Introduction to the protocol

Overview of the protocol

IMPORTANT

This is a Legacy product

This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit
which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product,
please contact Customer Support via email: [email protected].

Rapid Sequencing Kit features

This kit is recommended for users who:

require a short preparation time

have limited access to laboratory equipment

Introduction to Rapid Sequencing protocol (SQK-RAD004)

This protocol describes the step-by-step instructions to complete a rapid sequencing of genomic DNA using the Rapid Sequencing Kit
(SQK-RAD004). It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

- Extract your DNA, and check its length, quantity and purity.
The quality checks performed during the protocol are essential in ensuring experimental success.
- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- If not already installed, download the software for acquiring and analysing your data
- Check your flow cell(s) to ensure it has enough pores for a good sequencing run

Library preparation

You will need to:

- Tagment your DNA using the Fragmentation Mix in the kit
- Attach sequencing adapters supplied in the kit to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cell
Sequencing and analysis

You will need to:

- Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
- optional Start the EPI2ME software and select a workflow for further analysis, e.g. metagenomic analysis or drug resistance
mapping

IMPORTANT

Compatibility of this protocol

This protocol should only be used in combination with:

Rapid Sequencing Kit (SQK-RAD004)

R9.4.1 (FLO-MIN106) flow cells
Flow Cell Wash Kit (EXP-WSH004)

Equipment and consumables

Materials ~400 ng high molecular weight genomic DNA

 Rapid Sequencing Kit (SQK-RAD004)
Flow Cell Priming Kit (EXP-FLP002)

Consumables 1.5 ml Eppendorf DNA LoBind tubes

0.2 ml thin-walled PCR tubes
Nuclease-free water (e.g. ThermoFisher, AM9937)

Equipment Microfuge
P1000 pipette and tips
P100 pipette and tips
P20 pipette and tips
P10 pipette and tips
P2 pipette and tips
Timer
Thermal cycler or heat block at 30°C and 80°C

Optional Qubit fluorometer (or equivalent for QC check)

Equipment

For this protocol, you will need ~400 ng high molecular weight genomic DNA

Input DNA

How to QC your input DNA

It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor
quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.

For instructions on how to perform quality control of your DNA sample, please read theInput DNA/RNA QC protocol.

Chemical contaminants

Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which
can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the
Community.

Rapid Sequencing Kit contents

Name Acronym Cap colour No. of vials Fill volume per vial (µl)

Lambda DNA LMD Yellow 1 88

 Name Acronym Cap colour No. of vials Fill volume per vial (µl)

Fragmentation Mix FRA Brown 1 30

Rapid Adapter RAP Green 1 10

Sequencing Tether SQT Purple 1 10

Loading Beads LB Pink 1 360

Sequencing Buffer SQB Red 1 300

IMPORTANT

Please note that the Sequencing Tether (SQT) tube will NOT be used in this protocol.

Flow Cell Priming Kit contents (EXP-FLP002)

Name Acronym Cap colour No. of vials Fill volume per vial (μl)

Flush Buffer FB Blue 6 1,170

Flush Tether FLT Purple 1 200

Computer requirements and software

MinION Mk1B IT requirements

Sequencing on a MinION Mk1B requires a high-spec computer or laptop to keep up with the rate of data acquisition. Read more in the
MinION IT Requirements document.

MinION Mk1C IT requirements

The MinION Mk1C contains fully-integrated compute and screen, removing the need for any accessories to generate and analyse
nanopore data. Read more in the MinION Mk1C IT requirements document.

Software for nanopore sequencing

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data in real time and processes it into
basecalls. You will be using MinKNOW for every sequencing experiment. MinKNOW can also demultiplex reads by barcode, and
basecall/demultiplex data after a sequencing run has completed.
MinKNOW use
For instructions on how to run the MinKNOW software, please refer to the relevant section in the MinKNOW protocol.

EPI2ME (optional)

The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome,
barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-
basecalling.
EPI2ME installation and use
For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to the EPI2ME Platform
protocol.

Guppy (optional)

The Guppy command-line software can be used for basecalling and demultiplexing reads by barcode instead of MinKNOW. You can use
it if you would like to re-analyse old data, or integrate basecalling into your analysis pipeline.
Guppy installation and use
If you would like to use the Guppy software, please refer to the Guppy protocol.

