Experiment screen
At any time, the quick view shows how many reads you have obtained so far. After half an hour, you should already have obtained several thousand reads.
You can also monitor how the basecaller has progressed through the reads so far. Depending on sequencing system performance and computer specifications, there may be some lag between reads becoming available and being processed. This is indicated by the "Basecall statistics: Called x%". A small proportion of reads will fail - these are usually false positives and are discounted automatically.
This panel shows the current state of all the channels, arranged as they are located on the flow cells sensor array. Remember that the chip has 2048 wells in total, but a group of 512 channels are accessed at any one time. Each channel is classified by its current behaviour - whether it is currently sequencing, waiting for a strand, recovering, or unavailable.
The number of channels in each category depends on two factors:
- the performance of the sensor array (which is why we recommend a flow cell check)
- the performance of the sequencing sample
You should have a large number of channels in the 'Pore' (dark green) and 'Sequencing' (bright green) categories.
The majority of channels should be active, and therefore display as bright green (pore is sequencing) or dark green (pore is open and waiting for a DNA or RNA strand). If the sample added is of good quality, within a few minutes of the run starting you should see the channels going bright green as the pores capture the strands. If there is a problem with the sample preparation and there is little DNA/RNA available, they will stay dark green (empty pore).
You will see the channels flickering as they capture a strand (turn to bright green) and briefly return to the empty state (darker green) before the next strand. You may also notice some pores occasionally become Recovering (dark blue) - this is normal behaviour, and the software will automatically reactivate them when possible.
If you have a large group of channels clustered together which are light blue ('Inactive'), this may indicate an air bubble on the sensor array. In this case, these channels are permanently damaged, and it it is best not to attempt to remove the bubble as this may damage other channels as well.
The Translocation speed and Qscore graph allows you to monitor the performance of your flow cell. The speed and quality score values should stay within the target range (in green).
The cumulative output graph shows:
- the number of bases that have been sequenced and basecalled
- the number of reads that have been sequenced and basecalled; and whether the reads have passed of failed the quality filters
Temperature vs time graph
The temperature graph gives a real-time representation of the temperature below the flow cell. If the temperature reading drifts out of the target zone, please consult Technical Services, otherwise the quality of your data may be compromised.
Bias voltage v time graph
The bias voltage graph provides the running voltage in real-time. You will notice drops in the voltage at regular intervals and these will correspond to the channel scans that are defaulted to occur every one and a half hours. Here, each channel will be scanned to look for its availability for sequencing. The common voltage is reversed before and after each channel assessment for clearer results.
You can toggle between the graphs above in the MinKNOW GUI.
The trace viewer is an advanced option allowing the user to examine a pore and its sequencing at a fundamental level. You may skip this page if it is not of interest at this point.
However, if you are interested, the trace viewer allows you to observe for yourself the passing of a single DNA or RNA strand through a single nanopore. To view this live, a brief description is below.
Each channel in the flow cell array contains a nanopore which is a single molecule sensor; this plot allows you to look at these sensors in minute detail. On the vertical axis it shows the raw current flowing through each nanopore. As a strand passes through base-by-base, you will see "steps" as that current changes, depending on which bases are inside the pore. This signal known as a "squiggle" is fundamental to nanopore sequencing.
For more detail and a video overview of how nanopore sequencing works, you may wish to visit (https://nanoporetech.com/how-it-works)
The plot will show all channels at once, and when overlaid this can look confusing:
We recommend you choose a bright green channel from the panel on the 'Physical layout' page, hover over it to show its number, then select only that channel in the trace viewer.
Type your selected channel in the 'Selected channels' field. This will show just that one channel.
Change the 'Time seconds' field to '2'. This will effectively "zoom in" on the timescale, so 2 seconds of data are displayed, which will show the squiggle more clearly. The plot below is an example, in this case showing 2 seconds of channel 80.
If that channel is showing a DNA/RNA strand in the pore, it will look like the trace below. Each "step" in the current occurs as different bases move through the pore - hundreds of bases pass through the pore every second.
The pore activity plot summarises the channel states over time.
Each bar shows the sum of all channel activity in a particular amount of time. This time bucket defaults to 1 minute, and scales to 5 minutes automatically after reaching 48 buckets. However, bucket size can be adjusted in the "Bucket size" box to the right of the graph.
The graph populates over time, and can be used as a way to assess the quality of your sequencing experiment, and make an early decision whether to continue with the experiment or to stop the run.