoI/III and waiting until temperature down to 37C before adding ExoI/III
PLN (Polymerase/Ligase/ dNTP) mix solution Mix[edit]
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume (ul), total 150ul
|
Hemo KlenTaq |
100 |
% |
20 |
% |
30.00
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
15.00
|
dNTP |
1 |
mM |
100 |
uM |
15.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
15.00
|
H2O |
|
|
|
|
75.00
|
Total |
|
|
|
|
150.00
|
- Prepare PLN solution mix as follows: 20% (v/v) Hemo KlenTaq, 0.5 unit/μL Ampligase, 100 μM of dNTP mix, and 1x Ampligase Buffer.
- Aliquot 18.5ul of PLN mix to 8-tube strip and add 2.5 μL of KLN solution mix to each well with 10 μL multichannel pipette. Make sure that PLN mix is added under the mineral oil layer.
- Swirl pipette 10 times to mix the capture reaction without moving the plate from thermal cycler.
- Re-seal a 96-well plate with aluminum-seal.
- Continue to incubate at 55 ºC for 20 hours.
- Denature DNA at 94 ºC for 2 minutes.
Exonuclease digestion[edit]
- Prepare exonuclease I/III mix by mixing exonuclease I (20 units/μL) and exonuclease III (200 units/μL) in 1:1 ratio and aliquot equal volume to 8-tube strip
- Add 2 μL of exonuclease I/III mix to each well with multichannel pipette
- Mix the reaction by swirling pipette around the well 10 times.
- Incubate reaction at 37 ºC for 2 hours.
- Heat inactivate exonuclease I/III at 94 ºC for 5 minutes.
- Continue to qPCR without any purification or store at 4C
Amplification[edit]
- I normally do a quick test PCR in small volume 20ul with 10% v/v of captured DNA. This to monitor PCR cycle number and check if we get the right amplified product by running PCR product in 6% TBE gel without bead/colum purification. If the experiment has been optimized for a certain probe set, may not need to do quick qPCR check. This time I want to compare between the two enzymes, Phusion Flash High-Fidelity MM and KAPA HiFi Hotstart ReadyMix.
Quick PCR[edit]
- I randomly picked few samples from normal controls and cancer patient samples for a quick test PCR, including NC-1, NC-2, NC-3 NC-4, PCP-2, PCP-3, PCP-4, PCP-5, ^T1 from previous exp. as positive control and NTC.
Components
|
Stock conc.
|
Final conc. In PCR
|
1x rxn
|
13x rxn
|
Captured template |
100% |
10% |
2.00 |
0.00
|
10uM AmpF6.4Sol |
10uM |
0.2uM |
0.40 |
5.20
|
10uM AmpR6.3.IndX 8 |
10uM |
0.2uM |
0.40 |
5.20
|
2X Phusion Flash Hi-Fidelity MM or KAPA HiFi Hotstart ReadyMix |
2X |
1X |
10.00 |
130.00
|
50X SYBR |
50X |
0.4X |
0.16 |
2.08
|
H2O |
|
|
7.04 |
91.52
|
Total |
|
|
20.00 |
|
- - Aliquot 18ul + 2ul of captured DNA template
- Note: In PCR, I included one of positive control (PTC) of captured templates from gDNA of tumor tissue sample to compare the size of amplified amplicons.
- - 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x10-15 cycles -> 72C 3min
- Note that I just realized that KAPA HiFi Hotstart ReadyMix requires 2-3min for hot start activation. I paused the machine for 1 min a9 98C during denaturing step. I was not sure if this will result in not-fully active DNA polymerase for KAPA.
- - In this experiment, we stopped at 19 cycles
- - Loaded 5ul of PCR product in 6% TBE gel without purification (Run at 250 volts in 0.5X TBE buffer for 25min)
File:ZhangLab 2 2014-12-21 17hr 59min PhusionFlash-quickPCR-LMS-WGBS-5ul.jpg File:ZhangLab 2 2014-12-21 17hr 59min KAPAHiFi-quickPCR-LMS-WGBS-5ul.jpg
- The pattern of PCR products was consistent between the two enzymes. However, Phusion Flash Hi-Fidelity showed stronger intensity, so I will keep using this enzyme for the future. This should be note that a lower signal from KAPA HiFi Hotstart ReadyMix might be due to too short denaturation. However, I had no time to repeat experiment.
- I continued to amplified the rest of captured DNA in large volume (200ul --> split 2X of 100ul reaction) for 20 cycles using Phusion Flash Hi-Fidelity.
- I pooled 80ul from each well (total 160ul) and purified with 0.7X AMPure bead.
- Resuspended with 60ul H2O and quantified by PAGE quantification.