Check your flow cell

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be
done within three months of purchasing for MinION/GridION/PromethION flow cells, or within four weeks of purchasing for Flongle flow
cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result
is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the
flow cell check, please follow the instructions in the Flow Cell Check document.

Flow cell Minimum number of active pores covered by warranty

Flongle Flow Cell 50

MinION/GridION Flow 800

Cell

PromethION Flow Cell 5000

Library preparation
Library preparation
~10 minutes

Materials ~400 ng high molecular weight genomic DNA

Fragmentation Mix (FRA)
Rapid Adapter (RAP)

Consumables 0.2 ml thin-walled PCR tubes

Nuclease-free water (e.g. ThermoFisher, AM9937)
 Equipment Thermal cycler or heat block at 30°C and 80°C
P2 pipette and tips
P10 pipette and tips

DNA tagmentation

1 Thaw kit components at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated
by the table below:

Reagent 1. Thaw at room 2. Briefly spin 3. Mix well by pipetting

temperature down

Fragmentation Mix Not frozen ✓ ✓

(FRA)

Rapid Adapter (RAP) Not frozen ✓ ✓

Sequencing Buffer ✓ ✓ ✓*
(SQB)

Loading Beads (LB) ✓ ✓ Mix by pipetting or vortexing immediately

before use

Flush Buffer (FLB) - 1 ✓ ✓ ✓*

tube

Flush Tether (FLT) ✓ ✓ ✓

*Vortexing, followed by a brief spin in a microfuge, is recommended for Sequencing Buffer (SQB) and Flush Buffer (FLB).

Please note that the Sequencing Tether (SQT) tube will NOT be used in this protocol. It is provided in the kit for
potential future product compatibility.

2 Once thawed, keep all the kit components on ice.

3 Prepare the DNA in nuclease-free water.

Transfer ~400 ng genomic DNA into a DNA LoBind tube

Adjust the volume to 7.5 μl with nuclease-free water
Mix by flicking the tube to avoid unwanted shearing
Spin down briefly in a microfuge

4 In a 0.2 ml thin-walled PCR tube, mix the following:

Reagent Volume

400 ng template 7.5 μl

DNA

FRA 2.5 μl

Total 10 μl
5 Mix gently by flicking the tube, and spin down.

6 Incubate the tube at 30°C for 1 minute and then at 80°C for 1 minute. Briefly put the tube on ice to cool it down.

TIP

If heat blocks are used instead of a thermal cycler, incubation at both temperatures should be extended to 2
minutes.

400 ng tagmented DNA in 10 μl is taken into the next step.

Adapter attachment

7 Add 1 μl of RAP to the tube.

8 Mix gently by flicking the tube, and spin down.

9 Incubate the reaction for 5 minutes at room temperature.

END OF STEP

The prepared DNA library is used for loading into the flow cell. Store the library on ice until ready to load.

Priming and loading the SpotON Flow Cell

~10 minutes

Materials Flow Cell Priming Kit (EXP-FLP002)

Sequencing Buffer (SQB)
Loading Beads (LB)

Consumables 1.5 ml Eppendorf DNA LoBind tubes

Nuclease-free water (e.g. ThermoFisher, cat # AM9937)

Equipment MinION Mk1B

SpotON Flow Cell
P1000 pipette and tips
P100 pipette and tips
P20 pipette and tips
P10 pipette and tips
 IMPORTANT

Please note that the Sequencing Tether (SQT) tube will NOT be used in this protocol.

TIP

Priming and loading a flow cell

We recommend all new users watch the 'Priming and loading your flow cell' video before your first run.

1 Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at
room temperature before mixing the reagents by vortexing, and spin down at room temperature.

2 To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of
thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.

3 Open the MinION device lid and slide the flow cell under the clip.

Press down firmly on the flow cell to ensure correct thermal and electrical contact.

1a
Insert the flow cell into the
device under the clip and
press down firmly.

Key:

Storage buffer

Priming mix

DNA library

1b
Insert the flow cell into the
device under the clip and
press down firmly.

Optional Action
Complete a flow cell check to assess the number of pores available before loading the library.

