MinION: Protocol

V SCE_9075_v109_revV_25May2022

FOR RESEARCH USE ONLY

1. Overview of the protocol

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: support@nanoporetech.com. For further information on please see the product update page.

Sequence capture protocol features

Use this protocol if you:

Are not interested in analysing the entire genome
Want greater depth of coverage of a specific region
Want to analyse a large number of target regions

Introduction to sequence capture

This protocol describes how to carry out sequence capture of genomic DNA using the Ligation Sequencing Kit (SQK-LSK109) and the Agilent SureSelect method for the kit used (e.g. SureSelect Human All Exon). Other kits of exome probes are available depending on the exome region coverage required.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

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:

Fragment your DNA (this step is optional)
Ligate PCR adapters to the DNA ends and amplify the fragments
Hybridise the DNA to probes provided in the Agilent SureSelect Exome kit, and perform a pulldown
Elute and amplify the pulled-down fragments
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 Human Exome mapping

We recommend using the standard SureSelect Target Enrichment System protocol from Agilent Technologies with the following differences:

The DNA in this protocol is sheared with dsDNA Fragmentase rather than in a Covaris microTUBE, and is incubated at 16° C instead of the 37° C recommended by NEB
DNA purification in this protocol is carried out using Agencourt AMPure XP beads instead of QIAquick PCR purification
DNA End-prep is carried out using the NEBNext Ultra II End Repair/dA-Tailing Module instead of the Agilent method
Ligation of PCR adapters is carried out using the NEB Blunt/TA Ligase Master Mix instead of the Agilent method
No desalting of the capture solution is necessary
Long AMP Taq is used for amplification of the DNA post-hybridisation

This protocol is for the preparation of a single sample. Multiple samples can be prepared in parallel; each sample will require a separate pull-down and will need to be run on a separate flow cell.

Compatibility of this protocol

This protocol should only be used in combination with:

Ligation Sequencing Kit (SQK-LSK109)
Control Expansion (EXP-CTL001)
FLO-MIN106 (R9.4.1) flow cells
Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

材料

3.5 µg high molecular weight human genomic DNA
Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)
Ligation Sequencing Kit (SQK-LSK109)
Flow Cell Priming Kit (EXP-FLP002)
PCR Expansion (EXP-PCA001)

消耗品

NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
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)
NEBNext dsDNA Fragmentase (M0348L)
Cot-1 DNA (ThermoFisher Scientific 15279-011)
Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific, 65601)
Blocking oligo at 1 mM, sequence 5'-AGGTTAAACACCCAAGCAGACGCCGCAATATCAGCACCAACAGAAACAACC-3'
2x Long AMP Taq
Nuclease-free water (e.g. ThermoFisher, AM9937)
Freshly prepared 70% ethanol in nuclease-free water
1.5 ml Eppendorf DNA LoBind tubes
0.2 ml 薄壁のPCRチューブ
0.2 ml 96 well PCR plate

装置

Hula mixer（緩やかに回転するミキサー）
1.5 mlエッペンドルフチューブに最適のマグネット式ラック
小型遠心機
ボルテックスミキサー
Heating block at 37°C capable of taking 1.5 ml tubes
サーマルサイクラー
アイスバケツ（氷入り）
タイマー
SpeedVac
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

オプション装置

Standard gel electrophoresis equipment
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 3.5 µg high molecular weight human 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 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.

Third-party reagents

We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.

For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

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.

Ligation Sequencing Kit contents (SQK-LSK109)

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
DNA CS	DCS	Yellow	1	50
Adapter Mix	AMX	Green	1	40
Ligation Buffer	LNB	Clear	1	200
L Fragment Buffer	LFB	White cap, orange stripe on label	2	1,800
S Fragment Buffer	SFB	Grey	2	1,800
Sequencing Buffer	SQB	Red	2	300
Elution Buffer	EB	Black	1	200
Loading Beads	LB	Pink	1	360

