The sequencing software carries out several core tasks: data acquisition, real-time analysis and feedback, basecalling, data streaming, controlling the device, and ensuring that the platform chemistry is performing correctly to run the samples. The sequencing software takes the raw data and converts it into reads by recognition of the distinctive change in current that occurs when a DNA strand enters and leaves the pore. It then basecalls the reads and writes out the data into FASTQ and BAM files.

Please refer to the Q system GridION IT requirements document for details of the IT setup required to run a GridION Q.

MinKNOW can be accessed directly on the device or from another computer that remotely connects to the device used for sequencing, and summarises the states of the flow cells in the device.

• Blue tag: direct connection
• Purple tag: remote connection
  

Click Add host and enter the IP or hostname of a device to add it to the connection manager for remote access.

Default security settings for devices requires users to have a Nanopore account to remotely connect to a device.

To change the remote access availability, settings can be updated when directly connected to a device.

The MinKNOW Homepage enables you to navigate to:

a. The Start homepage b. Sequencing Overview of connected flow cells c. Recent and current Experiments d. System Messages e. Host Settings f. Connection Manager to connect with other available devices g. Start Sequencing experiment h. Post-run Analysis i. Flow Cell Check j. Hardware Check k. More includes option to generate .mmi from .fasta file or to import a sample sheet l. Guest/initials to logout

This page displays the inserted flow cell state and progress of a current sequencing experiment, including pausing, pore scan and basecalling.

Flow cells with no checks:

Sequencing states:

• Pore scanning:

• Basecalling catch up:

• Run paused:

During a sequencing run, Flow cell health will be displayed from after the first pore scan.

Select the flow cell to open the quick view of the current experiment.

The experiments page displays summary information for all experiments and device check data. You can control runs and see real-time information from sequencing flow cells.

All previous runs can be viewed here by altering the number of days available in "Experiments active in the last ... days".

All device reports and messages are displayed here.

A loading bar will be displayed under the flow cell during the checks.

A flow cell check must be carried out before loading a DNA or RNA library to assess the number of pores available.

Oxford Nanopore Technologies will replace any flow cell that falls below the warranty number of active pores within 12 weeks of purchase, provided the result is reported within two days of performing the flow cell check and the storage recommendations have been followed.

The minimum number of active pores covered by warranty is 800 for GridION Flow Cells.

A loading bar will be displayed under the flow cell during the checks.

The quality of the flow cell will be shown as one of the three outcomes on the Sequencing Overview page:

1. Yellow exclamation mark: The number of sequencing pores is below warranty.

2. Green tick: The number of sequencing pores is above warranty and ready for sequencing.

3. Question mark: A flow cell check has not been run on the flow cell during this MinKNOW session.

Note: the indicator of quality will only remain visible during the MinKNOW session when testing occurred. Once the sequencing device has been re-started, the status of the flow cell will be erased.

The experiments page displays summary information for all sequencing flow cells and device checks carried out on the device.

Previous runs can be viewed here until the MinKNOW service is restarted (e.g. after a device reboot).

From this page, you can control specific runs and identify real-time information, including flow cell health and reads.

• Run statistics: The total number of reads, estimated and basecalled bases across an experiment, and number of active and total runs
• Run time: The duration of the experiment
• Run state: The current state of the sequencing run: 'Active', 'Basecalling', 'Complete', 'Stopped with error'
• Health: The current flow cell health

The white panel displays a summary of sequencing experiments and the blue panel displays status information of a specific run.

For more status information of a specific run, click on the run to open the quick view, including current temperature and voltage.

Page configuration allows users to choose which graphs to generate in the quick view of an experiment.

To open page configuration, click on a run in the white panel to open the quick view and click on the Default, All, or Custom options to choose which graphs to display.

Tabs:

• Default: Default graphs available
• All: All graphs available
• Custom: Move graphs from 'Available pages' to 'Your pages' to display a custom order of graphs in the GUI. On this page, you can also choose graph order. Click the graph and drag, then click Save.

