ZWMS = 2, 1 and 0

[davisc]

After a run, you get a quality report of the loading of a particular cell, that is broken down into: 2, 1 and 0.

My question pertains to what constitutes a 2?

My understanding of the SMARTbell adapts and ligation biochemistry is such that every closed ss-circle will be flanked by two SMARTbells with the same adapter = that there are 2 priming sites per closed circle.

How is priming and amplification from BOTH priming sites prevented?

Does a '2' correspond to two separate circles loading into the ZWM (overloading) or two different sequences arising from a ZMW, possibly from independent priming off the redundant sites in the same circle (concentration of primer may be off)?

Thanks.

[rhall]

A loading value of 2 can indicate a number of issues, but the simplest most likely explanation is multiple polymerase loaded in a single ZMW, each bound to a separate SMRT bell. There is nothing stopping multiple ploymerases binding in a ZMW, hence the distribution of loading is a Poisson distribution.
There are 2 priming sites per SMRT bell, but it would be very unlikely energetically for 2 polymerases bound to the same piece of DNA to bind in the same ZMW, different ZMWs are too distant.

[davisc]

Is there any evidence to suggest that having 2 binding sites per amplicon/circle is either beneficial or detrimental to the sequencing reaction, even if the second polymerase is not located in the active ZMW but hanging out?