Hi! I've got two sets of replicate (x3) HiC libraries. I think I've set the min.inward, min.outward and max.frag thresholds for them correctly (or at least for the bulk of the datasets - not sure if I should set the threshold for replicate 3 individually?).
My question is: what is going on with these HiC libraries? Why do these peak heights look so different? Are any of the libraries "better" than the other ones? Are they usable?
Thanks!
OK, I've set the cutoff for outward pairs higher.
The three replicates are paired, in that Sample1"left" and Sample2"left' were generated at the same time, as were Sample1 and 2 "middle", and Sample 1 -2 "right". So that's why I think there could be some "wet-lab" variability (i.e. minor batch effects) between the three samples. The caveat is that I would hope that our signal would be robust enough to be stronger than these technical effects.
With normalisation, apart from the MA-plots, which other visualisations/metrics can I use to assess that the normalisation is occurring correctly?
I guess you could make some distance plots to check that the libraries have similar trends in the count size with respect to increasing distance between interacting loci. This should already be the case if you've normalised based on trends in the average abundance with
normOffsets
, given that distance is a monotonic function of the abundance.