Select Experiment Groups in the top right corner and swipe sideways to view all the graphs presenting data form all previous runs of the same experiment name, including cumulative output for individual and multiple flow cells.

Use the quick view to display information and graphs for specific runs. To open, select a specific run in the white panel and use the arrows to navigate between the graphs.

For more information about the graphs, refer to Check and monitoring in the 'Monitoring and troubleshooting your sequencing run' section.

You can pause a single flow cell in a particular position (a run) or every flow cell (an experiment).

Pausing works by dropping the voltage potential over the membrane to 0 mV to maintain a safe environment to add components, such as fuel, more DNA/RNA library or nucleases for a flow cell wash. Data acquisition will continue during this period, but because no DNA is going through pores at 0 mV, no sequencing data will be collected.

Click the yellow Pause button to open a dialogue box and select which flow cell(s) to pause, then click Pause.

The pore scan is used to assess the quality of the four wells in each channel to select the best performing pores. A new pore scan can be triggered every time a sequencing experiment is resumed after a pause (e.g. for a flow cell wash), or if the number of sequencing pores has significantly dropped during an experiment.

Navigate to the Experiments page, open a specific run, and click Start pore scan in the run controls. Confirm the flow cell to be scanned and click Start pore scan.

Sample sheet example:

FA026858,SQK-RBK110.96,barcoding_run,sequencing_20200522,barcode01,sample_id_5,test_sample

The sample sheet may describe flow cells being run at one or more than one position. The columns available are as follows, though some are optional:

For experiments that involve barcoding, an alias is associated with each barcode. Additional columns are available for barcoding runs:

The sample sheet contains one barcoding arrangement column:

Column titles are defined within the first row of the sample sheet. These must be defined in lowercase using the column title values listed above.

Sample sheet validation occurs against the hardware and between rows in the sample sheet to ensure validity. Validation occurs when the sample sheet is loaded. Validation and importing the sample sheet requires all the flow cells to be used in the experiment. Be careful to ensure the correct flow cells and positions are used.

Alongside a CSV file containing the sample information, users can also upload a JSON file containing the sequencing parameters for their experiments rather than inputting the information manually.

A JSON file in the correct format, containing all default settings, can be exported from MinKNOW and altered:

Run options tab:

• "runLengthHours": 72, (Run length. This must be an integer)
• "enrichDepleteAdaptiveSamplingEnabled": false,
• "enrichDepleteAdaptiveSamplingRefFile": null,
• "adaptiveSamplingChannelStart": 1,
• "adaptiveSamplingChannelEnd": 512,
• "enrichDepleteAdaptiveSamplingBedFile": null,
• "shouldEnrichAdaptiveSamplingRef": true,
• "barcodeBalancingEnabled": false,
• "barcodeBalancingCustomBarcodes": false,
• "barcodeBalancingBarcodeSelection": null,
• "minReadLength": 200, (Read length selection. This can be 20, 200 or 1000)
• "activeChannelSelection": true,
• "muxScanPeriod": 1.5,
• "groupChangePeriod": 16,
• "reservedPores": true,

Analysis tab:

• "basecallingEnabled": true, (Basecalling can be switched on/off as true/false)
• "modifiedBasecallingEnabled": false,
• "barcodingEnabled": true, (Barcoding can be switched on/off as true/false)
• "basecallModel": "dna_r9.4.1_450bps_hac.cfg", (Basecalling model depends on the flow cell and kit combinations. Make sure that the basecall model is stated as a ".cfg")
• "modifiedBasecallingContext": "",
• "trimBarcodesEnabled": true, (Trim barcodes can be switched on/off as true/false)
• "requireBarcodesBothEnds": false, (Requirement to have barcodes on both ends can be switched on/off as true/false)
• "detectMidStrandBarcodes": false, (Detect mid read barcodes on/off as true/false)
• "overrideMidBarcodingScore": false, (Override mid barcoding score can be switched on/off as true/false)
• "overrideBarcodingScore": false, (Override barcode score can be switched on/off as true/false)
• "minBarcodingScore": 60, (Selection of barcode score. This can be an integer between 40 to 100)
• "minBarcodingScoreMid": 50, (Selection of mid barcode score. This can be an integer between 40 to 100)
• "alignmentRefFile": null,
• "alignmentBedFile": null,