This step can be omitted if the flow cell has been checked previously.
See the flow cell check instructions in the MinKNOW protocol for more information.

4 Slide the priming port cover clockwise to open the priming port.

IMPORTANT

Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the
array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage
pores.

5 After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove
any bubbles:
1. Set a P1000 pipette to 200 µl
2. Insert the tip into the priming port
3. Turn the wheel until the dial shows 220-230 ul, to draw back 20-30 ul, or until you can see a small volume of buffer entering
the pipette tip
Note: Visually check that there is continuous buffer from the priming port across the sensor array.
6 Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait
for 5 minutes. During this time, prepare the library for loading by following the steps below.

7 Thoroughly mix the contents of the Loading Beads (LB) tubes by vortexing.

8 In a new tube, prepare the library for loading as follows:

Reagent Volume per flow cell

Sequencing Buffer (SQB) 34 µl

Loading Beads (LB), mixed immediately before 25.5 µl

use

Nuclease-free water 4.5 µl

DNA library 11 µl

Total 75 µl

Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer (SQB) and Loading Beads (LB) because
the fuel in the buffer will start to be consumed by the adapter.

IMPORTANT

The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they
are mixed immediately before use.
9 Complete the flow cell priming:
1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
2. Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air
bubbles.

5
Gently flip open the
SpotON
sample port cover.

10 Mix the prepared library gently by pipetting up and down just prior to loading.
11 Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each
drop flows into the port before adding the next.

12 Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port
and replace the MinION device lid.

Sequencing and data analysis

Data acquisition and basecalling

Overview of nanopore data analysis

For a full overview of nanopore data analysis, which includes options for basecalling and post-basecalling analysis, please refer to the
Data Analysis document.

How to start sequencing

The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software. It is assumed you
have already installed MinKNOW on your computer. There are multiple options for how to carry out sequencing:

1. Data acquisition and basecalling in real-time using MinKNOW on a computer

Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the
"Completing a MinKNOW run" section.

2. Data acquisition and basecalling in real-time using the GridION device

Follow the instructions in the GridION user manual.

3. Data acquisition and basecalling in real-time using the MinION Mk1C device

Follow the instructions in the MinION Mk1C user manual.

4. Data acquisition and basecalling in real-time using the PromethION device

Follow the instructions in the PromethION user manual or the PromethION 2 Solo user manual.

5. Data acquisition using MinKNOW on a computer and basecalling at a later time using MinKNOW or
Guppy

Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the
"Completing a MinKNOW run" section. When setting your experiment parameters, set the Basecalling tab to OFF. After the
sequencing experiment has completed, follow the instructions in the Post-run analysis section of the MinKNOW protocol or the Guppy
protocol starting from the "Quick Start Guide for Guppy" section.

Downstream analysis

Post-basecalling analysis

There are several options for further analysing your basecalled data:

1. EPI2ME platform

The EPI2ME platform is a cloud-based data analysis service developed by Metrichor Ltd., a subsidiary of Oxford Nanopore
Technologies. The EPI2ME platform offers a range of analysis workflows, e.g. for metagenomic identification, barcoding, alignment,
and structural variant calling. The analysis requires no additional equipment or compute power, and provides an easy-to-interpret
report with the results. For instructions on how to run an analysis workflow in EPI2ME, please follow the instructions in the EPI2ME
protocol, beginning at the "Starting data analysis" step.

2. EPI2ME Labs tutorials and workflows

For more in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in
EPI2ME Labs, which are available in the EPI2ME Labs section of the Community. The platform provides a vehicle where workflows
deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code
and example data.

3. Research analysis tools

Oxford Nanopore Technologies' Research division has created a number of analysis tools, which are available in the Oxford Nanopore
GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are
provided as-is, with minimal support.

4. Community-developed analysis tools

If a data analysis method for your research question is not provided in any of the resources above, please refer to theBioinformatics
section of the Resource centre. Numerous members of the Nanopore Community have developed their own tools and pipelines for
analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by
Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.

Ending the experiment

Materials Flow Cell Wash Kit (EXP-WSH004)

1 After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Wash Kit
instructions and store the washed flow cell at 2-8°C, OR

The Flow Cell Wash Kit protocol is available on the Nanopore Community.

