Please select your operating system here. You will be presented with instructions specific to your OS for downloading and installing the two pieces of software needed for nanopore sequencing.

Turn off sleep modes:

• Navigate to Start > 'Settings' > 'System' > 'Power & Sleep'
• Set 'Sleep modes' to Never

Ensure your computer is plugged into a power source.

Defer Windows upgrades:

• Navigate to Start > 'Settings' > ' Update & Security' > 'Windows Update' > 'Advanced options'
• Tick the Defer upgrades box

Note: on some versions of Windows, this will be called 'Pause updates'.

Check that you have sufficient disk space. A typical Control experiment will require approximately 40 GB of space. However, for a full sequencing experiment using your own DNA or RNA sample, a minimum of 1 TB storage space is recommended. Please see this FAQ for more information.

Turn off your anti-virus for the software download steps. You can enable it again once the software is installed on your computer.

Confirm with your IT department that you have the firewall settings described in this FAQ.

If you are working behind a proxy, follow the instructions in this FAQ, and speak to your IT department before implementing any changes.

Double-click the .exe file to run the MinKNOW installer.

Allow the installer to make changes to your computer.

In the pop-up window, click Next.

When the installer is complete, click Close.

You will see the MinKNOW icon on your Desktop. Double-click on the icon to open the software and login using your Community login credentials.

You will be prompted to enter your email address and Nanopore password:

If the error message “MinKNOW was not successfully installed…please run the installer manually" appears, please follow the instructions in this FAQ.

Click the EPI2ME executable and wait for the software to install.

When the installation is complete, you will see the Agent icon on the desktop. Double-click on the icon to open the software.

Click through the introductory pages, or skip the introduction. You will then be directed to a page prompting to enter a registration code.

Log in to https://epi2me.nanoporetech.com using your Nanopore Community username and password.

Enable pop-up notifications in your browser.

Find your registration code by clicking the link to the EPI2ME website on the Desktop Agent screen.

Open the Desktop Agent, then copy and paste your registration code from the EPI2ME website. Set a username for your EPI2ME profile under Profile description.

Check your box contains your MinION Mk1B device, Configuration Test Cell and USB 3.0 cable.

The MinION Mk1B arrives with the configuration test cell already inserted. The configuration test cell is of a similar shape to the flow cells, but white.

If the configuration test cell is separate, insert it into the MinION Mk1B and connect your MinION Mk1B to the computer.

Check that the MinION Mk1B device is recognised by your computer.

The MinION Mk1B status will be displayed on the Sequencing Overview page. The flow cell in the image below is ready for a hardware check.

Navigate to the Start homepage and select the Hardware Check.

Select Start.

The user will be automatically navigated to the System Overview page. A loading bar will be displayed under the flow cell during the checks.

The hardware check will complete after approximately one minute. A pass is indicated by a green check icon and a fail is an orange check.

Take out the configuration test cell from the MinION Mk1B. You do not need to disconnect the MinION Mk1B from the computer, or close any software.

Push the flow cell into the clip, ensuring good contact between the flow cell and the MinION Mk1B. Close the lid.

Close the MinION Mk1B lid.

Navigate to the Sequencing overview page to check the MinKNOW software has detected the inserted flow cell.

Navigate to the Start homepage and select Flow Cell Check.

The flow cell type will be recognised by the device. Click 'Start' to begin.

The user will be automatically navigated to the System Overview page. A loading bar will be displayed under the flow cell during the checks.

The flow cell will cycle through the 2048 wells to see how many pores are active on the sensor array. It will do this in four groups of 512 at a time.

Flow cell check completion will be indicated on the System Overview page with an icon on the flow cell with the number of pores available.

Keep these on ice:

• RT Adapter (RTA)
• RNA Calibration Strand (RCS)
• RNA Ligation Adapter (RLA)
• 10 mM dNTP solution

Defrost these at room temperature:

• NEBNext Quick Ligation Reaction Buffer
• T4 DNA Ligase

Once the contents of each tube have thawed, spin down the tubes in a microfuge and mix the contents by pipetting up and down for 10 full-volume pipette mixes.

Defrost these at room temperature:

• Wash Buffer (WSB)
• RNA Elution Buffer (REB)

Vortex both tubes, then spin them down and place on ice.

Keep the reagent tubes on ice when they are not in use.

