Noi/NOTES/2014-12-16

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LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient

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Continued from 2014-12-05

  • Conditions:
    • Probe set: MONOD V5 set A (LMS)
    • Probe:target ratio: 150:1 for LMS probe set.
      • Please note that I used very high amount of WGBS libraries for BSPP capture up to 600ng. This resulted in a very high amount of BSPP required for capture. I therefor reduced probe to target ratio from 200:1 to 125:1. The main reason is I do not have enough LMS BSPP to capture 50 samples with 600ng DNA each. For experiment I did for Illumina (Li Liu), I used 400ng of WGBS libraries, so I had enough probes to capture that amount. In order to be able to capture all unique molecule of ctDNA, I tried to increase WGBS libraries as much as possible. Ideally, Dr. Zhang wanted 1ug for capture.
    • Sample:
      • WGBS libraries of ctDNA from cancer patients prepared by KAPA Hyper Prep Kit 600ng.
      • WGBS libraries of cfDNA from normal control (re-amplified) prepare by using RRBS protocol but skipping DNA digestion/fragmentation 600ng

Probe calculation

Probe set 	        Probe size	Probe:target ratio
LMS                     73,574	              150:1
From 2014-12-08 exp., 400ng required 108ng (200:1). 
So 600ng (150:1) requires  ~100ng of LMS probes
  • Since the probe concentration a little low (8.25ng/ul), I will set up capture reaction in 300ul.

NOTE: WHAT ARE DIFFERENT FROM EXP. on 2014-12-08 (ILLUMINA, for Li Liu)

  • Template 600ng instead of 400ng
  • Probe: target ratio is 125:1 instead of 200:1
  • Capture reaction 30ul instead of 25ul

Precedures

  • I took 600ng of WGBS libraries in 1.5mL LoBind tube. Rationale: total volume of 600ng of WGBS libraries is too large to fit in 30ul capture reaction.
  • Speedvac at 60C for ~20-30min to dry up DNA in the tube.
  • Resuspend each tube with 14.88ul H2O which is the maximum volume of WGBS libraries to fit in 30ul capture reaction.
    • Make sure that we resuspend DNA on the bottom of the tube very well.
  • Transfer 14.88ul of DNA to 95-well plate
    • I re-organized the location of each sample on the well based on sample ID