The two pieces of software - MinKNOW and EPI2ME - that are used in nanopore sequencing can be used to judge the quality of your experiment.

This section contains guidance for how to read the MinKNOW pore activity plots and EPI2ME reports, including some examples of good and bad sequencing runs, and troubleshooting steps.

Pore activity plots in MinKNOW

It is recommended to observe the pore activity plot populating over the first 30 min-1 hr of the sequencing run. By this time, the channel state distribution will give an indication whether the DNA/RNA library is of a good quality, and whether the flow cell is performing well. Below are some examples of good and bad sequencing runs:

Good quality library

A good quality library will result in most of the pores being in the "Sequencing" state, and very few in "Pore", "Recovering" or "Inactive". A library that looks like this is likely to give a good sequencing throughput.

Channel blocking

Under certain conditions (usually the presence of contaminants in the library), pores may become blocked and therefore unable to sequence. This manifests itself as a build-up of "Recovering" pores over time.

Recommendation: Stop the sequencing run in MinKNOW. Then wash out the library from the flow cell using the instructions for the Flow Cell Wash Kit, which is included in your Starter Pack. Then prepare another library and load it on the flow cell.

Low pore occupancy

If there was insufficient starting material, or some sample has been lost during library prep, or the sequencing adapters did not ligate well to the strand ends, the pore activity plot will show a high ratio of "Pore" to "Sequencing" states, meaning that only a limited number of pores are sequencing at any one time.

Recommendation: Stop the sequencing run in MinKNOW. Then wash out the library from the flow cell using the instructions for the Flow Cell Wash Kit, which is included in your Starter Pack. Then prepare another library and load it on the flow cell.

High number of inactive channels

If the pore activity plot shows a high number of 'Inactive' channels building up over time, this could indicate that the channels or membranes have been damaged by e.g. air bubbles, osmotic imbalance, or the presence of detergents or surfactants in the library.

Recommendation: Check the channel panel: if the Inactive channels are all grouped in one part of the flow cell, this could indicate an air bubble that has been introduced during flow cell flushing or library loading. If the remaining channels are still sequencing, it is possible to carry on with the run. Do not try to move the air bubble, as this can damage even more channels.
If the Inactive channels are distributed throughout the flow cell:
Make a new batch of flow cell priming buffer. Flush the flow cell with the mixture, and load the library again.

EPI2ME report

The report shows a selection of metrics, which show whether your library preparation and sequence run has been conducted successfully. Your report should look similar to the example below.

Understanding the run report

Reads processed

This shows the percentage of currently available reads which have completed base calling. The figure below is the number of reads so far - this will increase as more reads are produced.

Coverage

This plot shows your reads mapped against the lambda genome (48,000 bases). Each read is shown in its correct position, where that individual sequence is found in the reference, giving the depth of coverage across the genome.

Accuracy

This histogram shows the accuracy of the reads, when each read is compared individually to the lambda reference. This is performed on a single strand basis, so these figures are for single molecule accuracy, though a consensus may be built from all the strands if desired.