MinION: Protocol

V GDX_9095_v109_revL_24Jan2020

FOR RESEARCH USE ONLY

1. Overview of the protocol

重要

This is a Legacy product

This kit has now been discontinued and we recommend all customers upgrade to the latest chemistry for their relevant kit which is available on the Store. For further information on please see the product update page.

Ligation Sequencing Kit XL features

This kit is recommended for users who:

would like to process multiple samples simultaneously, either with a multichannel pipette or a liquid-handling robot
want to optimise their sequencing experiment for throughput
would like to utilise upstream processes such as size selection, whole genome amplification, or enrichment for long reads

重要

Optional fragmentation and size selection

By default, the protocol contains no DNA fragmentation step, however in some cases it may be advantageous to fragment your sample. For example, when working with lower amounts of input gDNA (100 ng – 500 ng), fragmentation will increase the number of DNA molecules and therefore increase throughput. Instructions are available in the DNA Fragmentation section of Extraction methods.

Additionally, we offer several options for size-selecting your DNA sample to enrich for long fragments - instructions are available in the Size Selection section of Extraction methods.

Introduction to the Ligation Sequencing protocol for gDNA

This protocol describes how to carry out sequencing of multiple DNA samples simultaneously, using the Ligation Sequencing Kit XL (SQK-LSK109-XL). 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
Download the software for acquiring and analysing your data
Check your flow cell to ensure it has enough pores for a good sequencing run

Library preparation

You will need to:

Repair the DNA, and prepare the DNA ends for adapter attachment
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
Start the EPI2ME software and select a workflow for further analysis (this step is optional)

重要

Compatibility of this protocol

This protocol should only be used in combination with:

Ligation Sequencing Kit XL (SQK-LSK109-XL)
Control Expansion (EXP-CTL001)
FLO-MIN106 flow cells (R9.4.1)
Flow Cell Wash Kit (EXP-WSH004)
Sequencing Auxiliary Vials (EXP-AUX001)
PCR Barcoding Kits (EXP-PBC001 and EXP-PBC096)
Native Barcoding Kits (EXP-NBD104 and EXP-NBD114)

2. Equipment and consumables

材料

1 µg (又は100-200 fmol) のgDNA
1.5-3 µg (or 150-300 fmol) high molecular weight genomic DNA if using R10.3 flow cells
または、DNA断片化を行う場合は100 ng以上の高分子ゲノムDNA
Ligation Sequencing Kit XL (SQK-LSK109-XL)
Flow Cell Priming Kit XL (EXP-FLP002-XL)

消耗品

Agencourt AMPure XP beads (Beckman Coulter, A63881)
NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S or E7180L). Alternatively, you can use the NEBNext® products below:
NEBNext FFPE Repair Mix (NEB, M6630)
NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
NEBNext Quick Ligation Module (NEB, E6056)
Nuclease-free water (e.g. ThermoFisher, AM9937)
Freshly prepared 70% ethanol in nuclease-free water
1.5 ml Eppendorf DNA LoBind tubes
Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
OR 0.2 ml thin-walled PCR tubes
15 or 50 ml Falcon tubes

装置

Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
OR magnetic separator suitable for 0.2 ml PCR tube strips, e.g. DynaMag™-PCR Magnet (Thermo Fisher, #492025) or DynaMag™-96 Side Magnet (Thermo Fisher, #12331D)
小型遠心機
Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
ボルテックスミキサー
サーマルサイクラー
Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl, and tips
P1000 ピペット及びチップ
P200 ピペットとチップ
P100 ピペットとチップ
P20 ピペットとチップ
P10 ピペットとチップ
P2 ピペットとチップ
アイスバケツ（氷入り）
タイマー
Pipetting troughs

オプション装置

Agilent Bioanalyzer (or equivalent)
Qubit蛍光光度計（またはQCチェックのための同等品）
Eppendorf 5424 centrifuge (or equivalent)

For this protocol, you will need 1 µg (or 100-200 fmol) gDNA.

