[ningwang]

I've been told that with the Ion Fragment Kit, the maximum input fragment size should be 150 bp so that with adapters the fragment is no longer than 210 bp.

I thought the average read length depended on whether you're using the 314 or the 316 chip.

[ningwang]

Does de-phasing impose any limits on their read lengths?

This is my guess as well. If you look at the supplementary data submitted by Rothberg in his Nature article (from July?), the raw data gets pretty ugly after 60-80bps and requires some heavy phase-correction to clean up.

[ningwang]

I'd have to really think through the dephasing, and probably model it.

Probability that any given molecule will dephase in a given cycle is the 1-repetitive yield (I'll call this Pdephase). If you are going to model this, one option is to assume that dephased reads are lost forever. Alternatively, one can assume they can be rephased under the right circumstances (indeed, Ion claims their flow order encourages recovering dephased molecules).

I'm not convinced that fewer molecules leads to less dephasing until you get to very small numbers. The current beads carry 800K molecules per bead; even with very high repetitive yield you are going to lose some in each round and presumably that is close to a fixed proportion. Clearly, if you get to molecule numbers approach or go below 1/Pdephase then you need to model it as a stochastic process. I'm not sure what Pdephase is, but presumably you could crudely estimate it from the distribution of read lengths.

If you get to really small numbers (but not 1), then a possible issue is that the relative contribution of a dephased molecule in the population becomes large -- so it is rare that a bead has any dephased molecules but a small number of dephased molecules kills your signal-to-noise ratio.

First of all, I don't know if Pdephase is dependent on how many molecules you have per bead. We know that PGM uses 2um diameter beads, which gives you a surface area of 12.5um2. If they're loading 800k molecules per bead, you've got roughly 15-16nm2 per molecule. If that dense enough for one polymerase to impede with another? If so, I suppose having a lower density might reduce dephasing. But if 15-16nm2 per molecule is ample room, having fewer molecules/bead won't change your dephased ratio.

Plus if you go with fewer molecules, at what point are you pushing the limits of the ion sensors at the bottom of the well? Also in the scenario you described (small # of molecules), you might mask dephasing problems but you'll increase your indel error rate.

[krobison]

I thought the average read length depended on whether you're using the 314 or the 316 chip.

This premise of this thread is a bit outdated, as the protocols have made several major changes since I started it.

I believe read length is independent of the chip (to first approximation); it is the newer protocols for preparing the template & better base calling software that are enabling longer reads.