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

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 fragmentation

材料

3.5 µg high molecular weight human genomic DNA

消耗品

NEBNext dsDNA Fragmentase (M0348L)
Nuclease-free water (e.g. ThermoFisher, AM9937)
Agencourt AMPure XP beads (Beckman Coulter, A63881)
1.5 ml Eppendorf DNA LoBind tubes
Freshly prepared 70% ethanol in nuclease-free water
0.2 ml 薄壁のPCRチューブ
1.5 ml Eppendorf DNA LoBind tubes

装置

ボルテックスミキサー
Hula mixer（緩やかに回転するミキサー）
1.5 mlエッペンドルフチューブに最適のマグネット式ラック
アイスバケツ（氷入り）
小型遠心機
サーマルサイクラー

オプション装置

Agilent Bioanalyzer (or equivalent)
Eppendorf 5424 centrifuge (or equivalent)

Transfer 3.5 μg genomic DNA into a DNA LoBind tube
Adjust the volume to 16 μl or less with nuclease-free water
Mix thoroughly by inversion avoiding unwanted shearing
Spin down briefly in a microfuge

Mix the reagents in the order given below:

Reagent	Volume
DNA (1-16 µl)	x µl
10x Fragmentase Reaction Buffer v2	2 µl
Nuclease-free water	16 - x µl
dsDNA Fragmentase	2 µl
Total	20 µl

3.5 µg fragmented DNA in 47 µl is taken into the next step.

5. DNA repair and end-prep

材料

~ 3.5 µg fragmented DNA in 47 µl
DNA Control Sample (DCS)

消耗品

0.2 ml 薄壁のPCRチューブ
1.5 ml Eppendorf DNA LoBind 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

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
Hula mixer（緩やかに回転するミキサー）
マグネットラック
アイスバケツ（氷入り）

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.

Between each addition, pipette mix 10-20 times.

Reagent	Volume
DNA from the previous step	47 µl
DNA CS (optional)	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	60 µl

AMPure XP bead clean-up

It is recommended that the repaired/end-prepped DNA sample is 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.

Take forward approximately 700 ng of end-prepped DNA in 30 µl into adapter ligation.

6. Ligation of PCR adapters

材料

~ 3.5 µg fragmented DNA in 45 µl
PCR Adapter (PCA)

消耗品

Nuclease-free water (e.g. ThermoFisher, AM9937)
NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
Agencourt AMPure XP beads (Beckman Coulter, A63881)
Freshly prepared 70% ethanol in nuclease-free water
1.5 ml Eppendorf DNA LoBind tubes

装置

1.5 mlエッペンドルフチューブに最適のマグネット式ラック
Hula mixer（緩やかに回転するミキサー）
Qubit蛍光光度計（またはQCチェックのための同等品）
小型遠心機
ボルテックスミキサー

Reagent	Volume
End-prepped DNA	30 µl
PCR Adapters (PCA)	20 µl
NEB Blunt/TA Ligase Master Mix	50 µl
Total	100 µl

End-prep cleanup demo

Take forward 48 µl of the adapted DNA into the PCR reaction. However, at this point it is also possible to store the sample at 4°C overnight.

7. PCR

材料

Primer Mix (PRM)

消耗品

2x Long AMP Taq
Agencourt AMPure XP beads (Beckman Coulter, A63881)
Nuclease-free water (e.g. ThermoFisher, AM9937)
Freshly prepared 70% ethanol in nuclease-free water
1.5 ml Eppendorf DNA LoBind tubes
0.2 ml 薄壁のPCRチューブ

装置

サーマルサイクラー
アイスバケツ（氷入り）
1.5 mlエッペンドルフチューブに最適のマグネット式ラック
Hula mixer（緩やかに回転するミキサー）
ボルテックスミキサー
Qubit蛍光光度計（またはQCチェックのための同等品）

Reagent	Volume
LongAmp Taq 2X Master Mix	50 µl
PRM Adapters (10 μM)	2 µl
Template DNA	48 µl
Total	100 µl

Cycle step	Temperature	Time	No. of cycles
Initial denaturation	95 °C	3 mins	1
Denaturation	98 °C	20 secs	6 (b)
Annealing	62 °C (a)	15 secs (a)	6 (b)
Extension	65 °C (c)	3 mins	6 (b)
Final extension	65 °C	3 mins	1
Hold	4 °C	∞

a. This is specific to the Oxford Nanopore primer and should be maintained

b. Adjust accordingly if input quantities are altered

c. This temperature is determined by the type of polymerase that is being used (given here for LongAmp Taq polymerase)

Take forward 700–1000 ng amplified DNA into the hybridisation step.