Although 1 µg (or 100-200 fmol) gDNA is recommended, users can start with lower input quantities (down to 100 ng) if performing DNA fragmentation to increase the number of DNA molecules in the sample, or if amplifying the sample by PCR.

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 the Input 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.

NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing

For customers new to nanopore sequencing, we recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.

Please note, for our amplicon protocols, NEBNext FFPE DNA Repair Mix and NEBNext FFPE DNA Repair Buffer are not required.

Multichannel pipettes

For scaling up library prep using the Ligation Sequencing Kit XL, customers will need multichannel pipettes and appropriate pipette tips. Although the choice of brand is left to the user's discretion, our R&D team can recommend Rainin Pipet-Lite LTS L200-XLS+ (20–200 μl) and Pipet-Lit LTS L20-XLS+ (2–20 μl) pipettes and Rainin LTS pipette tips.

Ligation Sequencing Kit XL (SQK-LSK109-XL) contents

Name	Acronym	Cap colour	No. of tubes	Full volume (μl)
DNA CS	DCS	Yellow	1	52
Adapter Mix	AMX	Green	4	60
Ligation Buffer	LNB	Clear	4	300
L Fragment Buffer	LFB	White cap, orange stripe on label	4	6,000
S Fragment Buffer	SFB	Grey	4	6,000
Sequencing Buffer	SQB	Red	4	900
Elution Buffer	EB	Black	1	1,200
Loading Beads	LB	Pink	4	612

Flow Cell Priming Kit XL (EXP-FLP002-XL) contents

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
Flush Buffer	FB	Blue	4	15,400
Flush Tether	FLT	Purple	4	400

3. 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. For more information, refer to the MinION Mk1B 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. For more information refer to the MinION Mk1C IT requirements document.

MinION Mk1D IT requirements

Sequencing on a MinION Mk1D requires a high-spec computer or laptop to keep up with the rate of data acquisition. For more information, refer to the MinION Mk1D IT requirements document.

Software for nanopore sequencing

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.

For instructions on how to run the MinKNOW software, please refer to 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.

For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to this link.

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 12 weeks of purchasing for MinION/GridION/PromethION or within four weeks of purchasing 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 Cell	800
PromethION Flow Cell	5000

4. DNA repair and end-prep

材料

gDNA in 47 µl nuclease-free water
DNA Control Sample (DCS)

消耗品

Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
OR 0.2 ml thin-walled PCR tubes
Nuclease-free water (e.g. ThermoFisher, AM9937)
NEBNext® FFPE DNA Repair Mix (NEB, M6630)
NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
Agencourt AMPure XP beads (Beckman Coulter™, A63881)
Freshly prepared 70% ethanol in nuclease-free water

装置

Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
OR magnetic separator suitable for 0.2 ml PCR tube strips, e.g. DynaMag™-PCR Magnet (Thermo Fisher, #492025) or DynaMag™-96 Side Magnet (Thermo Fisher, #12331D)
Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl, and tips
P1000 ピペット及びチップ
P100 ピペットとチップ
P10 ピペットとチップ
サーマルサイクラー
小型遠心機
Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
ボルテックスミキサー
アイスバケツ（氷入り）
Pipetting troughs

重要

Optional fragmentation and size selection

Additionally, we offer several options for size-selecting your DNA sample to enrich for long fragments - instructions are available in the Size Selection section of Extraction methods.

For optimal performance, NEB recommend the following:

Thaw all reagents on ice.
Flick and/or invert the reagent tubes to ensure they are well mixed.
Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.
Always spin down tubes before opening for the first time each day.
The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
Note: It is important the buffers are mixed well by vortexing.
The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.

重要

It is important that the NEBNext FFPE DNA Repair Buffer and NEBNext Ultra II End Prep Reaction Buffer are mixed well by vortexing.

Check for any visible precipitate; vortexing for at least 30 seconds may be required to solubilise any precipitate.

重要

Do not vortex the NEBNext FFPE DNA Repair Mix or NEBNext Ultra II End Prep Enzyme Mix.

