Pore Occupancy: Definition, Improving Pore Occupancy

At a glance:

• What is Pore Occupancy?
• Why is Pore Occupancy Low?
• Strategies to Improve Pore Occupancy

What is Pore Occupancy?

Pore occupancy is a key determinant of sequencing efficiency and directly impacts the speed and success of data acquisition in the Oxford Nanopore Sequencing Technology Platform. Expressed as a percentage, it represents the proportion of active sequencing channels used during a sequencing run.

Technically, pore occupancy is defined as the ratio of lanes in sequencing to total lanes (i.e. lanes in sequencing plus lanes in wells) multiplied by 100. This measurement provides a quantitative view of the amount of "threadable" DNA or RNA, including adapters, that is present on the flow cell.

Why is Pore Occupancy Low?

Although it is important, maintaining optimal pore occupancy can be challenging. If you observe pore occupancy significantly below the 70% threshold within the first hour of a run, it is unlikely that the situation will improve over time. There are several factors that may contribute to suboptimal pore occupancy:

• Inaccurate conversion between mass and moles.
• Underestimation of input mass and fragment length, both of which can affect the final splice joining step and thus recovery.
• The quality and integrity of the starting materials and reagents used also significantly affects pore occupancy.
• Contaminant residues, including those from incompatible buffers, can reduce pore occupancy.
• For some sequencing technologies (e.g., Cas9 Targeted Enrichment Sequencing), the average pore occupancy is inherently low, typically between 5-15%.

Suboptimal pore occupancy can trigger a number of undesirable consequences. The most immediate effects are electrolyte utilization and the number of "good" pores available for sequencing. As pore occupancy decreases, the number of open-state pores increases, leading to accelerated electrolyte utilization. In addition, the number of good pores decreases rapidly, reducing the total output of the run and the lifetime of the flow cell.

Strategies to Improve Pore Occupancy

• Stop the run and prepare more libraries. Ensure that quantitation is accurate by using a fluorometric assay (e.g., Qubit) and loading the recommended amount of fmol onto the flow cell.
• Ensure efficient conversion between mass and moles. For example, the sequencing junction ligation step of the MinION library is optimized for 0.2 pmol end-prep DNA.
• Accurately estimate input mass and fragment length.
• Pay attention to the quality of reagents and starting materials. Good practice includes checking the integrity of reagents and maintaining the quality of starting materials. Another prevention strategy is to minimize contaminant residues. The use of incompatible buffers and other sources of contaminants can reduce pore occupancy.

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