Output tab:

• "dataOutputLocationType": 0,
• "offloadOrOutputLocationNavParams": "path": "/data/."
• "fastQEnabled": true, (FASTQ files can be switched on/off as true/false)
• "fastQReadsPerFile": 4000, (Number of reads per file. This is an integer)
• "fastQDataCompression": true, (FASTQ compression can be switched on/off as true/false)
• "fast5Enabled": true, (FAST5 files can be switched on/off as true/false)
• "fast5ReadsPerFile": 4000, (Number of reads per file. This is an integer)
• "fast5DataCompression": "vbz_compress", (Compression type. This can be "vbz_compress" or "zlib_compress")
• "selectedRawOutput": "fast5",
• "pod5Enabled": false,
• "pod5ReadsPerFile": 4000,
• "bamEnabled": false,
• "bamWriteMultiple": true,
• "readFilteringEnabled": true,
• "readFilteringMinQscore": 9,
• "readFilteringMinReadlength": null,
• "readFilteringMaxReadlength": null,
• "readSplittingEnabled": false,
• "overrideMinReadSplittingScore": false,
• "minReadSplittingScore": 58,
• "bulkFileEnabled": false,
• "bulkFileRaw": "1-512",
• "bulkFileEvents": "1-512",
• "bulkFileReadTable": "1-512",
• "bulkFileRawEnabled": false,
• "bulkFileEventsEnabled": true,
• "bulkFileReadTableEnabled": true

• Position defined using position_id does not exist
• Position defined using flow_cell_id cannot be found
• flow_cell_id and position_id are used but the flow_cell_id does not match the flow cell used
• flow_cell_product_code is used but the value doesn't match the flow cell used
• Different sample_id values are assigned against the same position whilst setting up a run
• flow_cell_product_code and kit do not match
• Zero or more than one sequencing kit listed in kit
• Barcode kits in kit do not exist as an option on the selected protocol
• type must be a valid selection from the list of valid types outlined above
• experiment_id must exist within each row and must all have the same value

When loading a FASTA file into MinKNOW for alignment, MinKNOW will generate a MiniMap index file as a first step. To save time when setting up a sequencing run with live alignment, you can create a MiniMap index from a FASTA file from the MinKNOW Start page, prior to set-up.

The Application settings are available on the Connection manager as well as the device homepage.

Below is an example from the Connection manager:

Tutorials are available here; they are also presented when logging into MinKNOW for the first time. Users can use Recap to view a specific tutorial or use Reset tutorial state to go through all the tutorials again.

Navigate here to logout from MinKNOW.

User email address used for login is displayed here, unless logged in as a guest.

Use Select language to change between English and Mandarin.

This tab opens the options where users can change device settings.

The Host settings have several features to co-ordinate the device. From this area, users are provided with information on system parameters, such as storage, date/time and IP address.

1. Device Settings: The devices can be shut down and rebooted from this page, and settings for the time and date can be altered. Disk space can be managed and peripherals may also be added and ejected from here. 2. Software: Users are able to download the latest software updates from this page. The option to update will only appear when an update is available. 3. File Manager: Stored data on the device can be managed and transferred. 4. Help: Device logs can be exported using the Export Logs function. Logs and temporary data can also be cleared by using the Clean up function. If troubleshooting of the device is required, a Repair problems function is available as well as a MinKNOW restart option.

Select Back to Main Menu to leave host settings.

• Device settings:

• Software

• File manager:

• Help:

Data can be managed and transferred from the file manager tab on the host settings.