8. Hybridisation

材料

Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)

消耗品

Cot-1 DNA (ThermoFisher Scientific 15279-011)
Nuclease-free water (e.g. ThermoFisher, AM9937)
0.2 ml 96-well PCR plate
Blocking oligo at 1 mM, sequence 5'-AGGTTAAACACCCAAGCAGACGCCGCAATATCAGCACCAACAGAAACAACC-3'

装置

SpeedVac
サーマルサイクラー
アイスバケツ（氷入り）
ボルテックスミキサー
小型遠心機

Reagent	Volume
DNA library	700-1000 ng
Cot-1 DNA	5 µg
Blocking oligo top	1 µl

The volume of the reaction can be variable, as the water is evaporated in step 2. After this, the DNA is reconstituted to a set volume.

Poke one or more holes in the lid with a narrow gauge needle. You can also break off the cap, cover with parafilm, and poke a hole in the parafilm.

Stage	Temperature	Time
Step 1	95 ºC	5 min
Step 2	65 ºC	5 min
Step 3	65 ºC	Hold

You will now need to prepare the Hybridisation Buffer mix and the RNase Block ready to be combined with the Capture Library reagent from the SureSelect kit. This will then be combined with the adapted, amplified DNA sample.

Reagent	Volume for 1 reaction
SureSelect Hyb 1 (orange cap or bottle)	6.63 µl
SureSelect Hyb 2 (red cap)	0.27 µl
SureSelect Hyb 3 (yellow cap or bottle)	2.65 µl
SureSelect Hyb 4 (black cap or bottle)	3.45 µl
Total	13 µl

In the event of precipitation, warm the Hybridisation Buffer at 65°C for 5 minutes. Otherwise, keep buffer at room temperature until it is used for the Hybridisation mix.

Capture library size	RNase block dilution (parts RNase block:water)	Volume of diluted RNase block per hybridisation reaction
≥3 Mb	25% (1:3)	2 μl
<3 Mb	10% (1:9)	5 μl

Please note that the 1:3 ratio of the RNase Block dilution is different to the SureSelect protocol.

Reagent	Volume for 1 reaction
Hybridisation buffer mixture	13 µl
25% RNase Block solution	2 µl
Capture library (red cap) ≥3 Mb	5 µl
Total	20 µl

If your capture library is <3 Mb prepare the Capture Library Hybridisation Mix as follows:

Reagent	Volume for 1 reaction
Hybridisation buffer mixture	13 µl
10% RNase Block solution	5 µl
Capture library (red cap) <3 Mb	2 µl
Total	20 µl

Do not keep solutions containing the Capture Library at room temperature for longer than 10 mins.

Wells must be sealed to minimise evaporation, to avoid a negative impact on your results.

9. Pull-down

材料

Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)

消耗品

Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific, 65601)
0.2 ml 96 well PCR plate
Nuclease-free water (e.g. ThermoFisher, AM9937)

装置

サーマルサイクラー
ボルテックスミキサー
1.5 mlエッペンドルフチューブに最適のマグネット式ラック
Multichannel pipette and tips
Plate mixer
Centrifuge capable of taking 96-well plates
アイスバケツ（氷入り）

It is important to maintain bead suspensions at 65°C during the washing procedure below to ensure specificity of capture. Make sure that the SureSelect Wash Buffer 2 is pre-warmed to 65°C before use. Do not use a tissue incubator, or other devices with significant temperature fluctuations, for the incubation steps.

