Noi/NOTES/2014-8-3: Difference between revisions
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>Noi mNo edit summary |
>Noi mNo edit summary |
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| Poorly differentiated carcinoma, possibly adenoSCC||0.203||30.00||6.09||YES||7-P-10 | | 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 | |||
====<u>Pancreas</u>==== | |||
{| {{table}} border = 1 | |||
| align="center" style="width:120px;background:#f0f0f0;"|'''Zhang lab ID''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Klenow,exo-:dA:dC:dG Mix''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Total''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''# in strip''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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 | |||
|} | |||
====<u>Colon</u>==== | |||
{| {{table}} border = 1 | |||
| align="center" style="width:120px;background:#f0f0f0;"|'''Zhang lab ID''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Klenow,exo-:dA:dC:dG Mix''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Total''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''# in strip''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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 | |||
|} | |||
====<u>Lung</u>==== | |||
{| {{table}} border = 1 | |||
| align="center" style="width:120px;background:#f0f0f0;"|'''Zhang lab ID''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Klenow,exo-:dA:dC:dG Mix''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Total''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''# in strip''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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 | |||
|} | |||
==== <u>Klenow exo- and dA:dG:dC reaction mix</u> ==== | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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<br> | |||
<br> | |||
== 2) Methylated adaptor ligation == | |||
* Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul | |||
==== <u>Ligation reaction mix</u> ==== | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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 |
Revision as of 18:46, 3 August 2014
Preparation of WGBS libraries of small fragment DNA in plasma samples (no DNA fragmentation step)
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