For R9.4.1 flow cells, transfer 1 μg (or 100-200 fmol) genomic DNA into a separate well of a 96-well plate or a 0.2 ml PCR tube strip
For R10.3 flow cells, transfer 1.5-3 μg (or 150-300 fmol) genomic DNA into a separate well of a 96-well plate or a 0.2 ml PCR tube strip
Adjust the volume to 47 μl with nuclease-free water
Mix thoroughly by pipetting up and down, or by flicking the tube
If necessary, seal and spin down briefly in an appropriate centrifuge

Between each addition, pipette mix 10-20 times.

Reagent	Volume
DNA CS	1 µl
NEBNext FFPE DNA Repair Buffer	3.5 µl
NEBNext FFPE DNA Repair Mix	2 µl
Ultra II End-prep reaction buffer	3.5 µl
Ultra II End-prep enzyme mix	3 µl
Total	13 µl
Total including DNA	60 µl

ヒント

For ease, make a master mix of these reagents prior to adding to the DNA samples:

Combine the repair and end-prep reagents in the correct ratio and mix well by gently pipetting the entire volume up and down 10 times (ensure the total volume is enough to accommodate 13 µl being added to each DNA sample, with an excess to allow for pipetting losses).
Add 13 µl of the master mix to each DNA sample. This can be done by pre-aliquoting the master mix and transferring 13 µl into each sample tube simultaneously, using a multichannel pipette.

AMPure XP bead clean-up

It is recommended that the repaired/end-prepped DNA samples are subjected to the following clean-up with AMPure XP beads. This clean-up can be omitted for simplicity and to reduce library preparation time. However, it has been observed that omission of this clean-up can: reduce subsequent adapter ligation efficiency, increase the prevalence of chimeric reads, and lead to an increase in pores being unavailable for sequencing. If omitting the clean-up step, proceed to the next section (“Adapter Ligation and Clean-Up”).

重要

Resuspend and transfer the beads to the pipetting trough immediately before use to ensure beads do not settle.

チェックポイント

Quantify 1 µl of each eluted sample using a Qubit fluorometer.

最終ステップ

Take forward the repaired and end-prepped DNA into the adapter ligation step. However, at this point it is also possible to store the samples at 4° C overnight.

5. Adapter ligation and clean-up

材料

Adapter Mix (AMX)
Ligation Buffer (LNB)
Long Fragment Buffer (LFB)
Short Fragment Buffer (SFB)
Elution Buffer (EB)

消耗品

Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
OR 0.2 ml thin-walled PCR tubes
NEBNext Quick Ligation Module (NEB, E6056)
Agencourt AMPure XP beads (Beckman Coulter™, A63881)

装置

Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
OR magnetic separator suitable for 0.2 ml PCR tube strips, e.g. DynaMag™-PCR Magnet (Thermo Fisher, #492025) or DynaMag™-96 Side Magnet (Thermo Fisher, #12331D)
小型遠心機
Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
ボルテックスミキサー
Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl, and tips
P1000 ピペット及びチップ
P100 ピペットとチップ
P20 ピペットとチップ
P10 ピペットとチップ
Pipetting troughs

重要

Although the recommended 3rd party ligase is supplied with its own buffer, the ligation efficiency of Adapter Mix (AMX) is higher when using Ligation Buffer supplied within SQK-LSK109-XL.

重要

Depending on the wash buffer (LFB or SFB) used, the clean-up step after adapter ligation is designed to either enrich for DNA fragments of >3 kb, or purify all fragments equally.

To enrich for DNA fragments of 3 kb or longer, use Long Fragment Buffer (LFB)
To retain DNA fragments of all sizes, use Short Fragment Buffer (SFB)

ヒント

Each vial of Adapter Mix (AMX), Ligation Buffer (LNB) and S Fragment Buffer (SFB)/L Fragment Buffer (LFB) provides enough reagent for the preparation of 12 samples. Prepare the appropriate number of tubes/bottles of each reagent.

ヒント

Once Short Fragment Buffer (SFB), Long Fragment Buffer (LFB) and Elution Buffer (EB) are thawed, they can be aliquoted and stored for up to one month at 4°C.