TIP

We recommend you to wash the flow cell as soon as possible after you stop the run. However, if this is not
possible, leave the flow cell on the device and wash it the next day.

2 Follow the returns procedure to flush out the flow cell ready to send back to Oxford Nanopore.

Instructions for returning flow cells can be foundhere.

All flow cells must be flushed with deionised water before returning the product.
 IMPORTANT

If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting
Guide that can be found in the online version of this protocol.

Troubleshooting
Issues during DNA/RNA extraction and library
preparation

Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

We also have an FAQ section available on theNanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email
([email protected]) or via LiveChat in the Nanopore Community.

Low sample quality

Observation Possible cause Comments and actions

Low DNA purity (Nanodrop reading The DNA extraction The effects of contaminants are shown in the Contaminants
for DNA OD 260/280 is <1.8 and OD method does not Know-how piece. Please try an alternative extraction method
260/230 is <2.0–2.2) provide the required that does not result in contaminant carryover.
purity
Consider performing an additional SPRI clean-up step.

Low RNA integrity (RNA integrity The RNA degraded Try a different RNA extraction method. For more info on RIN,
number <9.5 RIN, or the rRNA band during extraction please see the RNA Integrity Number Know-how piece.
is shown as a smear on the gel)

RNA has a shorter than expected The RNA degraded Try a different RNA extraction method. For more info on RIN,
fragment length during extraction please see the RNA Integrity Number Know-how piece.

We recommend working in an RNase-free environment, and to

keep your lab equipment RNase-free when working with RNA.

Low DNA recovery after AMPure bead clean-up

Observation Possible cause Comments and actions

Low DNA loss due to a 1. AMPure beads settle quickly, so ensure they are well resuspended before adding them to
recovery lower than intended the sample.
AMPure beads-to-
sample ratio 2. When the AMPure beads-to-sample ratio is lower than 0.4:1, DNA fragments of any size
will be lost during the clean-up.
 Observation Possible cause Comments and actions

Low DNA fragments are The lower the AMPure beads-to-sample ratio, the more stringent the selection against short
recovery shorter than fragments. Please always determine the input DNA length on an agarose gel (or other gel
expected electrophoresis methods) and then calculate the appropriate amount of AMPure beads to
use.

Low The wash step used DNA will be eluted from the beads when using ethanol <70%. Make sure to use the correct
recovery ethanol <70% percentage.
after end-
prep

Issues during the sequencing run

Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

We also have an FAQ section available on theNanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email
([email protected]) or via LiveChat in the Nanopore Community.

Fewer pores at the start of sequencing than after Flow Cell Check

Observation Possible Comments and actions

cause

MinKNOW An air bubble After the Flow Cell Check it is essential to remove any air bubbles near the priming port before
reported a was introduced priming the flow cell. If not removed, the air bubble can travel to the nanopore array and
lower number into the irreversibly damage the nanopores that have been exposed to air. The best practice to
of pores at the nanopore array prevent this from happening is demonstrated in this video.
start of
sequencing
than the
number
reported by
the Flow Cell
Check
 Observation Possible Comments and actions
cause

MinKNOW The flow cell is Stop the sequencing run, remove the flow cell from the sequencing device and insert it again,
reported a not correctly checking that the flow cell is firmly seated in the device and that it has reached the target
lower number inserted into temperature. If applicable, try a different position on the device (GridION/PromethION).
of pores at the the device
start of
sequencing
than the
number
reported by
the Flow Cell
Check

MinKNOW Contaminations The pore count during the Flow Cell Check is performed using the QC DNA molecules present
reported a in the library in the flow cell storage buffer. At the start of sequencing, the library itself is used to estimate
lower number damaged or the number of active pores. Because of this, variability of about 10% in the number of pores is
of pores at the blocked the expected. A significantly lower pore count reported at the start of sequencing can be due to
start of pores contaminants in the library that have damaged the membranes or blocked the pores.
sequencing Alternative DNA/RNA extraction or purification methods may be needed to improve the purity
than the of the input material. The effects of contaminants are shown in the Contaminants Know-how
number piece. Please try an alternative extraction method that does not result in contaminant
reported by carryover.
the Flow Cell
Check

MinKNOW script failed

Observation Possible Comments and actions

cause

MinKNOW shows Restart the computer and then restart MinKNOW. If the issue persists, please collect the
"Script failed" MinKNOW log files and contact Technical Support.