File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel1.jpg File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel2.jpg
File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel3.jpg File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel4.jpg
File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel5.jpg File:ZhangLab 2 2014-12-28 04hr 25min PQ-gel6.jpg
PAGE size selection & Sequencing library information[edit]
- Alan will do PAGE size selection
- There are high variations of PAGE quantification between different gel, so I ask Alan to do size selection separately for samples quantified in different gel. There should be total 6 library pools.
WHERE I KEEP INDIVIDUAL SEQUENCING LIBRARIES[edit]
File:NP photo 5-1.JPG
Tube ID: NP-BSPP_V5A_NC.p1-Dec16[edit]
Sequencing Name
|
Sample ID
|
ZhangLab Index
|
Seq(RC)
|
Conc. (ng/ul)
|
Volume for 70ng (ul)
|
NP-BSPP-V5A-Dec16-NC-1_41 |
NC-1 |
41 |
GGGAAC |
5.23 |
13.39
|
NP-BSPP-V5A-Dec16-NC-2_42 |
NC-2 |
42 |
GAAAAG |
5.42 |
12.92
|
NP-BSPP-V5A-Dec16-NC-3_43 |
NC-3 |
43 |
AGTGCC |
9.44 |
7.42
|
NP-BSPP-V5A-Dec16-NC-5_44 |
NC-5 |
44 |
GCATCC |
4.74 |
14.77
|
NP-BSPP-V5A-Dec16-NC-6_45 |
NC-6 |
45 |
ACTACG |
5.54 |
12.64
|
NP-BSPP-V5A-Dec16-NC-7_46 |
NC-7 |
46 |
CATTTC |
4.44 |
15.76
|
NP-BSPP-V5A-Dec16-NC-8_47 |
NC-8 |
47 |
CTCTCC |
4.01 |
17.46
|
NP-BSPP-V5A-Dec16-NC-9_48 |
NC-9 |
48 |
ACGTTA |
5.49 |
12.76
|
Total 107.12
6X Dye 22.00
Sum 129.12
Per well 32.28 (4 wells)
Tube ID: NP-BSPP_V5A_NC.p2-Dec16[edit]
Sequencing Name
|
Sample ID
|
ZhangLab Index
|
Seq(RC)
|
Conc. (ng/ul)
|
Volume for 70ng (ul)
|
NP-BSPP-V5A-Dec16-NC-12_49 |
NC-12 |
49 |
CTGTGT |
2.41 |
29.09
|
NP-BSPP-V5A-Dec16-NC-13_50 |
NC-13 |
50 |
ACCTTT |
2.14 |
32.66
|
NP-BSPP-V5A-Dec16-NC-14_51 |
NC-14 |
51 |
TATCGC |
2.70 |
25.91
|
NP-BSPP-V5A-Dec16-NC-15_52 |
NC-15 |
52 |
GACACG |
2.65 |
26.45
|
NP-BSPP-V5A-Dec16-NC-16_53 |
NC-16 |
53 |
TTCTAC |
2.60 |
26.95
|
NP-BSPP-V5A-Dec16-NC-17_54 |
NC-17 |
54 |
ACGTCC |
3.37 |
20.80
|
NP-BSPP-V5A-Dec16-NC-18_55 |
NC-18 |
55 |
TCGACT |
2.26 |
31.01
|
NP-BSPP-V5A-Dec16-NC-19_56 |
NC-19 |
56 |
TCAGAC |
3.55 |
19.72
|
Total 212.59
6X Dye 45.00
Sum 257.59
Per well 64.40 (4 wells)
Tube ID: NP-BSPP_V5A_NC.p3-Dec16[edit]
Sequencing Name
|
Sample ID
|
ZhangLab Index
|
Seq(RC)
|
Conc. (ng/ul)
|
Volume for 100ng (ul)
|
NP-BSPP-V5A-Dec16-NC-20_57 |
NC-20 |
57 |
TCCTTC |
5.50 |
18.17
|
NP-BSPP-V5A-Dec16-NC-21_58 |
NC-21 |
58 |
CAGCAT |
6.45 |
15.52
|
NP-BSPP-V5A-Dec16-NC-22_59 |
NC-22 |
59 |
GATAGA |
6.02 |
16.61
|