Between each addition, pipette mix 10-20 times.

Reagent	Volume
Ligation Buffer (LNB)	25 µl
NEBNext Quick T4 DNA Ligase	10 µl
Adapter Mix (AMX)	5 µl
Total	40 µl
Total including DNA	100 µl

ヒント

For ease, you can pre-aliquot the reagents separately into empty PCR tubes, from which the reagents are transferred to the DNA samples using a multichannel pipette.

Ensure excess volumes of the reagents are present in these tubes. Leftover reagent should be recovered. For example, for twelve separate DNA samples, aliquot:

Reagent	Volume
Ligation Buffer (LNB)	30 µl into each of 12 clean PCR tubes
NEBNext Quick T4 DNA Ligase	12 µl into each of 12 clean PCR tubes
Adapter Mix (AMX)	6 µl into each of 12 clean PCR tubes

Alternatively, you can make a master mix of these reagents (allowing up to a 20% excess of each reagent), and add 40 μl of this to each DNA sample. However, ligation efficiency may be compromised if the master mix is not used within 10 minutes.

重要

If you have omitted the AMPure purification step after DNA repair and end-prep, do not incubate the reaction for longer than 10 minutes.

重要

Resuspend and transfer the beads to the pipetting trough immediately before use to ensure beads do not settle.

重要

It is essential that beads are resuspended fully and not simply moved around the tubes through use of the magnet.

ヒント

Ensure the beads are fully resuspended. Brief centrifugation can be used to help to draw liquid droplets and beads to the bottom of the wells/tubes during and after resuspension.

チェックポイント

Quantify 1 µl of each eluted sample using a Qubit fluorometer.

重要

We recommend loading the final prepared library onto a flow cell following one of our recommendations depending on the flow cell type:

R9.4.1 flow cells, load 5-50 fmol
R10.3 flow cells, load 25-75 fmol

Loading more than the maximal recommended amount of DNA can have a detrimental effect on output as higher quantities of DNA results in a larger number of ligated DNA ends with loaded motor protein. This depletes fuel in the Sequencing Buffer, regardless of whether or not the DNA fragments are being sequenced. This leads to fuel depletion and speed drop-off early in the sequencing run. Dilute the libraries in Elution Buffer if required.

If you are using the Flongle for sample prep development, we recommend loading 3-20 fmol instead.

最終ステップ

The prepared libraries are used for loading into the flow cells. Store the libraries on ice until ready to load.

ヒント

Library storage recommendations

We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short term storage or repeated use, for example, reloading flow cells between washes. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes. For further information, please refer to the DNA library stability Know-How document.

オプショナルステップ

If quantities allow, the libraries may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.

Additional buffer for doing this can be found in the Sequencing Auxiliary Vials expansion (EXP-AUX001), available to purchase separately. This expansion also contains additional vials of Sequencing Buffer (SQB) and Loading Beads (LB), required for loading the libraries onto flow cells.

6. Priming and loading the SpotON flow cell

材料

Loading Beads (LB)
Sequencing Buffer (SQB)
Flow Cell Priming Kit XL (EXP-FLP002-XL)

消耗品

Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
OR 0.2 ml thin-walled PCR tubes
1.5 ml Eppendorf DNA LoBind tubes
15 or 50 ml Falcon tubes
Nuclease-free water (e.g. ThermoFisher, AM9937)

装置

MinIONかGridION のデバイス
MinIONとGridIONのFlow Cell ライトシールド
Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl, and tips
P1000 ピペット及びチップ
P100 ピペットとチップ
P20 ピペットとチップ
P10 ピペットとチップ

ヒント

Each vial provides enough reagent for the preparation of 12 samples. Thaw the appropriate number of vials of each reagent.

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

オプショナルステップ

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.

重要

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.

Set a P1000 pipette to 200 µl
Insert the tip into the priming port
Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, 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.

重要

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.