Pore occupancy below 40%

Observation Possible cause Comments and actions

Pore Not enough library was loaded 5–50 fmol of good quality library can be loaded on to a MinION Mk1B/GridION flow
occupancy on the flow cell cell. Please quantify the library before loading and calculate mols using tools like
<40% the Promega Biomath Calculator, choosing "dsDNA: µg to pmol"

Pore The Ligation Sequencing Kit Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford
occupancy was used, and sequencing Nanopore Technologies Ligation Buffer (LNB, provided in the SQK-LSK109 kit) at
close to 0 adapters did not ligate to the the sequencing adapter ligation step, and use the correct amount of each
DNA reagent. A Lambda control library can be prepared to test the integrity of the
third-party reagents.

Pore The Ligation Sequencing Kit Ethanol can denature the motor protein on the sequencing adapters. Make sure
occupancy was used, and ethanol was the LFB or SFB buffer was used after ligation of sequencing adapters.
close to 0 used instead of LFB or SFB at
the wash step after sequencing
adapter ligation

Pore No tether on the flow cell Tethers are adding during flow cell priming (FLT tube). Make sure FLT was added
occupancy to FB before priming.
close to 0
 Observation Possible cause Comments and actions

Shorter than expected read length

Observation Possible cause Comments and actions

Shorter than Unwanted fragmentation Read length reflects input DNA fragment length. Input DNA can be
expected read length of DNA sample fragmented during extraction and library prep.

1. Please review the Extraction Methods in the Nanopore Community for

best practice for extraction.
2. Visualise the input DNA fragment length distribution on an agarose gel
before proceeding to the library prep.

In the image above, Sample 1 is of high molecular weight, whereas Sample

2 has been fragmented.

3. During library prep, avoid pipetting and vortexing when mixing reagents.
Flicking or inverting the tube is sufficient.

Large proportion of recovering pores

Observation Possible Comments and actions

cause
 Observation Possible Comments and actions
cause

Large proportion of Contaminants Some contaminants can be cleared from the pores by the unblocking function
recovering pores (shown as are present in built into MinKNOW. If this is successful, the pore status will change to "single
dark blue in the channels the sample pores". If the portion of recovering pores (unavailable pores in the extended
panel and duty time plot) view) stays large or increases:

1. A nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) can be
performed, or
2. Run several cycles of PCR to try and dilute any contaminants that may be
causing problems.

The duty time plot above shows an increasing proportion of "recovering" pores
over the course of a sequencing experiment

Large proportion of inactive pores

Observation Possible Comments and actions

cause

Large proportion of inactive pores (shown as Air bubbles Air bubbles introduced through flow cell priming and
light blue in the channels panel and duty time have been library loading can irreversibly damage the pores. Watch
plot. Pores or membranes are irreversibly introduced into the Priming and loading your flow cell video for best
damaged) the flow cell practice

Large proportion of inactive pores Certain Known compounds, include polysaccharides, typically
compounds co- associate with plant genomic DNA.
purified with
DNA 1. Please refer to the Plant leaf DNA extraction method.
2. Clean-up using the QIAGEN PowerClean Pro kit.
3. Perform a whole genome amplification with the
original gDNA sample using the QIAGEN REPLI-g kit.

Large proportion of inactive pores Contaminants The effects of contaminants are shown in the
are present in Contaminants Know-how piece. Please try an alternative
the sample extraction method that does not result in contaminant
carryover.

Reduction in sequencing speed and q-score later into the run

 Observation Possible cause Comments and actions

Reduction in Fast fuel consumption is typically seen when the flow Add more fuel to the flow cell by following the
sequencing speed cell is overloaded with library (please see the instructions in the MinKNOW protocol. In future
and q-score later appropriate protocol for your DNA library to see the experiments, load lower amounts of library to the
into the run recommendation). flow cell.