The hybrid capture protocol uses the SureSelect Target Enrichment Box 1 reagents (stored at room temperature), as well as Dynabeads MyOne Streptavidin T1 magnetic beads.

Captured DNA remains on the streptavidin beads during the post-capture amplification step.

10. Elution and amplification of DNA

材料

Primer Mix (PRM)

消耗品

0.2 ml 薄壁のPCRチューブ
2x Long AMP Taq
Nuclease-free water (e.g. ThermoFisher, AM9937)
Agencourt AMPure XP beads (Beckman Coulter, A63881)

装置

サーマルサイクラー
アイスバケツ（氷入り）
1.5 mlエッペンドルフチューブに最適のマグネット式ラック
Hula mixer（緩やかに回転するミキサー）
ボルテックスミキサー

Reagent	Volume
2x Long AMP Taq	50 µl
PRM Adapters (10 μM)	2 µl
Template DNA	48 µl
Total	100 µl

Cycle step	Temperature	Time	No. of cycles
Initial denaturation	95 °C	3 mins	1
Denaturation	98 °C	20 secs	14 (b)
Annealing	62 °C (a)	15 secs (a)	14 (b)
Extension	65 °C (c)	3 mins	14 (b)
Final extension	65 °C	3 mins	1
Hold	4 °C	∞

a. This is specific to the Oxford Nanopore primer and should be maintained

b. Adjust accordingly if input quantities are altered

c. This temperature is determined by the type of polymerase that is being used (given here for LongAmp Taq polymerase)

If the yield is under 500 ng, perform an additional amplification. Otherwise, proceed to Library preparation.

11. Further amplification of low-yield samples (optional)

材料

Primer Mix (PRM)

消耗品

0.2 ml 薄壁のPCRチューブ
2x Long AMP Taq
Nuclease-free water (e.g. ThermoFisher, AM9937)
Agencourt AMPure XP beads (Beckman Coulter, A63881)

装置

サーマルサイクラー
アイスバケツ（氷入り）
1.5 mlエッペンドルフチューブに最適のマグネット式ラック
Hula mixer（緩やかに回転するミキサー）
ボルテックスミキサー

Perform the second round of amplification only if the DNA yield from the previous round is under 500 ng. Otherwise, proceed to Library preparation.

Reagent	Volume
2x Long AMP Taq	50 µl
PRM Adapters (10 μM)	2 µl
Template DNA	48 µl
Total	100 µl

Cycle step	Temperature	Time	No. of cycles
Initial denaturation	95 °C	3 mins	1
Denaturation	98 °C	20 secs	11 (b)
Annealing	62 °C (a)	15 secs (a)	11 (b)
Extension	65 °C (c)	3 mins	11 (b)
Final extension	65 °C	3 mins	1
Hold	4 °C	∞

a. This is specific to the Oxford Nanopore primer and should be maintained

b. Adjust accordingly if input quantities are altered

c. This temperature is determined by the type of polymerase that is being used (given here for LongAmp Taq polymerase)

If the yield is over 500 ng, take the amplified DNA into Library preparation.

12. End-prep

材料

DNA CS
500 ng–1 µg captured DNA in 48 µl

消耗品

NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
Freshly prepared 70% ethanol in nuclease-free water
1.5 ml Eppendorf DNA LoBind tubes
Nuclease-free water (e.g. ThermoFisher, AM9937)
Agencourt AMPure XP beads (Beckman Coulter, A63881)

装置

サーマルサイクラー
1.5 mlエッペンドルフチューブに最適のマグネット式ラック
Hula mixer（緩やかに回転するミキサー）
ボルテックスミキサー
アイスバケツ（氷入り）

オプション装置

Qubit蛍光光度計（またはQCチェックのための同等品）

End-prep demo

Mix the following reagents in a 1.5 ml Eppendorf DNA LoBind tube:

Reagent	Volume
~1 µg DNA (fragmented genomic DNA, amplicon or cDNA)	45 µl
Ultra II End-prep reaction buffer	7 µl
Ultra II End-prep enzyme mix	3 µl
DNA CS	5 µl
Total	60 µl

If using the barcoding approach the pooled input DNA should be ~1 µg in 45 µl, whether genomic, amplicon or cDNA.