Reagent	Volume
Sequencing Buffer (SQB)	37.5 µl
Loading Beads (LB), mixed immediately before use	25.5 µl
DNA library	12 µl
Total	75 µl

ヒント

For ease, make a master mix of Sequencing Buffer (SQB) and Loading Beads (LB) before adding to the DNA samples:

Combine the Sequencing Buffer (SQB) and Loading Beads (LB) in the correct ratio and mix well by vortexing. Allow up to a 20% excess of each reagent.
Add 63 µl of the master mix to each DNA sample and mix well by pipetting.

It is imperative that the master mix is mixed well immediately before any transfer to prevent unequal distribution of Loading Beads between samples.

Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

重要

Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.

We recommend leaving the light shield on the flow cell when library is loaded, including during any washing and reloading steps. The shield can be removed when the library has been removed from the flow cell.

Carefully place the leading edge of the light shield against the clip. Note: 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.

注意

The MinION Flow Cell Light Shield is not secured to the flow cell and careful handling is required after installation.

最終ステップ

Close the device lid and set up a sequencing run on MinKNOW.

7. Data acquisition and basecalling

How to start sequencing

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

MinKNOW can be used and set up to sequence in multiple ways:

On a computer either directly or remotely connected to a sequencing device.
Directly on a GridION or PromethION 24/48 sequencing device.

For more information on using MinKNOW on a sequencing device, please see the device user manuals:

To start a sequencing run on MinKNOW:

1. Navigate to the start page and click Start sequencing.

2. Fill in your experiment details, such as name and flow cell position and sample ID.

3. Select the sequencing kit used in the library preparation on the Kit page.

4. Configure the sequencing and output parameters for your sequencing run or keep to the default settings on the Run configuration tab.

Note: If basecalling was turned off when a sequencing run was set up, basecalling can be performed post-run on MinKNOW. For more information, please see the MinKNOW protocol.

5. Click Start to initiate the sequencing run.

Data analysis after sequencing

After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.

After sequencing and basecalling, the data can be analysed. For further information about options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

In the Downstream analysis section, we outline further options for analysing your data.

8. Flow cell reuse and returns

材料

Flow Cell Wash Kit (EXP-WSH004)

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

ヒント

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.

Instructions for returning flow cells can be found here.

重要

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.

9. Downstream analysis

Post-basecalling analysis

There are several options for further analysing your basecalled data:

1. EPI2ME workflows

For in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in EPI2ME. 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.

2. 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.

3. 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 the resource centre and search for bioinformatics tools for your application. 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.

10. 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 the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

Low sample quality

Observation	Possible cause	Comments and actions
Low DNA purity (Nanodrop reading for DNA OD 260/280 is <1.8 and OD 260/230 is <2.0–2.2)	The DNA extraction method does not provide the required purity	The effects of contaminants are shown in the Contaminants document. Please try an alternative extraction method that does not result in contaminant carryover. Consider performing an additional SPRI clean-up step.
Low RNA integrity (RNA integrity number <9.5 RIN, or the rRNA band is shown as a smear on the gel)	The RNA degraded during extraction	Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page.
RNA has a shorter than expected fragment length	The RNA degraded during extraction	Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page. 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 recovery	DNA loss due to a lower than intended AMPure beads-to-sample ratio	1. AMPure beads settle quickly, so ensure they are well resuspended before adding them to the sample. 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.
Low recovery	DNA fragments are shorter than expected	The lower the AMPure beads-to-sample ratio, the more stringent the selection against short fragments. Please always determine the input DNA length on an agarose gel (or other gel electrophoresis methods) and then calculate the appropriate amount of AMPure beads to use.
Low recovery after end-prep	The wash step used ethanol <70%	DNA will be eluted from the beads when using ethanol <70%. Make sure to use the correct percentage.