Set up a thermal cycler with the program 50°C for 50 minutes, then 70°C for 10 minutes, then hold at 4°C. Alternatively, set two heat blocks at 50°C and 70°C.

Transfer 3 µl of NEBNext Quick Ligation Reaction Buffer into a clean 0.2 ml PCR tube (Tube 1).

Add 8.5 µl RNA Calibration Strand (RCS) to Tube 1.

Add 1 µl RNaseOUT to the tube.

Add 1 µl RT Adapter (RTA) to the tube.

Add 1.5 µl T4 DNA Ligase to the tube.

Mix the contents of the tube by pipetting up and down. Spin down briefly in a microfuge.

Incubate Tube 1 for 10 minutes at room temperature. This is the adapter ligation reaction (Tube 1).

Take a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 2), and add 9 µl nuclease-free water.

Add 2 µl of 10 mM dNTPs to Tube 2.

Add 8 µl first strand buffer from the SuperScript III kit to Tube 2.

Add 4 µl of 0.1 M DTT to Tube 2.

Mix the contents of the tube by flicking the tube with your finger. Spin down briefly in a microfuge.

Add the contents of the 1.5 ml tube (Tube 2) to the 0.2 ml PCR tube (Tube 1) containing the RT adapter ligated RNA.

Mix the contents of the tube by pipetting up and down. Spin down briefly in a microfuge.

Add 2 µl of SuperScript III reverse transcriptase to the 0.2 ml tube (Tube 1).

Mix the contents of the tube by pipetting up and down. Spin down briefly in a microfuge.

Incubate in heat blocks or a thermal cycler at 50°C for 50 minutes, then at 70°C for 10 minutes, then hold at 4°C for approximately 30-60 seconds. Take the tube out from the thermal cycler or heat block, and spin down briefly in a microfuge.

Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 3). Tube 3 is the reverse transcription reaction.

Resuspend the Agencourt RNAClean XP beads by vortexing.

Add 72 µl of resuspended RNAClean XP beads to the reverse transcription reaction (Tube 3), and mix by pipetting up and down.

Put the tube in a Hula mixer, and leave to incubate for 5 minutes.

While the RNA sample is incubating, transfer 140 µl of pure ethanol to a clean 1.5 ml Eppendorf DNA LoBind tube.

Add 50 µl nuclease-free water to the ethanol.

Mix the tube of diluted ethanol by vortexing. Spin down briefly in a microfuge.

Take Tube 3 with the reverse transcription reaction off the Hula mixer, and spin down in a microfuge.

Place the tube in a magnetic rack, and wait for the beads to collect in a pellet near the magnet, and the solution to become clear.

Keep the tube on the magnet to pipette off and discard the supernatant. Take care to not disturb the pellet.

Add 150 µl of the freshly-prepared ethanol to Tube 3, without taking the tube off the magnetic rack or disturbing the pellet.

Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and form a pellet.

Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet.

After this, pipette off and discard the ethanol.

Take the tube off the magnetic rack and spin down briefly in a microfuge.

Place the tube back in the magnetic rack, and wait for the beads to collect in a pellet near the magnet. There may be a small amount of residual liquid at the bottom of the tube. Pipette off and discard this liquid.

Remove the tube from the magnetic rack, add 20 µl nuclease-free water to the tube, and resuspend the beads by flicking the tube.

Incubate for 5 minutes at room temperature.

Place the tube back in the magnetic rack, and wait for the beads to collect in a pellet near the magnet.

Pipette off the 20 µl of eluate from Tube 3, and transfer it to a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 4). Discard Tube 3 with the pellet and retain Tube 4 with the eluate.

Add 8 µl of NEBNext Quick Ligation Reaction Buffer to your reverse-transcribed RNA (Tube 4).

Add 6 µl of RNA Ligation Adapter (RLA) to Tube 4.

Add 3 µl nuclease-free water to the tube.

Add 3 µl T4 DNA Ligase to the tube.

Mix the contents of the tube by pipetting up and down. Spin down briefly in a microfuge.

Incubate Tube 4 for 10 minutes at room temperature. This is the adapter ligation reaction (Tube 4).

Resuspend the Agencourt RNAClean XP beads by vortexing.

Add 16 µl of resuspended RNAClean XP beads to the adapter ligation reaction (Tube 4), and mix by pipetting up and down.

Put the tube in a Hula mixer, and leave to incubate for 5 minutes.