Navigate through the tabs to view the data stored:

• Internal tab: Data stored on the connected sequencing device
• Removable tab: Data stored on a connected removable storage device e.g. USB drive
• Network tab: Data stored on a connected network drive. The network drive must be mounted prior.

Click the icon in the bottom right corner to open Create New Directory and type in a directory name. Click Create.

Navigate to Device Settings to view available disk space on the device. Any peripherals plugged in, including USB, can be ejected using the Eject option.

A network drive can also be connected to the device by selecting Add Network Drive and filling in the required credentials for connecting using a Samba (SMB) server or a Network File System (NFS).

Network drives for data storage can be mounted from either the Device Settings or the File Manager in the Network tab:

• Device Settings:

• File Manager:

On opening the MinKNOW software, the tutorials will start to navigate you through the user interface.

Tutorials may be skipped if you are familiar with the user interface.

To skip tutorials, select the three dots and choose to either skip all tutorials or just a section.

Tutorials can be reviewed again by selecting Reset tutorial state in the Tutorials panel of Application Settings.

Our device FAQs are located here.

For the PromethION 24/48, PromethION 2i, GridION and PromethION 2 Solo connected to a GridION:

• Click Export in the Export Logs section.

When you have successfully exported the logs, you will be notified of where they are located below the button. The logs will be downloaded as a TGZ file in the logs directory.

You can change the location of the logs by using the file manager, where you can easily retrieve them without needing SSH:

• Click on the file next to Logs exported to: in the Export Logs panel. This will open where the logs are located in the File Manager of MinKNOW.
• Use the control panel in the lower right corner of the File Manager to move the log files.

If the PromethION 2 Solo is connected to a stand-alone computer

Log files for each sequencing experiment can be found in:

• Windows: C:\data\logs

• macOS: /private/var/log/MinKNOW

• Linux: /var/log/minknow

In the "Help" page, click Clean up in the Clean Up Files section and confirm in the in the pop-up.

Note: Run data will not be deleted. Logs and temporary data will be removed.

Click Run repair in the troubleshooting section of the Help page and confirm in the pop-up. MinKNOW will attempt to repair the following issues:

• Ensure required directories under "/data" exist and have the correct ownership and permissions
• Ensure required files under "/data" have the correct ownership and permissions
• Ensure the GPU is working at maximum performance
• Ensure that Bluetooth is disabled

Basecalling models can be selected at two stages in the MinKNOW software:

• Setting up an experiment in MinKNOW - the instructions for selecting a model can be found in the section 'Starting a sequencing run' of this protocol.
• Using post-run basecalling in MinKNOW - the instructions for how to basecall data once an experiment has finished can be found in the section 'Post-run analysis' of this protocol.

Select the High accuracy basecalling model.

Please note than only the High Accuracy (HAC) model is recommended for use with the Q system. While the other unverified (Fast and SUP) models can be used, issues have been observed when using multiple SUP models simultaneously and it is strongly advised you avoid running these alongside important experiments.

Select Options in the Output Format panel of the Output tab.

• FAST5: The number of basecalls that MinKNOW will write in a single file.

  • By default, 4000 reads are written out per file
  • The files can be compressed with zlib or VBZ (note: VBZ compression will require a VBZ plugin for compatibility with existing tools. This is optimised for nanopore data, with improved compression over zlib)

• FASTQ: The number of files that MinKNOW will write in a single folder.

  • By default, 4000 files are written into a single folder
  • The files can be uncompressed, or compressed with Gzip to ~55% of original size

• BAM: Files containing aligned reads (one BAM file per input FASTQ file).

Users can change reads per file:

• Fewer reads per file: reads become more quickly available. However, too few reads per file means MinKNOW may not keep up with writing out files in real-time.
• More reads per file: reduces the number of files especially for amplicon/cDNA experiments that produce a large number of reads. Some downstream analysis tools may have an upper limit on the uploaded file size.

Chosen options can be saved by selecting Save for future use.