11. 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 the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

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

Observation	Possible cause	Comments and actions
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	An air bubble was introduced into the nanopore array	After the Flow Cell Check it is essential to remove any air bubbles near the priming port before priming the flow cell. If not removed, the air bubble can travel to the nanopore array and irreversibly damage the nanopores that have been exposed to air. The best practice to prevent this from happening is demonstrated in this video.
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	The flow cell is not correctly inserted into the device	Stop the sequencing run, remove the flow cell from the sequencing device and insert it again, checking that the flow cell is firmly seated in the device and that it has reached the target temperature. If applicable, try a different position on the device (GridION/PromethION).
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	Contaminations in the library damaged or blocked the pores	The pore count during the Flow Cell Check is performed using the QC DNA molecules present in the flow cell storage buffer. At the start of sequencing, the library itself is used to estimate the number of active pores. Because of this, variability of about 10% in the number of pores is expected. A significantly lower pore count reported at the start of sequencing can be due to contaminants in the library that have damaged the membranes or blocked the pores. Alternative DNA/RNA extraction or purification methods may be needed to improve the purity of the input material. The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

MinKNOW script failed

Observation	Possible cause	Comments and actions
MinKNOW shows "Script failed"		Restart the computer and then restart MinKNOW. If the issue persists, please collect the MinKNOW log files and contact Technical Support. If you do not have another sequencing device available, we recommend storing the flow cell and the loaded library at 4°C and contact Technical Support for further storage guidance.

Pore occupancy below 40%

Observation	Possible cause	Comments and actions
Pore occupancy <40%	Not enough library was loaded on the flow cell	Ensure you load the recommended amount of good quality library in the relevant library prep protocol onto your flow cell. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol"
Pore occupancy close to 0	The Ligation Sequencing Kit was used, and sequencing adapters did not ligate to the DNA	Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford Nanopore Technologies Ligation Buffer (LNB, provided in the sequencing kit) at the sequencing adapter ligation step, and use the correct amount of each reagent. A Lambda control library can be prepared to test the integrity of the third-party reagents.
Pore occupancy close to 0	The Ligation Sequencing Kit was used, and ethanol was used instead of LFB or SFB at the wash step after sequencing adapter ligation	Ethanol can denature the motor protein on the sequencing adapters. Make sure the LFB or SFB buffer was used after ligation of sequencing adapters.
Pore occupancy close to 0	No tether on the flow cell	Tethers are adding during flow cell priming (FLT/FCT tube). Make sure FLT/FCT was added to FB/FCF before priming.

Shorter than expected read length

Observation	Possible cause	Comments and actions
Shorter than expected read length	Unwanted fragmentation of DNA sample	Read length reflects input DNA fragment length. Input DNA can be 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 unavailable pores

Observation	Possible cause	Comments and actions
Large proportion of unavailable pores (shown as blue in the channels panel and pore activity plot) The pore activity plot above shows an increasing proportion of "unavailable" pores over time.	Contaminants are present in the sample	Some contaminants can be cleared from the pores by the unblocking function built into MinKNOW. If this is successful, the pore status will change to "sequencing pore". If the portion of unavailable pores 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.

Large proportion of inactive pores

Observation	Possible cause	Comments and actions
Large proportion of inactive/unavailable pores (shown as light blue in the channels panel and pore activity plot. Pores or membranes are irreversibly damaged)	Air bubbles have been introduced into the flow cell	Air bubbles introduced through flow cell priming and library loading can irreversibly damage the pores. Watch the Priming and loading your flow cell video for best practice
Large proportion of inactive/unavailable pores	Certain compounds co-purified with DNA	Known compounds, include polysaccharides, typically associate with plant genomic 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/unavailable pores	Contaminants are present in the sample	The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative 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 sequencing speed and q-score later into the run	For Kit 9 chemistry (e.g. SQK-LSK109), fast fuel consumption is typically seen when the flow cell is overloaded with library (please see the appropriate protocol for your DNA library to see the recommendation).	Add more fuel to the flow cell by following the instructions in the MinKNOW protocol. In future experiments, load lower amounts of library to the flow cell.