NP-BSPP-V5A-Dec16-NC-23_60 |
NC-23 |
60 |
ACAGAT |
6.10 |
16.38
|
NP-BSPP-V5A-Dec16-NC-25_61 |
NC-25 |
61 |
CTCTAT |
5.68 |
17.61
|
NP-BSPP-V5A-Dec16-NC-27_62 |
NC-27 |
62 |
TTTAGC |
5.85 |
17.08
|
NP-BSPP-V5A-Dec16-NC-29_63 |
NC-29 |
63 |
CCTGGT |
5.82 |
17.18
|
NP-BSPP-V5A-Dec16-NC-30_64 |
NC-30 |
64 |
AGTTGG |
7.19 |
13.90
|
Total 132.44
6X Dye 30.00
Sum 162.44
Per well 27.07 (4 wells)
Tube ID: NP-BSPP_V5A_PCP-Dec16[edit]
Sequencing Name
|
Sample ID
|
ZhangLab Index
|
Seq(RC)
|
Conc. (ng/ul)
|
Volume for 70ng (ul)
|
NP-BSPP-V5A-Dec16-PCP-2_65 |
PCP-2 |
65 |
TTCCTT |
3.06 |
22.90
|
NP-BSPP-V5A-Dec16-PCP-3_66 |
PCP-3 |
66 |
TGGAGG |
2.32 |
30.13
|
NP-BSPP-V5A-Dec16-PCP-4_67 |
PCP-4 |
67 |
GACGTG |
2.55 |
27.46
|
NP-BSPP-V5A-Dec16-PCP-5_68 |
PCP-5 |
68 |
GTTATG |
3.56 |
19.65
|
NP-BSPP-V5A-Dec16-PCP-6_69 |
PCP-6 |
69 |
ATATGG |
1.99 |
35.16
|
NP-BSPP-V5A-Dec16-PCP-7_70 |
PCP-7 |
70 |
GACTTC |
2.25 |
31.16
|
NP-BSPP-V5A-Dec16-PCP-8_71 |
PCP-8 |
71 |
TCTTTG |
2.07 |
33.81
|
NP-BSPP-V5A-Dec16-PCP-9_72 |
PCP-9 |
72 |
CTGCCA |
4.03 |
17.36
|
Total 217.63
6X Dye 45.00
Sum 262.63
Per well 65.66 (4 wells)
Tube ID: NP-BSPP_V5A_6P-Dec16[edit]
Sequencing Name
|
Sample ID
|
ZhangLab Index
|
Seq(RC)
|
Conc. (ng/ul)
|
Volume for 80ng (ul)
|
NP-BSPP-V5A-Dec16-6P-1_73 |
6P-1 |
73 |
GGACTC |
4.68 |
17.08
|
NP-BSPP-V5A-Dec16-6P-2_74 |
6P-2 |
74 |
TGGCGA |
3.99 |
20.05
|
NP-BSPP-V5A-Dec16-6P-3_75 |
6P-3 |
75 |
CGACTT |
6.79 |
11.77
|
NP-BSPP-V5A-Dec16-6P-4_76 |
6P-4 |
76 |
CCTATT |
4.62 |
17.31
|
NP-BSPP-V5A-Dec16-6P-5_77 |
6P-5 |
77 |
ACGGGT |
4.63 |
17.26
|
NP-BSPP-V5A-Dec16-6P-8_78 |
6P-8 |
78 |
CGTGTT |
5.58 |
14.35
|
NP-BSPP-V5A-Dec16-6P-9_79 |
6P-9 |
79 |
CCAAGC |
3.99 |
20.05
|
NP-BSPP-V5A-Dec16-6P-10_80 |
6P-10 |
80 |
TGGTAA |
4.87 |
16.42
|
Total 134.30
6X Dye 30.00
Sum 164.30
Per well 41.07
Tube ID: NP-BSPP_V5A_7P-Dec16[edit]
Sequencing Name
|
Sample ID
|
ZhangLab Index
|
Seq(RC)
|
Conc. (ng/ul)
|
Volume for 70ng (ul)
|
NP-BSPP-V5A-Dec16-7P-1_81 |
7P-1 |
81 |
ACCTGG |
3.42 |
20.48
|
NP-BSPP-V5A-Dec16-7P-2_82 |
7P-2 |
82 |
CAAACG |
1.60 |
43.62
|
NP-BSPP-V5A-Dec16-7P-3_83 |
7P-3 |
83 |
GTGGTC |
3.86 |
18.13
|
NP-BSPP-V5A-Dec16-7P-5_84 |
7P-5 |
84 |
TCTTGT |
3.22 |
21.74
|
NP-BSPP-V5A-Dec16-7P-6_85 |
7P-6 |
85 |
TGCGGT |
3.25 |
21.54
|
NP-BSPP-V5A-Dec16-7P-7_86 |
7P-7 |
86 |
TACCCA |
4.18 |
16.75
|
NP-BSPP-V5A-Dec16-7P-8_87 |
7P-8 |
87 |
CGGAAT |
3.48 |
20.13
|
NP-BSPP-V5A-Dec16-7P-10_88 |
7P-10 |
88 |
ACATTC |
3.69 |
18.98
|
Total 181.37
6X Dye 40.00
Sum 221.37
Per well 55.34 (4 wells)