Noi/NOTES/2011-11-15: Difference between revisions
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* Continued from library preparation on 2011/09/18: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-9-18#PCR_Amplification_with_barcoded_primers_.28AmpF6.4Sol_and_AmpR6.3_IndX_No.1-46.29]] and library normalization on 2011/11/17: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-10-17]] | * Continued from library preparation on 2011/09/18: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-9-18#PCR_Amplification_with_barcoded_primers_.28AmpF6.4Sol_and_AmpR6.3_IndX_No.1-46.29]] and library normalization on 2011/11/17: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-10-17]] | ||
*Note: The rest of libraries were normalized and pooled for sequencing (some of them were repeated on 2011/10/17) | *Note: The rest of libraries were normalized and pooled for sequencing (some of them were repeated on 2011/10/17) | ||
* Libraries from two families from HAPMAP plate, the 9 replicates for probe normalization and PGP1iPS treated with different bisulfite conversion kits (Zymoresearch and Imprint from Sigma)<br> | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''# of reads''' | |||
| align="center" style="background:#f0f0f0;"|'''Indx''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Well ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Relative ratio to 4 million reads''' | |||
| align="center" style="background:#f0f0f0;"|'''original volume for 60ng''' | |||
| align="center" style="background:#f0f0f0;"|'''calculated new volume''' | |||
| align="center" style="background:#f0f0f0;"|'''Well ID''' | |||
|- | |||
| 3,457,940||s_7_1_Indx10||GM11995 (wellID G7)||B3||0.864||10.80||12.49||B3 | |||
|- | |||
| 4,062,805||s_7_1_Indx11||GM11993 (wellID F2)||C3||1.016||11.09||10.92||C3 | |||
|- | |||
| 3,733,084||s_7_1_Indx12||**GM10861 (wellID A9)||D3||0.933||11.46||12.27||D3 | |||
|- | |||
| 3,663,235||s_7_1_Indx13||GM11992 (wellID C2 )||E3||0.916||10.32||11.27||E3 | |||
|- | |||
| 2,928,739||s_7_1_Indx14||GM11994 (wellID G9 )||F3||0.732||22.00||30.05||F3 | |||
|- | |||
| 7,213,122||s_7_1_Indx15||GM10860 (wellID F10)||G3||1.803||10.99||6.09||G3 | |||
|- | |||
| 2,853,891||s_7_1_Indx16||GM12813 (wellID E8)||H3||0.713||6.98||9.79||H3 | |||
|- | |||
| 3,678,983||s_7_1_Indx17||GM12802 (wellID E7)||A5||0.920||7.88||8.56||A5 | |||
|- | |||
| 3,067,241||s_7_1_Indx18||GM12815 (wellID G12)||B5||0.767||7.48||9.75||B5 | |||
|- | |||
| 3,763,585||s_7_1_Indx19||GM12812 (wellID F11)||C5||0.941||10.20||10.84||C5 | |||
|- | |||
| 3,246,362||s_7_1_Indx20||GM12814 (wellID A7)||D5||0.812||6.53||8.05||D5 | |||
|- | |||
| 3,833,426||s_7_1_Indx21||GM12801 (wellID D9)||E5||0.958||7.08||7.39||E5 | |||
|- | |||
| 3,177,943||s_7_1_Indx22||NA12156 (wellID B3)-1||F5||0.794||7.43||9.35||F5 | |||
|- | |||
| 3,349,096||s_7_1_Indx23||NA12156 (wellID B3)-2||G5||0.837||6.45||7.70||G5 | |||
|- | |||
| 3,604,801||s_7_1_Indx24||NA12156 (wellID B3)-3||H5||0.901||6.49||7.21||H5 | |||
|- | |||
| 4,379,684||s_7_1_Indx25||NA12156 (wellID B3)-4||A7||1.095||8.51||7.77||A7 | |||
|- | |||
| 3,557,582||s_7_1_Indx26||NA12156 (wellID B3)-5||B7||0.889||10.12||11.38||B7 | |||
|- | |||
| 4,331,433||s_7_1_Indx27||NA12156 (wellID B3)-6||C7||1.083||7.49||6.92||C7 | |||
|- | |||
| 2,945,843||s_7_1_Indx28||NA12156 (wellID B3)-7||D7||0.736||7.62||10.34||D7 | |||
|- | |||
| 3,444,795||s_7_1_Indx29||NA12156 (wellID B3)-8||E7||0.861||9.90||11.50||E7 | |||
|- | |||
| 3,429,469||s_7_1_Indx30||NA12156 (wellID B3)-9||F7||0.857||7.39||8.62||F7 | |||
|- | |||
| 6,808,403||s_7_1_Indx41||**PGP1iPS||A11||1.702||14.16||8.32||A11 | |||
|- | |||
| 5,281,331||s_7_1_Indx42||PGP1iPS-IMPRINT||B11||1.320||17.42||13.19||B11 | |||
|- | |||
| 6,256,272||s_7_1_Indx94||UCLA-RO_Indx94||in tube||1.564||4.06||2.60||tube | |||
|}<br> | |||
* Perform PAGE size selection in 5-well 6% TBE gel (2 5-well gels) | |||
[[File:ZhangLab_2 2011-11-15 13hr 30min_PAGE-SS_NP_BSPP-HAPMAP-norm.jpg| 400px]] | |||
* Note: The gel had curve while running, so I cut the band following the curve. | |||
