AlanFung:LabNotes/2015/2015-1-15

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Revision as of 20:53, 16 January 2015 by >Alan6017518 (→‎Library Concentration)
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SeqCap Epi probe pool (UMR v1) hybridization capture on normal and cancer patient WGBS libraries

SeqCap Epi User Guide

  • Protocol: Dropbox/ZhangLab/Capture/Noi SeqCap Epi probe pool protocol

Background

  • Noi did the capture experiment: Noi/NOTES/2014-12-25.
  • The libraries were sequenced in one HiSeq PE100bp rapid run 141214_SN1001.
  • Dinh mapped the reads: Dinh/Dinh_2015/NOTES/2015-1-7.
  • Dr. Zhang analyzed the Hiseq Rapid run data, mapping rate is good. But specificity and enrichment factors are much lower than the first experiment
  • Kun did the comparison: Kun:LabNotes/MONOD/2015-1-9.
  • Kun did additional comparison for the two sets of data, he is sure the first batch of data has good on-target rates but low library complexity, and the second batch was the opposite.
  • Kun:LabNotes/MONOD/2015-1-9#Further_comparison_of_the_two_batches_of_library_preparation_and_capturing_experiments.
    • Conclusion 1 Plasma DNA needs to be end-polished using the standard library prep protocol such as the one implemented in the Kapa Hyper Prep kit.
    • Conclusion 2 The Tang et al scRRBS protocol is based on some assumptions of the DNA fragment ends, which are not applicable to all plasma DNA fragments.
  • I just need to repeat the capture experiments on those libraries made in the second batch, then we should be fine.
Experiment End Repair Capture Hiseq Run Specificity Enrichment Factors Complexity Clonal Rates Conclusion
#1 Tang et al RRBS 141104 141112 higher higher lower higher lower complexity and higher clonal rates due to Tang et assumption the DNA fragment ends
#2 Kapa Hyper Prep kit 141225 141216 lower lower higher lower lower specificity and enrichment due to capture condition

Task

Locate remaining WGBS libraries, and the NimbleGen capturing reagents

Noi recently re-amplified WGBS libraries of cancer patients and normal control
The tubes of the most recent batches should be labeled in December 2014
They all should be in PCR rack (3 of 8-tube strip for cancer patient and 3 of 8-tube strip for normal control) with the cover. 
Please do not use the sequencing libraries in the yellow PCR rack as those libraries are sequencing libraries from BSPP capture.
Library location -20C 
WGBS from ctDNA of cancer patients were generated using KAPA Hyper Prep Kit
They were generated on 2014-12-13 and should be located in the PCR rack with cover

Repeat capturing with optimized capturing conditions

Library Concentration

(The tube should be labeled ~2014-12-13) (The tube should be labeled ~2014-12-16)
Sample Conc. (ng/ul) TruSeq Indx Volume for 100ng (ul) Sample Conc. (ng/ul) TruSeq Indx Volume for 100ng (ul)
PCP-9 26.13 8 3.83 NC-1 13.73 1 7.28
PCP-4 21.73 3 4.60 NC-2 16 2 6.25
PCP-8 19.07 7 5.24 NC-3 16.93 4 5.91
PCP-6 20.13 5 4.97 NC-5 13.07 5 7.65
PCP-7 22.8 6 4.39 NC-6 15.33 6 6.52
PCP-2 22.13 1 4.52 NC-7 13.73 7 7.28
PCP-3 21.47 2 4.66 NC-8 15.87 8 6.30
PCP-5 21.47 4 4.66 NC-9 16.53 10 6.05
6P-1 26 9 3.85 NC-12 14.8 13 6.76
6P-8 24.8 14 4.03 NC-13 13.19 14 7.58
6P-10 22.27 16 4.49 NC-14 14.93 15 6.70
6P-9 20.27 15 4.93 NC-15 13.6 16 7.35
6P-5 24.67 13 4.05 NC-16 13.87 27 7.21
6P-2 19.6 10 5.10 NC-17 13.47 18 7.42
6P-3 19.87 11 5.03 NC-18 14.67 19 6.82
6P-4 19.2 12 5.21 NC-19 13.47 20 7.42
7P-8 22.8 25 4.39 NC-20 12.79 11 7.82
7P-3 19.07 20 5.24 NC-21 12.31 12 8.12
7P-10 25.33 27 3.95 NC-22 16 21 6.25
7P-7 29.6 23 3.38 NC-23 13.6 22 7.35
7P-6 26.93 22 3.71 NC-24 11.97 3 8.35
7P-1 14.67 18 6.82 NC-27 14.67 9 6.82
7P-2 19.73 19 5.07 NC-29 12.96 23 7.72
7P-5 18 21 5.56 NC-30 15.47 25 6.46
Total volume: 111.67 Total Volume: 169.41
- Re-purified the two pools with 1X AMOPure bead
- Resuspended each library with 33ul H2O
- Measure conc. with Qubit dsDNA HS assay using 1ul of purified library pool. Total volume left ~32ul
- Normal WGBS library pool: conc. = ng/ul --> total amount in 32ul = ng
- Cancer patient WGBS library pool: conc. = ng/ul --> total amount in 32ul = ng
- The total amount of the two library pools is close to 1ug as recommended by User guide.
- Continue to SeqCap hybridization capture