Take Tube 4 with the reverse transcription reaction off the Hula mixer, and spin down in a microfuge.

Place the tube in a magnetic rack and wait for 5 minutes for the beads to collect in a pellet near the magnet and the solution to become clear.

Keep the tube on the magnet to pipette off and discard the supernatant. Take care to not disturb the pellet.

Add 150 µl of Wash Buffer (WSB) to Tube 4. Close the tube lid, and resuspend the beads by flicking the tube with your finger.

Return the tube to the magnetic rack, allow the beads to pellet, then pipette off and discard the buffer.

Place the tube in a magnetic rack and wait for 5 minutes for the beads to collect in a pellet near the magnet and the solution to become clear.

Keep the tube on the magnet to pipette off and discard the supernatant. Take care to not disturb the pellet.

Repeat the previous three steps: Add 150 µl of Wash Buffer (WSB) to Tube 4. Close the tube lid, and resuspend the beads by flicking the tube with your finger.

Return the tube to the magnetic rack, allow the beads to pellet for 5 minutes, then pipette off and discard the buffer.

Take the tube off the magnetic rack and spin down briefly in a microfuge.

Place Tube 2 back in the magnetic rack, and wait for the beads to collect in a pellet near the magnet. There may be a small amount of residual liquid at the bottom of the tube. Pipette off and discard this liquid.

Remove the tube from the magnetic rack, add 13 µl RNA Elution Buffer (REB) to the tube, and resuspend the beads by flicking the tube.

Incubate for 10 minutes at room temperature.

Place the tube in a magnetic rack and wait for 5 minutes for the beads to collect in a pellet near the magnet and the solution to become clear.

Pipette off the 13 µl of eluate from Tube 4 and transfer it to a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 5). Discard Tube 4 with the pellet.

Remove 1 µl of eluate from Tube 5 and use the Qubit fluorometer DNA HS assay to quantify the amount of RNA in the sample.

Make sure the contents of the Flow Cell Flush (FCF) and RNA Flush Tether (RFT) tubes are fully defrosted. Mix the tubes by vortexing and then spin down in a microfuge.

Take a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 6) and add 1,170 µl of Flow Cell Flush (FCF).

Add 30 µl of RNA Flush Tether (RFT) to Tube 6.

Mix the contents of the tube by vortexing and then spin down briefly in a microfuge.

Open the priming port by sliding the cover clockwise so that the port is visible.

Remove inlet channel air plug

• Set a P1000 pipette to 500 µl; insert the tip into the priming port
• Keeping the tip inserted in the port, twist the wheel of the pipette until you reach 520-530 µl, or until you see a small amount of the yellow buffer in the tip. This removes any air plug that has developed in the port during shipping and storage, and a tiny volume of buffer to wet the port
• Remove the pipette tip from the priming port, and throw away the tip

Prime your flow cell

• Using a P1000 pipette, slowly load 800 µl of the priming mix into the priming port
• Do not eject to the last stop of the pipette; take care to not introduce air bubbles
• Close the priming port
• Wait 5 minutes

The pre-sequencing mix is a mix of your RNA library, Sequencing Buffer (SB) and Library Solution (LIS) that is loaded into the flow cell.

Please ensure you have completed the Flow Cell Check section before preparing your pre-sequencing mix.

Take the 1.5 ml tube containing your control RNA library and add 37.5 µl Sequencing Buffer (SB).

Add 25.5 µl RNA Library Solution (LIS) to the tube.

• Mix the contents of the tube thoroughly by flicking the tube with your finger
• Spin down briefly in a microfuge

Open the priming port by sliding the cover clockwise so that the port is visible.

Open the sample port by gently lifting the sample port cover. You will still need to use the priming port, so keep this open.

Using a P1000 pipette, slowly load 200 µl of the Priming Mix into the priming port. Take care to not introduce air bubbles. Pipette slowly, to avoid the buffer bubbling out of the sample port. Carry out this step immediately before loading the library.

Using a P200 pipette, hold the pipette tip just above the sample port and slowly load 75 µl, drop by drop, of the Pre-Sequencing Mix (Tube 5).

• Close the sample port with the cover ensuring the bung enters into the port
• Close the priming port
• Close the MinION Mk1B lid

Place the light shield onto the flow cell. Carefully place the leading edge of the light shield against the clip. Do not force the light shield underneath the clip. Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.