* After gel purification resuspend the libraries in total volume 30ul. | |||
[[File:ZhangLab_2 2011-11-16 12hr 36min.jpg| 300px]] | |||
* 1ul of sequencing libraries were analyzed by PAGE quantification, conc.: ng/ul or nM | |||
* Concentration was also verified by Qubit ds-DNA HS, conc.: ng/ul or nM | |||
* Library ID: NP-BSPP_HAPMAP_Nov15,2011, HL110 run --> PE 100 by GAIIx flowcell | |||
* Note: HL110 run in Zhang's lab failed (in lane 1 & 5), these libraries were sequenced again HL111 run (PE 110) by HiSeq. | |||
* Mapping: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-12-25]] |
Latest revision as of 22:31, 25 December 2011
Normalization of libraries from HL104 run (Library ID: NP-BSPP-Ind1_45-Sep18) to repeat sequencing by GAIIx flowcell[edit]
- Continued from library preparation on 2011/09/18: [[1]] and library normalization on 2011/11/17: [[2]]
- Note: The rest of libraries were normalized and pooled for sequencing (some of them were repeated on 2011/10/17)
- Libraries from two families from HAPMAP plate, the 9 replicates for probe normalization and PGP1iPS treated with different bisulfite conversion kits (Zymoresearch and Imprint from Sigma)
# of reads | Indx | Sample ID | Well ID | Relative ratio to 4 million reads | original volume for 60ng | calculated new volume | Well ID |
3,457,940 | s_7_1_Indx10 | GM11995 (wellID G7) | B3 | 0.864 | 10.80 | 12.49 | B3 |
4,062,805 | s_7_1_Indx11 | GM11993 (wellID F2) | C3 | 1.016 | 11.09 | 10.92 | C3 |
3,733,084 | s_7_1_Indx12 | **GM10861 (wellID A9) | D3 | 0.933 | 11.46 | 12.27 | D3 |
3,663,235 | s_7_1_Indx13 | GM11992 (wellID C2 ) | E3 | 0.916 | 10.32 | 11.27 | E3 |
2,928,739 | s_7_1_Indx14 | GM11994 (wellID G9 ) | F3 | 0.732 | 22.00 | 30.05 | F3 |
7,213,122 | s_7_1_Indx15 | GM10860 (wellID F10) | G3 | 1.803 | 10.99 | 6.09 | G3 |
2,853,891 | s_7_1_Indx16 | GM12813 (wellID E8) | H3 | 0.713 | 6.98 | 9.79 | H3 |
3,678,983 | s_7_1_Indx17 | GM12802 (wellID E7) | A5 | 0.920 | 7.88 | 8.56 | A5 |
3,067,241 | s_7_1_Indx18 | GM12815 (wellID G12) | B5 | 0.767 | 7.48 | 9.75 | B5 |
3,763,585 | s_7_1_Indx19 | GM12812 (wellID F11) | C5 | 0.941 | 10.20 | 10.84 | C5 |
3,246,362 | s_7_1_Indx20 | GM12814 (wellID A7) | D5 | 0.812 | 6.53 | 8.05 | D5 |
3,833,426 | s_7_1_Indx21 | GM12801 (wellID D9) | E5 | 0.958 | 7.08 | 7.39 | E5 |
3,177,943 | s_7_1_Indx22 | NA12156 (wellID B3)-1 | F5 | 0.794 | 7.43 | 9.35 | F5 |
3,349,096 | s_7_1_Indx23 | NA12156 (wellID B3)-2 | G5 | 0.837 | 6.45 | 7.70 | G5 |
3,604,801 | s_7_1_Indx24 | NA12156 (wellID B3)-3 | H5 | 0.901 | 6.49 | 7.21 | H5 |
4,379,684 | s_7_1_Indx25 | NA12156 (wellID B3)-4 | A7 | 1.095 | 8.51 | 7.77 | A7 |
3,557,582 | s_7_1_Indx26 | NA12156 (wellID B3)-5 | B7 | 0.889 | 10.12 | 11.38 | B7 |
4,331,433 | s_7_1_Indx27 | NA12156 (wellID B3)-6 | C7 | 1.083 | 7.49 | 6.92 | C7 |
2,945,843 | s_7_1_Indx28 | NA12156 (wellID B3)-7 | D7 | 0.736 | 7.62 | 10.34 | D7 |
3,444,795 | s_7_1_Indx29 | NA12156 (wellID B3)-8 | E7 | 0.861 | 9.90 | 11.50 | E7 |
3,429,469 | s_7_1_Indx30 | NA12156 (wellID B3)-9 | F7 | 0.857 | 7.39 | 8.62 | F7 |
6,808,403 | s_7_1_Indx41 | **PGP1iPS | A11 | 1.702 | 14.16 | 8.32 | A11 |
5,281,331 | s_7_1_Indx42 | PGP1iPS-IMPRINT | B11 | 1.320 | 17.42 | 13.19 | B11 |
6,256,272 | s_7_1_Indx94 | UCLA-RO_Indx94 | in tube | 1.564 | 4.06 | 2.60 | tube |
- Perform PAGE size selection in 5-well 6% TBE gel (2 5-well gels)
File:ZhangLab 2 2011-11-15 13hr 30min PAGE-SS NP BSPP-HAPMAP-norm.jpg
- Note: The gel had curve while running, so I cut the band following the curve.
- After gel purification resuspend the libraries in total volume 30ul.
File:ZhangLab 2 2011-11-16 12hr 36min.jpg
- 1ul of sequencing libraries were analyzed by PAGE quantification, conc.: ng/ul or nM
- Concentration was also verified by Qubit ds-DNA HS, conc.: ng/ul or nM
- Library ID: NP-BSPP_HAPMAP_Nov15,2011, HL110 run --> PE 100 by GAIIx flowcell
- Note: HL110 run in Zhang's lab failed (in lane 1 & 5), these libraries were sequenced again HL111 run (PE 110) by HiSeq.
- Mapping: [[3]]