Temperature fluctuation

Observation Possible Comments and actions

cause

Temperature The flow cell Check that there is a heat pad covering the metal plate on the back of the flow cell. Re-insert the
fluctuation has lost contact flow cell and press it down to make sure the connector pins are firmly in contact with the device.
with the device If the problem persists, please contact Technical Services.

Failed to reach target temperature

Observation Possible cause Comments and actions

MinKNOW The instrument was MinKNOW has a default timeframe for the flow cell to reach the target temperature.
shows placed in a location that Once the timeframe is exceeded, an error message will appear and the sequencing
"Failed to is colder than normal experiment will continue. However, sequencing at an incorrect temperature may lead
reach target room temperature, or a to a decrease in throughput and lower q-scores. Please adjust the location of the
temperature" location with poor sequencing device to ensure that it is placed at room temperature with good
ventilation (which leads ventilation, then re-start the process in MinKNOW. Please refer to this FAQ for more
to the flow cells information on MinION Mk 1B temperature control.
overheating)

Guppy – no input .fast5 was found or basecalled

Observation Possible cause Comments and actions

No input .fast5 input_path did not point to The --input_path has to be followed by the full file path to the .fast5 files to be
was found or the .fast5 file location basecalled, and the location has to be accessible either locally or remotely
basecalled through SSH.

No input .fast5 The .fast5 files were in a To allow Guppy to look into subfolders, add the--recursive flag to the
was found or subfolder at the input_path command
basecalled location

Guppy – no Pass or Fail folders were generated after basecalling

Observation Possible cause Comments and actions

No Pass or Fail The -- The --qscore_filtering flag enables filtering of reads into Pass and Fail folders inside the
folders were qscore_filtering output folder, based on their strand q-score. When performing live basecalling in MinKNOW,
generated after flag was not a q-score of 7 (corresponding to a basecall accuracy of ~80%) is used to separate reads
basecalling included in the into Pass and Fail folders.
command

Guppy – unusually slow processing on a GPU computer

 Observation Possible cause Comments and actions

Unusually slow The --device flag The --device flag specifies a GPU device to use for accelerate basecalling. If not included in
processing on a wasn't included the command, GPU will not be used. GPUs are counted from zero. An example is --device
GPU computer in the command cuda:0 cuda:1, when 2 GPUs are specified to use by the Guppy command.

MinIT – the MinKNOW interface is not shown in the web browser

Observation Possible cause Comments and actions

The MinKNOW Browser compatibility Always use Google Chrome as the browser to view MinKNOW. Alternatively,
interface is not issue instead of typing //mt-xxxxxx (x is a number) in the address bar, type in in the
shown in the web generic IP address, 10.42.0.1, which identifies the MinIT Wi-Fi router.
browser

The MinKNOW The MinIT Wi-Fi was not Make sure the computer or mobile device is using the MinIT Wi-Fi. It should be
interface is not used for connecting to the shown as MT-xxxxxx (x is a number) on the underside label on the MinIT:
shown in the web computer or mobile
browser device

Disable the Ethernet connection from the computer or mobile device as

needed. If necessary, contact your IT department to determine if the MinIT Wi-
Fi is blocked (MinIT generic IP: 10.42.0.1). Please white-list MinIT as needed.

The MinKNOW The MinIT was not on the Make sure that the wall sockets used by the Ethernet cables from the MinIT and
interface is not same network that the computer belong to the same local network.
shown in the web computer was connected
browser to.

MinIT – the MinIT software cannot be updated

Observation Possible Comments and actions

cause

The MinIT The Please consult your IT department, as the MinIT software requires access to thefollowing AWS IP ranges.
software firewall Access to the following IP addresses is also needed:
cannot be is 178.79.175.200
updated blocking 96.126.99.215
IPs for
update

The MinIT The Occassionaly, the MinIT software admin page displays "updates available" even when the software is
software device already up-to-date. Please compare the version listed on the admin page with the one on the Software
cannot be already Downloads page. Alternatively, SSH into the MinIT through a SSH Client (e.g. Bitvise or Putty, as
updated has the described in the MinIT protocol) on a Windows computer or the terminal window on a Mac, run the
latest command, dpkg -l | grep minit, to find out the version of the MinIT software and sudo apt update if an
version update is needed. If the issue still persists, please contact Technical Services with details of the error.
of the
software