Your MinION Mk1B and flow cell should still be connected. Check your MinION Mk1B is plugged in to your computer's USB 3.0 port, and the flow cell is clipped firmly into the MinION Mk1B device.

Check the MinKNOW software is still open. If you have closed it, double-click the icon on the desktop and login.

Select Start Sequencing on the home page.

On the New experiment pop-up screen, select the running parameters for your experiment from the individual tabs.

Select positions The positions tab will show the chosen flow cell. Assign an experiment name and sample ID. The other tabs will not become available until an experiment name has been provided.

Kit selection The kit selection tab will provide a selection of available kits. Check the Control button, then click on SQK-RNA004.

Click Skip to final review.

On the final page, there will be a summary of all the options chosen for the experiment. Click Start.

When sequencing is initiated, the software will move to the channel screen. The flow cell will cycle through all 2048 channels to select the best set of 512 to start the run.

Once the best combination of channels is selected, sequencing will begin on those channels automatically.

You will be indicated sequencing has started when flow cell health is displayed and a progress bar will appear beneath the flow cell.

Now your sequencing run is started, it is a good time to get EPI2ME running in the background. This allows reads to be sent into the workflow as soon as they have been acquired and basecalled.

If you have closed the EPI2ME Agent from earlier, please reopen it:

Click New analysis and select FASTQ RNA control experiment.

If prompted, select a folder containing FASTQ files to be analysed. If a MinKNOW-generated folder structure is detected in the default input location, this will be shown in the Experiments & samples tab. You can change the default input location in the Settings page (cog icon) under Data sources.

Alternatively, you will be directed to the Folders tab, where you can select your input folder.

Adjust the run-time parameters of your analysis: Select No under "Are you uploading human DNA/RNA?". Leave all the other settings at their default values, then click Accept & Start.

EPI2ME will start the workflow.

While your experiment is running, you can track how it is progressing using the MinKNOW software. To view your experiment metrics, click on the sequencing flow cell to open the quick view and a summay of the run. Use the arrows to move between real-time data, including channel panel and channel scan.

Within the first half an hour you should have most active pores sequencing and already have obtained several thousand reads.

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.

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 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.

You can see further information about your run using EPI2ME. Once MinKNOW has aquired and basecalled the runs, they will be picked up by EPI2ME and sent down the chosen workflow.

For this experiment, we have already chosen the 'FASTQ RNA Control Experiment' workflow. This will take the basecalled reads and align them to the reference yeast enolase 2 gene. You can use the report to confirm that you have produced a successful library and examine some basic genomic statistics such as depth and distribution of coverage, accuracy, etc.

Click View Report in the EPI2ME Agent window

If necessary, you may need to click Accept cookies.

The EPI2ME report will appear in a new browser window.

You can see your attributes (e.g. "RNA_control") have appeared at the top. Click the 'RNA alignment' tab, which will show you your reads as they appear, and map them to the enolase 2 gene.

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 duty time 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.

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.

RNA adapter

Opening up the "More" view gives more granular detail about channel states. For RNA in particular, this view may show a large proportion of pores sequencing Adapter. This happens because RNA strands are usually shorter than DNA, and the adapter takes up a larger proportion of the strand. Additionally, the RNA sequencing chemistry is optimised for sequencing RNA, whereas the adapter is DNA, and is processed slower. As long as the 'basic' pore activity plot view shows the majority of pores in "Sequencing", a high proportion of Adapter should not be a problem.

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 basecalling. 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 enolase 2 gene (~1400 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 gene.

Accuracy

This histogram shows the accuracy of the reads, when each read is compared individually to the 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.

When the sequencing run finishes, MinKNOW will return to the home screen. You can view a summary of information from your run by selecting "Export PDF Report":

• If you are ready to start planning your own sequencing experiment, our interactive Protocol Builder tool allows you to plan your end-to-end workflow, which includes selecting the right DNA/RNA extraction protocol and data analysis method for your application.
• For more information, visit the Nanopore Community, where you can find recommended library preparation protocols. We also provide DNA and RNA extraction methods for various sample types.
• For any questions regarding your order, or technical questions about your experiment, please contact Support.
• Finally, you can also join forums on the Nanopore Community, where our enthusiastic and diverse community of users discuss their experiment plans and results, share hints and tips, and form collaborations.

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