Temperature fluctuation

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

Failed to reach target temperature

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

Guppy – no input .fast5 was found or basecalled

Observation	Possible cause	Comments and actions
No input .fast5 was found or basecalled	input_path did not point to the .fast5 file location	The --input_path has to be followed by the full file path to the .fast5 files to be basecalled, and the location has to be accessible either locally or remotely through SSH.
No input .fast5 was found or basecalled	The .fast5 files were in a subfolder at the input_path location	To allow Guppy to look into subfolders, add the --recursive flag to the command

Guppy – no Pass or Fail folders were generated after basecalling

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

Guppy – unusually slow processing on a GPU computer

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

Device:

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Documentation

Nanopore Learning

Ligation sequencing gDNA (SQK-LSK109-XL) (GDX_9095_v109_revL_24Jan2020)

Introduction to the protocol

Library preparation

Sequencing and data analysis

Troubleshooting

ドキュメントのバージョン

MinION: Protocol

V GDX_9095_v109_revL_24Jan2020

Contents

Introduction to the protocol

Library preparation

Sequencing and data analysis

Troubleshooting

1. Overview of the protocol

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This is a Legacy product

Ligation Sequencing Kit XL features

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Optional fragmentation and size selection

Introduction to the Ligation Sequencing protocol for gDNA

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Compatibility of this protocol

2. Equipment and consumables

材料

消耗品

装置

オプション装置

For this protocol, you will need 1 µg (or 100-200 fmol) gDNA.

Input DNA

How to QC your input DNA

Chemical contaminants

NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing

Multichannel pipettes

Ligation Sequencing Kit XL (SQK-LSK109-XL) contents

Flow Cell Priming Kit XL (EXP-FLP002-XL) contents

3. Computer requirements and software

MinION Mk1B IT requirements

MinION Mk1C IT requirements

MinION Mk1D IT requirements

Software for nanopore sequencing

MinKNOW

EPI2ME (optional)

Check your flow cell

4. DNA repair and end-prep

材料

消耗品

装置

重要

Optional fragmentation and size selection

Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.

重要

It is important that the NEBNext FFPE DNA Repair Buffer and NEBNext Ultra II End Prep Reaction Buffer are mixed well by vortexing.

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Do not vortex the NEBNext FFPE DNA Repair Mix or NEBNext Ultra II End Prep Enzyme Mix.

Prepare the DNA in nuclease-free water per sample:

To each sample, add the following:

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For ease, make a master mix of these reagents prior to adding to the DNA samples:

Mix well by gently pipetting the entire volume within each well/tube up and down 10 times, or by flicking the tubes, and spin down.

Seal the plate, or close the tube lids.

Using a thermal cycler, incubate the samples at 20°C for 5 minutes and 65°C for 5 mins.

AMPure XP bead clean-up

Resuspend the AMPure XP beads by vortexing and transfer to a pipetting trough. Ensure that the volume transferred is enough for 60 µl to be added to each DNA sample, with an excess to allow for dead volume within the pipetting trough.

重要

Resuspend and transfer the beads to the pipetting trough immediately before use to ensure beads do not settle.

Keep the DNA samples in their original wells/PCR tubes. Add 60 µl of resuspended AMPure XP beads to each sample and mix by pipetting at least 100 µl up and down ten times. Retain any unused beads.

Incubate for 5 minutes at room temperature.

Prepare fresh 70% ethanol in nuclease-free water and pour into a pipetting trough. Allow enough for 500 µl per sample, with an excess to allow for dead volume within the pipetting trough. After the bead washing steps, discard any unused ethanol.

Pellet the beads on a magnet for at least 2 minutes, or until the supernatant is clear. Keep the plate/tube strip on the magnet and pipette off the supernatant.

Keeping the plate/tube strip on the magnet, wash each pellet of beads with 200 µl of the freshly-prepared 70% ethanol without disturbing the pellets. Remove the 70% ethanol using a pipette and discard.

Repeat the previous step.

Seal the plate, or close the tube lids. Spin down and place the plate/tube strip back on the magnet. Pipette off any residual ethanol.

Pour nuclease-free water into a pipetting trough. Allow enough for 61 µl per sample, with an excess to allow for dead volume within the pipetting trough.

Remove the plate/tube strip from the magnetic rack and resuspend each pellet in 61 µl nuclease-free water from the pipetting trough. Pipette the entire volume up and down ten times).

Seal the plate or close the tube lids, and incubate for 2 minutes at room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 min.