Refuelling is the replenishment of motor fuel in the sequencing experiment through the addition of Flush Buffer (FB) from the Flow Cell Priming Kit (EXP-FLP002). The translocation speed graph in MinKNOW can indicate whether it is necessary to top up fuel.

If the DNA translocation speed drops below 300 bases per second, you may start to see a reduction in quality of data reflected in the Qscore. We therefore recommend topping up the flow cell with fuel, using the Flush Buffer (FB) from the Flow Cell Priming Kit. Please follow the instructions below if you wish to top up the fuel during an experiment.

Instructions for refuelling a GridION Flow Cell:

• Remove one tube of Flush Buffer (FB) from the freezer and thaw by bringing to room temperature

• Pause the experiment on the GridION position that is being refuelled:

  a. Navigate to Experiments and select the experiment running b. Click Pause c. A dialogue box will open. Choose the flow cell to pause and click Pause to confirm

• After opening the priming port, check for a small bubble under the cover. Draw back a small volume to remove the bubble (a few µl):

  a. Set a P1000 pipette to 200 µl b. Insert the tip into the priming port c. Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip

Visually check that there is continuous buffer from the priming port across the sensor array.

Complete the flow cell refuelling:

• Load 250 µl of the FB into the flow cell via the priming port (i.e. not the SpotON port), avoiding the introduction of air bubbles
• Close the priming port and replace the GridION lid
• Unpause the experiment on the relevant GridION position by clicking Resume
• (optional) Click Start pore scan and choose your flow cell, to pick a new set of the best channels for the remainder of the sequencing experiment.

Translocation speed and Qscore over time

Below is a graph that shows what is expected for translocation speed after the addition of FB to the flow cell in a previous version of MinKNOW.

As the speed drops below 300 bases per second, the Qscore begins to decline for the reads processed through the nanopores at this speed. After refuelling at the 17.5 hour mark, the speed begins to increase and returns to an improved rate (~400 bases per second), which is similar to the speed at the start of the experiment. After the addition of fuel, the quality of the data increases and returns to Qscores equivalent to those seen at the start of the run.

Refuelling multiple times in a run

You can refuel a sequencing run multiple times over an experiment. When you should refuel will depend on when the translocation speed drops below 300 bases per second on the speed graph in the MinKNOW UI.

During a sequencing experiment, the MinKNOW Sequencing Overview page shows a flow cell icon with coloured bars. The bars represent the combined health of all pores in a flow cell, and indicate how well the flow cell is performing. The colours are:

• Light green: sequencing
• Dark green: open pore
• Dark blue: pore recovering
• Light blue: pore inactive

This information is identical to the last bar of the pore activity plot (described later).

The channel states pannel gives an overview of the states the flow cell pores are in to give the user an idea of how well the sequencing run is performing in real time. A good library will be indicated by a higher proportion of light green channels in "Sequencing" than are in "Pore available". The combination of "Sequencing" and "Pore available" indicates the number of active pores at any point in time. A low proportion of "Sequencing" channels will reduce the output of the run.

Clicking on the Show Detailed button reveals a more detailed array of channel states:

• Sequencing: Pore currently sequencing.
• Pore Available: Pore available for sequencing.
• Adapter: Pore currently sequencing adapter.
• Active feedback: Channel ejecting analyte.
• No pore: No pore detected in channel.
• Multiple: Multiple pores detected. Unavailable for sequencing.
• Unavailable: Pore unavailable for sequencing.
• Unclassified: Pore status unknown.
• Saturated: The channel has switched off due to current levels exceeding hardware limitations.
• Out of range-high: Current is positive but unavailable for sequencing.
• Out of range-low: Current is negative but unavailable for sequencing.
• Zero: Pore currently unavailable for sequencings.

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 48 minutes. However, bucket size can be adjusted in the "Bucket size" box in Display Settings.

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.

To see the more detailed view of channel states, click Show detailed.

