Revision as of 18:47, 3 August 2014 by >Noi
Preparation of WGBS libraries of small fragment DNA in plasma samples (no DNA fragmentation step)
Calendar
Select plasma DNA samples for WGBS
Pancreas (UCSD-004)
Diagnosis
|
Conc. (ng/uL)
|
Volume (ul)
|
Yields (ng)
|
Selected for WGBS
|
Zhang lab ID
|
Adenocarcinoma, moderately to poorly differentiated, c/w pT3N0MX |
0.473 |
30.00 |
14.19 |
|
PC-P-1
|
Adenocarcinoma, ductal type, moderately to poorly differentiated, pT3N1MX |
0.852 |
30.00 |
25.56 |
YES |
PC-P-2
|
Poorly differentiated adenocarcinoma, ductal type, pT3N1MX |
1.360 |
30.00 |
40.80 |
YES |
PC-P-3
|
Adenocarcinoma, ductal type, moderately to poorly differentiated, involving head and body of pancreas and invading into duodenal submucosa and peripancreatic soft tissue, c/w pT3N1MX |
0.340 |
30.00 |
10.20 |
YES |
PC-P-4
|
Poorly differentiated pancreatic ductal adenocarcinoma, invading into duodenum, consistent with pT3N0MX |
0.813 |
30.00 |
24.39 |
YES |
PC-P-5
|
Invasive pancreatic duct adenocarcinoma, moderately to poorly differentiated, Stage: ypT3N1MX, Stage IIB |
0.767 |
30.00 |
23.01 |
YES |
PC-P-6
|
Invasice ductal adenocarcinoma of the pancreas, moderately to poorly differentiated, T3N1MX, stage IIB |
0.644 |
30.00 |
19.32 |
YES |
PC-P-7
|
Metastatic pancreatic adenocarcinoma |
0.524 |
30.00 |
15.72 |
YES |
PC-P-8
|
Metastatic adenocarcinoma |
0.238 |
30.00 |
7.14 |
YES |
PC-P-9
|
Adenocarcinoma, moderately to poorly differentiated, c/w pT3N0MX |
0.370 |
30.00 |
11.10 |
|
PC-P-10
|
Colon (UCSD-006)
Diagnosis
|
Conc. (ng/uL)
|
Volume (ul)
|
Yields (ng)
|
Selected for WGBS
|
Zhang lab ID
|
Poorly differentiated adenocarcinoma, consistent with recurrent colonic adenocarcinoma |
0.200 |
30.00 |
6.00 |
YES |
6-P-1
|
Metastatic malignant hemangiopericytoma/ malignant solitary fibrous tumor |
1.100 |
30.00 |
33.00 |
YES |
6-P-2
|
Sigmoid colon, rectum, and anus, resection. Recurrent invasive moderately- to poorly- differentiated |
1.570 |
30.00 |
47.10 |
YES |
6-P-3
|
Metastatic adenocarcinoma. |
0.889 |
30.00 |
26.67 |
YES |
6-P-4
|
Metastatic mucinous adenocarcinoma. |
0.487 |
30.00 |
14.61 |
YES |
6-P-5
|
Metastatic moderately-differentiated adenocarcinoma with mucinous features. |
0.249 |
30.00 |
7.47 |
YES |
6-P-6
|
Adenocarcinoma |
0.247 |
30.00 |
7.41 |
|
6-P-7
|
Metastatic moderately-differentiated adenocarcinoma with municous features |
0.226 |
30.00 |
6.78 |
YES |
6-P-8
|
Adenocarcinoma with municous features, moderately differentiated, pT4aN2b |
0.408 |
30.00 |
12.24 |
YES |
6-P-9
|
Mucinous adenocarcinoma, moderately differentiated |
0.333 |
30.00 |
9.99 |
|
6-P-10
|
Lung (UCSD-007)
Diagnosis
|
Conc. (ng/uL)
|
Volume (ul)
|
Yields (ng)
|
Selected for WGBS
|
Zhang lab ID
|
Invasive moderately differentiated adenocarcinoma, pT26N0 |
0.692 |
30.00 |
20.76 |
YES |
7-P-1
|
Poorly differentiated squamous cell carcinoma. Consistent with pT2N0MX, stage IB |
1.140 |
30.00 |
34.20 |
YES |
7-P-2
|
Metastatic melanoma |
0.208 |
30.00 |
6.24 |
YES |
7-P-3
|
Squamous cell carcinoma, clear cell variant, moderately differentiated |
0.202 |
30.00 |
6.06 |
|
7-P-4
|
Squamous cell carcinoma, pT1bN0 |
1.110 |
30.00 |
33.30 |
YES |
7-P-5
|
Adenocarcinoma, mix subtype, including 60% bronchioalveolar carcinoma, mucinous subtype with 40% invasive carcinoma, acinar and papillary subtypes |
0.233 |
30.00 |
6.99 |
YES |
7-P-6
|
Invasive squamous carcinoma, moderately differentiated, consistent with pT1N0MX |
0.229 |
30.00 |
6.87 |
YES |
7-P-7
|
Invasive adenocarcinoma, poorly differentiated |
