Rapid Sequencing Kit V14 - Lambda control (SQK-RAD114)
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MinION: Protocol
Rapid Sequencing Kit V14 - Lambda control (SQK-RAD114) V RSL_9176_v114_revC_16Nov2022
The fastest and simplest protocol for genomic DNA involving:
- ~10 mins library prep
- fragmentation
- no third-party ligase needed
This is an Early Access product For more information about our Early Access programmes, please see this article on product release phases.
For Research Use Only
FOR RESEARCH USE ONLY
Contents
Introduction to the protocol
Library preparation
Sequencing and data analysis
Troubleshooting
Overview
The fastest and simplest protocol for genomic DNA involving:
- ~10 mins library prep
- fragmentation
- no third-party ligase needed
This is an Early Access product For more information about our Early Access programmes, please see this article on product release phases.
For Research Use Only
1. Overview of the protocol
IMPORTANT
This is an Early Access product
For more information about our Early Access programmes, please see this article on product release phases.
Please ensure you always use the most recent version of the protocol.
Rapid Sequencing Kit features
This kit is recommended for users who:
- require a short preparation time
- have limited starting input
- have limited access to laboratory equipment
Introduction to Rapid Lambda Control protocol (SQK-RAD114)
This protocol describes the step-by-step instructions to complete a Lambda Control experiment using the Rapid Sequencing Kit V14 (SQK-RAD114) on a MinION flow cell. The control experiment uses a high-quality DNA sample supplied in the kit. It allows you to become familiar with nanopore sequencing before preparing your own library.
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:
- 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 V14 (SQK-RAD114)
- Control Expansion (EXP-CTL001)
- R10.4.1 MinION flow cells (FLO-MIN114)
- Flow Cell Wash Kit (EXP-WSH004)
- MinION Mk1C - MinION Mk1C IT requirements document
- MinION Mk1B - MinION Mk1B IT requirements document
- GridION - GridION IT requirements document
2. Equipment and consumables
Materials
- Rapid Sequencing Kit V14 (SQK-RAD114)
- Control Expansion (EXP-CTL001)
Consumables
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
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 blocks
Optional equipment
- Qubit fluorometer (or equivalent for QC check)
For this protocol, you will need 100 ng Lambda DNA (LMD) supplied in the Control Expansion (EXP-CTL001) pack.
Rapid Sequencing Kit V14 (SQK-RAD114) contents
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format.
Single-use tubes format:
Bottle format:
Control Expansion (EXP-CTL001) contents
Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome. Lambda DNA (LMD) fragment length is 48.5 kb.
3. Computer requirements and software
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.
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 Mk1B IT Requirements document.
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. Library preparation
Materials
- Lambda DNA (LMD)
- Fragmentation Mix (FRA)
- Rapid Adapter (RA)
- Adapter Buffer (ADB)
Consumables
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
Equipment
- Thermal cycler or heat blocks
- P2 pipette and tips
- P10 pipette and tips
DNA tagmentation
Thaw the 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 temperature | 2. Briefly spin down | 3. Mix well by pipetting |
|---|---|---|---|
| Fragmentation Mix (FRA) | Not frozen | ✓ | ✓ |
| Rapid Adapter (RA) | Not frozen | ✓ | ✓ |
| Adapter Buffer (ADB) | Not frozen | ✓ | ✓ |
| Lambda DNA (LMD) | ✓ | ✓ | ✓ |
Once thawed, keep all the kit components on ice.
Prepare the Lambda DNA in nuclease-free water.
- Transfer 2 µl Lambda DNA into a 1.5 ml Eppendorf DNA LoBind tube
- Adjust the volume to 10 µl with nuclease-free water
- Mix by flicking the tube to avoid unwanted shearing
- Spin down briefly in a microfuge
In a 0.2 ml thin-walled PCR tube, mix the following:
| Reagents | Volume (µl) |
|---|---|
| Lambda DNA | 10 |
| Fragmentation Mix (FRA) | 1 |
| Total | 11 |
Mix gently by flicking the tube, and spin down.
Incubate the tube at 30°C for 2 minutes and then at 80°C for 2 minutes. Briefly put the tube on ice to cool it down.