If condensation is observed in the tube after the thermocycling, briefly spin down the tube contents in a microfuge.

Take forward approximately 700 ng of end-prepped DNA in 30 µl nuclease-free water into adapter ligation. However, at this point, it is also possible to store the sample at 4°C overnight.

13. Adapter ligation and clean-up

材料

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

消耗品

NEBNext Quick Ligation Module (NEB, E6056)
1.5 ml Eppendorf DNA LoBind tubes
Agencourt AMPure XP beads (Beckman Coulter™, A63881)
Nuclease-free water (e.g. ThermoFisher, AM9937)

装置

マグネットラック
小型遠心機
ボルテックスミキサー
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

Although the recommended third-party ligase is supplied with its own buffer, the ligation efficiency of Adapter Mix (AMX) is higher when using Ligation Buffer supplied within the Ligation Sequencing Kit.

Reagent	Volume
DNA sample from the previous step	30 µl
Nuclease-free water	30 µl
Ligation Buffer (LNB)	25 µl
NEBNext Quick T4 DNA Ligase	10 µl
Adapter Mix (AMX)	5 µl
Total	100 µl

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

Dispose of the pelleted beads

The prepared library is used for loading into the ﬂow cell. Store the library on ice or at 4°C until ready to load.

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.

If quantities allow, the library 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.

14. Priming and loading the SpotON flow cell

材料

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

消耗品

1.5 ml Eppendorf DNA LoBind tubes
Nuclease-free water (e.g. ThermoFisher, AM9937)

装置

MinION Mk1B or Mk1C
SpotON Flow Cell
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

Priming and loading a flow cell

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

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

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.

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.

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 per flow cell
Sequencing Buffer (SQB)	37.5 µl
Loading Beads (LB), mixed immediately before use	25.5 µl
DNA library	12 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.

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.

15. 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.

16. 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.

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.

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.

17. 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.

18. 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.

19. 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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すべての製品

Documentation

Nanopore Learning

Ligation sequencing gDNA - sequence capture (SQK-LSK109) (SCE_9075_v109_revV_25May2022)

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 SCE_9075_v109_revV_25May2022

Contents

Introduction to the protocol

Library preparation

Sequencing and data analysis

Troubleshooting

1. Overview of the protocol

This is a Legacy product

Sequence capture protocol features

Introduction to sequence capture

We recommend using the standard SureSelect Target Enrichment System protocol from Agilent Technologies with the following differences:

This protocol is for the preparation of a single sample. Multiple samples can be prepared in parallel; each sample will require a separate pull-down and will need to be run on a separate flow cell.

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 3.5 µg high molecular weight human genomic DNA.

Input DNA

How to QC your input DNA

Chemical contaminants

Third-party reagents

NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing

Ligation Sequencing Kit contents (SQK-LSK109)

Flow Cell Priming Kit contents (EXP-FLP002)

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 fragmentation

材料

消耗品

装置

オプション装置

Prepare the DNA in nuclease-free water.

Fragment DNA to ~1-2 kb as follows:

Vortex for 3 sec.

Incubate the reaction for 10 minutes at 16° C.

Resuspend the AMPure XP beads by vortexing.

Add 36 µl of the AMPure XP beads to the reaction and mix gently by pipetting.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.

Prepare 500 μl of fresh 70% ethanol in nuclease-free water.

Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend pellet in 48 µl nuclease-free water. Incubate for 2 minutes at room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 48 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1 µl of fragmented and repaired DNA using a Qubit fluorometer

3.5 µg fragmented DNA in 47 µl is taken into the next step.

5. DNA repair and end-prep

材料

消耗品

装置

Thaw DNA Control Sample (DCS) at room temperature, spin down, mix by pipetting, and place on ice.

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.

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

Ensure the components are thoroughly mixed by pipetting, and spin down.

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

AMPure XP bead clean-up

Resuspend the AMPure XP beads by vortexing.

Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.

Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.