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What is pore occupancy and how to improve it?
What is pore occupancy and how to improve it?
Updated over a week ago

Pore occupancy is defined as the percentage of the sequencing channels = channels in sequencing/ (channels in sequencing + channels in pore) * 100.

During the sequencing run, you can observe the pore occupancy by reviewing the flow cell health diagram, Channels Panel, or Duty Time Plot in the MinKNOW GUI.

A good pore occupancy indicates that there is a good amount of "threadable" DNA or RNA (DNA or RNA +adapters) on the flow cell.

If you are observing a pore occupancy significantly below the 70% threshold within the first hour of the run, it is unlikely to improve over time.

For best results please stop the run and prepare more library. Ensure that quantification is accurate by using a fluorometric method, such as Qubit, and that the recommended fmol amount is loaded onto the flow cell.

A less than optimal pore occupancy will lead to a higher than expected number of pores in the open state, which results in an increased rate of electrolyte utilization and rapid depletion in the number of “good” pores. This will negatively impact the total output of the run and the longevity of the flow cell.

Additional information and tips can be found in the MinKNOW protocol.

Note: If you are performing Cas9 targeted enrichment sequencing then the average pore occupancy will be between 5-15%.

A low pore occupancy can be caused by (but not limited to):

1. Incorrect conversion between mass and moles. For example, for MinION libraries, the sequencing adapter ligation step is optimized for 0.2 pmol of end-prep DNA. This convertor can be used for the calculation.

2. Sub-optimal estimation of input mass and fragment length can affect the final adapter ligation step and therefore the recovery.

3. Integrity of reagents used.

4. Quality of starting material.

5. Contaminant carryover (including non-compatible buffers).

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