Yes, Pairs are always better to measure start points and distinct molecules but with single ended reads we just assume if any single replicated start point is identical it was PCR induced. This is conservative and we only count unique placements in the genome..yet again an underestimate. The above example was 5 cycles of PCR with long extensions.
We have also performed no amplification libraries which eliminate the PCR replication problem. Taqman is another angle we use to measure at the linker step and I'll have to check our notes but I believe we are getting 300M-500M positive beads with 1ng of 200bp library. ABout 15% of our total beads (3B total) amplify which suggest few have 2 molecules in the bubble based on poisson. Not far off from your estimate of 5M molecules per 1pg of 200bp library or 5B per ng.
In our case the DNA was not size selected so we cant blame the gel extraction or Ampure.
We spent some time looking into peroxide formation during the shearing step and monitoring heat. Curious if any one has used the NEB preCR kit or their Fpg and Hogg1 repair enzymes to repair other forms of DNA damage like 8-oxoG or glycosic bond breaks which may be induced by this method.
Alternatively, DNAse based forms of digesting DNA may be less caustic?
Despite leaning high on beads in the emPCR this currently cant get a bead in every reactor without alot of clumping. Probably only 20-25% of the reactors populated. Raindance like techniques have been contemplated but would take days to make Billions of reactors.
We have also performed no amplification libraries which eliminate the PCR replication problem. Taqman is another angle we use to measure at the linker step and I'll have to check our notes but I believe we are getting 300M-500M positive beads with 1ng of 200bp library. ABout 15% of our total beads (3B total) amplify which suggest few have 2 molecules in the bubble based on poisson. Not far off from your estimate of 5M molecules per 1pg of 200bp library or 5B per ng.
In our case the DNA was not size selected so we cant blame the gel extraction or Ampure.
We spent some time looking into peroxide formation during the shearing step and monitoring heat. Curious if any one has used the NEB preCR kit or their Fpg and Hogg1 repair enzymes to repair other forms of DNA damage like 8-oxoG or glycosic bond breaks which may be induced by this method.
Alternatively, DNAse based forms of digesting DNA may be less caustic?
Despite leaning high on beads in the emPCR this currently cant get a bead in every reactor without alot of clumping. Probably only 20-25% of the reactors populated. Raindance like techniques have been contemplated but would take days to make Billions of reactors.
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