• Sequencing: Pore currently sequencing.
• Adapter: Pore currently sequencing adapter.
• Pore available: Pore available for sequencing.
• Unavailable: Pore unavailable for sequencing.
• Active feedback: Channel ejecting analyte.
• No pore: No pore detected in channel.
• Out of range-high: Current is positive but unavailable for sequencing.
• Out of range-low: Current is negative but unavailable for sequencing.
• Multiple: Multiple pores detected. Unavailable for sequencing.
• Saturated: The channel has switched off as current level exceed hardware limitations.
• Zero: Pore currently unavailable for sequencing.
• Channel disabled: Channel is disabled and awaiting another pore scan.

The cumulative histogram shows reads compared to bases. Use the options below to choose the axis legends:

• Y-axis: Estimated bases or basecalled bases
• X-axis: Read length or read counts

Read count - this shows the number of reads vs read length. This enables you to understand how the read lengths vary in number and size.

Read length - this shows the total number of bases vs the read length.

The N50 value is presented (only for the whole set of passed reads) in the top left corner of the histogram.

Each histogram's X-axis (read length) can be zoomed in using the scaled bar under the histogram. Use Reset to refocus the zoom bar and histogram for the entire 'passed read' dataset. You can see the number of bases by hovering over the bar in question.

Reads that are outliers in terms of length can be removed from the graph by ticking the Hide outliers box below the histogram.

Select Split by read end reason to view split reads and hover over for further information. This is useful for adaptive sampling which is further explained in the Adaptive Sampling info sheet.

• Device changed MUX/Pore scan initiated: The strand ended because there was a scheduled pore scan that interrupted the read.
• Read completed: The strand ended naturally as it passed through the pore.
• Read blocked: The strand ended because it was deemed of low quality and purposefully rejected.
• Adaptive sampling voltage reversal/Adaptive sampling rejection: The strand was rejected by adaptive sampling because it did not align to the target sequence when enriching, or the strand matched to the target sequence when depleting.

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

If there are multiple flow cells running under the same experiment name, you will see the Experiment view. This gives output information on all assigned flow cells, plus a running cumulative total of bases or reads sequenced.

The cumulative output graph shows the running total number of reads or Gbases sequenced by the multi-flow cell platform like GridION.

The output generated by each flow cell to make the total cumulative output can be represented by individual output plots.

Both graphs can be switched between in the MinKNOW GUI. Reads, bases (estimated or basecalled), sample ID or flow cell ID can be selected to tailor the output graph, as required.

Temperature vs time graph

The temperature graph gives a real-time representation of the heatsink temperature of 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 vs time graph

The bias voltage graph provides the running voltage in real-time. MinKNOW will automatically adjust the applied voltage and will naturally drift to lower voltages as the electrochemistry in the flow cell is depleted.

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.

Note: These graphs are only present if basecalling is turned on.

Translocation speed vs time

The translocation graph gives a real-time representation of the speed at which DNA/RNA strands pass through the pore. If the translocation speed drops below this window, data quality and output may be compromised as strands take longer to move through the pore.

Quality score vs time

The Quality score graph gives a live representation of the median strand Q-score over time.

The Barcode Read Counts graph shows the breakdown of barcoded reads, if barcoding was used for the experiment. The default view only shows reads that have passed the quality score filters. However, selecting the Display failed box will show all reads.

The X-axis of the histogram also has a zoom function using the scaled bar underneath. Use Reset to refocus the zoom bar and graph.

Note that there is a small amount of cross-talk between barcodes. Some barcoded reads may appear on the graph even if the barcode in question was not used for the experiment.

Note: This graph is only present if barcoded is turned on.

The alignment hits graph will populate when alignment and basecalling is set-up to run during sequencing. This bar graph shows the number of reads and bases that align to each of the entries in the user reference .fasta file or minimap index file. An entry in the reference file will only appear on the graph once a single read has aligned to it.

The X-axis of the histogram also has a zoom function using the scaled bar underneath. Use the Reset to refocus the zoom bar and graph.

A target alignment hits view is also available in the same graph by clicking Alignment coverage. This is populated by the .BED targets if a .BED file is supplied with the alignment reference.