CHECKPOINT
The tagmented Lambda DNA in 11 μl is taken into the next step.
Adapter attachment
In a fresh 1.5 ml Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows and pipette mix:
| Reagents | Volume |
|---|---|
| Rapid Adapter (RA) | 1.5 μl |
| Adapter Buffer (ADB) | 3.5 μl |
| Total | 5 μl |
Add 1 μl of diluted Rapid Adapter (RA) to the tagmented DNA.
Mix gently by flicking the tube, and spin down.
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.
5. Priming and loading the MinION and GridION Flow Cell
Materials
- Flow Cell Flush (FCF)
- Flow Cell Tether (FCT)
- Library Solution (LIS)
- Library Beads (LIB)
- Sequencing Buffer (SB)
Consumables
- MinION and GridION Flow Cell
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
Equipment
- MinION or GridION device
- MinION and GridION Flow Cell Light Shield
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
IMPORTANT
Please note, this kit is only compatible with R10.4.1 flow cells (FLO-MIN114).
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.
Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.
IMPORTANT
For optimal sequencing performance and improved output on MinION R10.4.1 flow cells (FLO-MIN114), we recommend adding Bovine Serum Albumin (BSA) to the flow cell priming mix at a final concentration of 0.2 mg/ml.
Note: We do not recommend using any other albumin type (e.g. recombinant human serum albumin).
To prepare the flow cell priming mix with BSA, combine Flow Cell Flush (FCF) and Flow Cell Tether (FCT), as directed below. Mix by pipetting at room temperature.
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format. Please follow the instructions for your kit format.
Single-use tubes format: Add 5 µl Bovine Serum Albumin (BSA) at 50 mg/ml and 30 µl Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).
Bottle format: In a suitable tube for the number of flow cells, combine the following reagents:
| Reagent | Volume per flow cell |
|---|---|
| Flow Cell Flush (FCF) | 1,170 µl |
| Bovine Serum Albumin (BSA) at 50 mg/ml | 5 µl |
| Flow Cell Tether (FCT) | 30 µl |
| Total volume | 1,205 µl |
Open the MinION or GridION device lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.
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.
Slide the flow cell 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.
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
- 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.
Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.
IMPORTANT
The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
We recommend using the Library Beads (LIB) for most sequencing experiments. However, the Library Solution (LIS) is available for more viscous libraries.
Thoroughly mix the contents of the Library Beads (LIB) by pipetting.
In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:
| Reagent | Volume per flow cell |
|---|---|
| Sequencing Buffer (SB) | 37.5 µl |
| Library Beads (LIB) mixed immediately before use, or Library Solution (LIS), if using | 25.5 µl |
| DNA library | 12 µl |
| Total | 75 µl |
Complete the flow cell priming:
- 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.
Mix the prepared library gently by pipetting up and down just prior to loading.
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.
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.
IMPORTANT
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.
Place the light shield onto the flow cell, as follows:
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.
CAUTION
The MinION Flow Cell Light Shield is not secured to the flow cell and careful handling is required after installation.
END OF STEP
Close the device lid and set up a sequencing run on MinKNOW.
6. 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, MinION Mk1C 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.
7. Flow cell reuse and returns
Materials
- Flow Cell Wash Kit (EXP-WSH004)
After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Flow Cell Wash Kit protocol and store the washed flow cell at 2-8°C.
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.
Alternatively, follow the returns procedure to flush out the flow cell ready to send back to Oxford Nanopore.
Instructions for returning flow cells can be found here.
Note: 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.
8. 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.
9. 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. |
10. Issues during the sequencing run using a Rapid-based sequencing kit
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 the correct concentration of good quality library is loaded on to a MinION/GridION flow cell. To check the concentration, please refer to the library preparation protocol. 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 Rapid Sequencing Kit V14/Rapid Barcoding Kit V14 was used, and sequencing adapters did not attach to the DNA | Make sure to closely follow the protocol and use the correct volumes and incubation temperatures. A Lambda control library can be prepared to test the integrity of reagents. |
| Pore occupancy close to 0 | No tether on the flow cell | Tethers are adding during flow cell priming (FCT tube). Make sure FCT was added to 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. |
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 FAQ for more information on MinION temperature control. |