Noi/NOTES/2011-10-17

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Normalization of libraries from HL104 run (Library ID: NP-BSPP-Ind1_45-Sep18) to repeat sequencing by GAIIx flowcell[edit]

  • Continued from the library preparation on 2011/09/18: [[1]]
  • Since the HiSeq at Cancer center (HL104) failed for the second run, and we need to get the data from this set very urgen, Dr. Zhang suggested to do sequencing in our lab by GAIIx. The GAIIx is 1/6 of a Hiseq, so we need to prioritize the libraries to be sequenced and normalized based on the number of reads we got form the first read of HL104.
  • The prioritized libraries
    • Nine libraries from Sergio
    • Penn libraries
    • Two of HFF libraries
    • Three of Hues libraries
  • Note: Some libraries from Penn have very poor quality and amplification, so I don't have more sample for KEBR028A (B4-plate) and TZHZ018 (F1-plate). For KEBR040 (D4-plate), I have some left, but the total amount will be less than other libraries have been normalized.
# of reads Indx Sample ID Well ID Relative ratio to 4 million reads original volume for 60ng calculated new volume Well ID
2,995,193 s_7_1_Indx1 1. H9 A1 0.75 14.35 9.58 A1
9,894,673 s_7_1_Indx2 **2. H9+Activin 100ng/mL 5days B1 2.47 50.82 10.27 B1
7,258,959 s_7_1_Indx3 3. H9+BMP4 50ng/mL 5days C1 1.81 30.16 8.31 C1
7,027,403 s_7_1_Indx4 4. HUViPS4F1 D1 1.76 33.61 9.57 D1
3,708,801 s_7_1_Indx5 5. HUViPS4F1+Activin 100ng/mL 5days E1 0.93 22.28 12.02 E1
5,662,790 s_7_1_Indx6 6. HUViPS4F1+BMP4 50ng/mL 5days F1 1.42 11.63 4.11 F1
6,962,223 s_7_1_Indx7 7. HUViPS4F3 G1 1.74 32.74 9.40 G1
6,062,184 s_7_1_Indx8 8. HUViPS4F3+Activin 100ng/mL 5days H1 1.52 10.85 3.58 H1
4,602,067 s_7_1_Indx9 9. HUViPS4F3+BMP4 50ng/mL 5days A3 1.15 20.60 8.95 A3
3,006,781 s_7_1_Indx32 **2HFF P6 H7 0.75 8.54 5.68 H7
4,612,655 s_7_1_Indx33 **218 HFF P7 A9 1.15 15.33 6.65 A9
956,419 s_7_1_Indx34 KEBR028A (B4-plate) B9 0.24 NA NA B9
2,828,802 s_7_1_Indx36 KEBR040 (D4-plate) D9 0.71 26.40 18.67 D9
164,146 s_7_1_Indx37 TZHZ018 (F1-plate) E9 0.04 NA NA E9
3,210,402 s_7_1_Indx38 TZHZ031A (tube) F9 0.80 9.45 5.89 F9
3,996,257 s_7_1_Indx39 TZHZ041A (tube) G9 1.00 9.44 4.72 G9
3,205,833 s_7_1_Indx40 TZHZ075 (H01-plate) H9 0.80 10.59 6.61 H9
5,605,295 s_7_1_Indx43 **Hues1 C11 1.40 20.71 7.39 C11
5,028,360 s_7_1_Indx44 **Hues3 D11 1.26 18.50 7.36 D11
4,495,384 s_7_1_Indx45 **Hues8 E11 1.12 15.01 6.68 E11


  • Note: I don't have enough Inx2 sample, so I do the PCR to amplify and add to the pool.
  • Perform PAGE size selection in 5-well 6% TBE gel
  • Total volume = 145.5ul, add 30ul of 6x loading dye. Loaded 22ul/well (2 gels)

File:ZhangLab 2 2011-10-17 19hr 18min PAGE-SS normBSPPlib.jpg

  • After gel purification resuspend the libraries in to tal volume 24ul.

File:ZhangLab 2 2011-10-18 12hr 00min PQ-BSPP-norm.jpg

  • 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 6.79 ng/ul or 29.7nM
  • Concentration was also verified by Qubit ds-DNA HS, conc.: ng/ul or nM
  • Library ID: NP-BSPP_Sep18-norm, HL106 run --> PE 110 by GAIIx flowcell
  • The rest libraries were normalized, pooled and sequenced on 2011/11/16: [[2]]