The alignment and barcode heatmap is only available when alignment and demultiplexing is performed with basecalling during sequencing. The heatmap graph shows the alignment hits split per barcode. The colour gradient shows bases and reads which are the more popular barcode and alignment hit combinations. Use the options below to view reads or bases and the slide bar above to focus on specific regions.

Note: These graphs are only present if barcoding and alignment are both turned on.

The Traceviewer displays the current levels from individual channels. By default, it is set to show 10 channels. This number can be changed through the selection boxes beneath the viewer. Additional parameters that can be altered:

• Time: The length of time plotted on one screen
• Maximum: The highest current level to be shown on the y axis

Please note that viewing a high number of channels in the traceviewer may impact the speed at which the GUI is able to function.

As the sequencing protocol starts, a pore scan begins before the sequencing. There are four groups of active pores, and the pore scan allows MinKNOW to pick the best-performing pores in each group, maximising the data output in the initial stages of the run. The software also instantly switches to a new channel in the group if a channel is in the “Saturated” state, or after ~5 minutes if a channel is “Recovering”.

The pore activity plot feature in the MinKNOW software can be used to judge the quality of your experiment. The pore activity plot shows the distribution of channel states over time, grouped by time chunks, or 'buckets'. The basic view shows the five main channel states: Sequencing, Pore, Recovering, Inactive, and Unclassified. Clicking the "More" button shows a more detailed breakdown of channel states.

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.

The software instantly switches to a new channel in the group if a channel is in the “Saturated” or “Multiple” state, or after ~5 minutes if a channel is “Recovering”. This feature maximises the number of channels sequencing at the start of the experiment; however this may also result in an artificially high number of "Sequencing" or "Pore" channels in the pore activity plot. For this reason, we recommend referring to the pore scan plot, which shows the true distribution of channel states at the point of the most recent pore scan.

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 output.

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 "Unavailable" pores over time.

Recommendation:

• If, despite the channel blocking, the library is still producing a sufficient number reads to answer your biological question, you can carry on with the sequencing experiment.
• Otherwise, stop the sequencing run in MinKNOW. Then wash out the library from the flow cell using the instructions for the Flow Cell Wash Kit. Then prepare another library and load it on the flow cell.

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

Pore activity:

Pore scan:

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:

• Check that the heat tape on the underside of the flow cell is intact.
• Make sure that the input DNA is in either TE buffer or nuclease-free water, and that the buffer contains no detergents or surfactants.
• Make a new batch of flow cell priming buffer (a mixture of Flush Buffer/Flow Cell Flush and Flush Tether/Flow Cell Tether). Flush the flow cell with the mixture and load the library again.

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 plot will show a high ratio of "Pore available" to "Sequencing" states, meaning that only a limited number of pores are sequencing at any one time.

Recommendation:

• Check the amount of DNA/RNA in your prepared library, for example by using the Qubit fluorometer. We recommend preparing a fresh library and reloading your flow cell with the recommended loading input from the relevant protocol
• If your library is at a low concentration, prepare the library again using a higher amount of starting material.
• Ensure you are adding the flush tether to your priming mix during flow cell priming and loading before adding your library.
• For ligation-based protocols, ensure you are following the protocol to ligate the sequencing adapter correctly and performing any clean-up steps after adapter ligation with short or long fragment buffer rather than ethanol.

The GridION's computer requires a stepwise, processed shut down, otherwise you may face problems e.g. when recognising flow cells.

Follow the instructions in this section to ensure you do not face errors with your GridION from incorrectly shutting down your device.

A user can basecall and demultiplex their data directly in MinKNOW after a sequencing experiment has finished, or re-analyse old data using the latest basecalling models. Reads can also be aligned against a reference post-run.

Note: Both barcoding and alignment can be run on .fastq or .fast5 reads when coupled with basecalling.

A user can barcode their data directly in MinKNOW after a sequencing experiment has finished, using FASTQ data.