Saturated Pores for Nanopore Operation

Overview

Nanopore sequencing has revolutionized the field of genomics by providing a direct, real-time method for decoding DNA or RNA sequences. A very useful diagnostic of a nanopore run is the state of the pores: whether they are being sequenced ("single pore"), saturated, unavailable, or multiple. Saturated pores pile up and these pores are "clogged" and cannot be sequenced.

Consequences of Saturated Pores in Nanopore Sequencing

During a sequencing run, the presence of inactive channels on the working time graph often indicates nanopore saturation, a problematic state that reflects increasing membrane stress. Saturated pores occur when an excessive accumulation of molecules within the nanopore surpasses its capacity to effectively process them. This overload, known as saturation, significantly hampers the efficiency of the sequencing process by reducing the number of active channels available for DNA or RNA translocation.

### Factors Contributing to Pore Saturation

Several factors can trigger the membrane stresses that lead to pore saturation:

- **Variations in Environmental Conditions**: Changes in the salt concentration or pH levels within the sequencing environment can alter the fluid dynamics of the nanopore. Such fluctuations may disrupt the stability of the nanopore's operation, making it more susceptible to saturation as the ionic balance is disturbed.

- **Presence of Surfactants**: Surfactants, while often necessary for various processing steps, can increase the viscosity of the fluid environment within the nanopore. This increased viscosity can hinder the smooth passage of molecules, contributing to the clogging of pores over time.

- **Contaminants and Osmotic Imbalances**: Contaminants play a critical role in inducing osmotic imbalances. These imbalances can significantly affect the nanopore by disturbing the equilibrium necessary for molecular dynamics. Contaminants can lead to erratic molecular movements, resulting in molecular spillage where molecules prematurely exit the nanopore or face impeded translocation through the nanopore.

Saturated pores not only reduce the throughput of sequencing data but also complicate data analysis by introducing errors and inconsistencies. Therefore, understanding and mitigating these factors are crucial for enhancing the reliability and accuracy of nanopore sequencing. Implementing rigorous quality control and monitoring protocols can help minimize the risk of saturation, ensuring that the sequencing process remains efficient and productive.

Strategies to Mitigate Saturated Pores

Addressing pore saturation requires a multifaceted approach. An alternative extraction method that minimizes contaminant residues can be used. In addition, stringent quality control (QC) of the input DNA/RNA according to guidelines can ensure that potential contaminants are minimized, thereby reducing the chance of pore saturation.

It is also critical to monitor pore status throughout the sequencing process. Visualization tools such as multiplexer scan plots can reveal the onset and progression of pore saturation. These metrics can guide necessary adjustments to experimental protocols and ensure the robustness of the sequencing process.

Learn more: whole genome resequencing