0.167 |
30.00 |
5.01 |
YES |
7-P-8
|
Non-small-cell carcinoma |
0.231 |
30.00 |
6.93 |
|
7-P-9
|
Poorly differentiated carcinoma, possibly adenoSCC |
0.203 |
30.00 |
6.09 |
YES |
7-P-10
|
1) End-repair/dA-tailing
- Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG (final conc. 1:0.1:0.1mM) in total volume 20ul
Pancreas
Zhang lab ID
|
Conc. (ng/ul)
|
Volume for 1ng
|
H2O
|
Klenow,exo-:dA:dC:dG Mix
|
Total
|
# in strip
|
Exp ID
|
PC-P-2 |
0.852 |
1.17 |
8.83 |
10.00 |
20.00 |
#4_1 |
wPC-P-2
|
PC-P-3 |
1.360 |
0.74 |
9.26 |
10.00 |
20.00 |
#4_2 |
wPC-P-3
|
PC-P-4 |
0.340 |
2.94 |
7.06 |
10.00 |
20.00 |
#4_3 |
wPC-P-4
|
PC-P-5 |
0.813 |
1.23 |
8.77 |
10.00 |
20.00 |
#4_4 |
wPC-P-5
|
PC-P-6 |
0.767 |
1.30 |
8.70 |
10.00 |
20.00 |
#4_5 |
wPC-P-6
|
PC-P-7 |
0.644 |
1.55 |
8.45 |
10.00 |
20.00 |
#4_6 |
wPC-P-7
|
PC-P-8 |
0.524 |
1.91 |
8.09 |
10.00 |
20.00 |
#4_7 |
wPC-P-8
|
PC-P-9 |
0.238 |
4.20 |
5.80 |
10.00 |
20.00 |
#4_8 |
wPC-P-9
|
Colon
Zhang lab ID
|
Conc. (ng/ul)
|
Volume for 1ng
|
H2O
|
Klenow,exo-:dA:dC:dG Mix
|
Total
|
# in strip
|
Exp ID
|
6-P-1 |
0.200 |
5.00 |
5.00 |
10.00 |
20.00 |
#6_1 |
w6-P-1
|
6-P-2 |
1.100 |
0.91 |
9.09 |
10.00 |
20.00 |
#6_2 |
w6-P-2
|
6-P-3 |
1.570 |
0.64 |
9.36 |
10.00 |
20.00 |
#6_3 |
w6-P-3
|
6-P-4 |
0.889 |
1.12 |
8.88 |
10.00 |
20.00 |
#6_4 |
w6-P-4
|
6-P-5 |
0.487 |
2.05 |
7.95 |
10.00 |
20.00 |
#6_5 |
w6-P-5
|
6-P-6 |
0.249 |
4.02 |
5.98 |
10.00 |
20.00 |
#6_6 |
w6-P-6
|
6-P-8 |
0.226 |
4.42 |
5.58 |
10.00 |
20.00 |
#6_7 |
w6-P-8
|
6-P-9 |
0.408 |
2.45 |
7.55 |
10.00 |
20.00 |
#6_8 |
w6-P-9
|
Lung
Zhang lab ID
|
Conc. (ng/ul)
|
Volume for 1ng
|
H2O
|
Klenow,exo-:dA:dC:dG Mix
|
Total
|
# in strip
|
Exp ID
|
7-P-1 |
0.692 |
1.45 |
8.55 |
10.00 |
20.00 |
#7_1 |
w7-P-1
|
7-P-2 |
1.140 |
0.88 |
9.12 |
10.00 |
20.00 |
#7_2 |
w7-P-2
|
7-P-3 |
0.208 |
4.81 |
5.19 |
10.00 |
20.00 |
#7_3 |
w7-P-3
|
7-P-5 |
1.110 |
0.90 |
9.10 |
10.00 |
20.00 |
#7_4 |
w7-P-5
|
7-P-6 |
0.233 |
4.29 |
5.71 |
10.00 |
20.00 |
#7_5 |
w7-P-6
|
7-P-7 |
0.229 |
4.37 |
5.63 |
10.00 |
20.00 |
#7_6 |
w7-P-7
|
7-P-8 |
0.167 |
5.99 |
4.01 |
10.00 |
20.00 |
#7_7 |
w7-P-8
|
7-P-10 |
0.203 |
4.93 |
5.07 |
10.00 |
20.00 |
#7_8 |
w7-P-10
|
NTC_1 |
0.000 |
0.00 |
10.00 |
10.00 |
20.00 |
NA |
ntc1
|
NTC_2 |
0.000 |
0.00 |
10.00 |
10.00 |
20.00 |
NA |
ntc2
|
Klenow exo- and dA:dG:dC reaction mix
Components
|
1x rxn
|
28x rxn
|
10X Tango buffer |
2.00 |
56.00
|
dA:dC:dG (20:2:2mM) |
1.00 |
28.00
|
Klenow fragment, exo- (5U/ul) |
1.00 |
28.00
|
H2O |
6.00 |
168.00
|
Total |
10.00 |
|
-Aliquot 32.50 of Klwnow, exo- and dA:dC:dG reaction mix to 8-tube strip
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 10ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA with multichannel pipette. Pipette few times
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- - 75C, 10min --> hold at 4C (heat lid on)
- No AMPure bead purification
2) Methylated adaptor ligation
- Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul
Ligation reaction mix
Components
|
1x rxn
|
28x rxn
|
dA-tailed DNA |
20.00 |
0.00
|
10X Tango buffer |
0.50 |
14.00
|
HC T4 DNA ligase (30units/ul) |
1.00 |
28.00
|
10mM ATP |
1.25 |
35.00
|
H2O |
1.25 |
35.00
|
Total |
24.00 |
|
- - Aliquot 13ul of ligation reaction mix to 8-tube strip
- - Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- - Add 4ul of ligation reaction mix with multichannel pipette
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 20h (no heat lid)
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- - Continue to bisulfite conversion