Ligation sequencing gDNA - Cas9 enrichment (SQK-LSK109)

Version for device: MinION

1. Important This is a Legacy product

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

2. Features of using Cas9 targeted sequencing

   We recommend CRISPR/Cas targeted sequencing if the user:

   • Wishes to sequence multiple human gene targets (up to 100 in a single panel) to high coverage (>100x) on a single MinION Mk1B flow cell
   • Wishes to sequence up to a 50 kb Region of Interest (ROI), using up to 100 target sites, in a single assay*
   • Has 1-10 µg of available gDNA
   • Wishes to gain insight into methylation patterns or other modified bases
   • Has gene targets that are highly repetitive, or wishes to evaluate the number of repeats in an expansion, where traditional amplification methods or sequencing-by-synthesis methods could yield a biased result
   • Wishes to sequence long gene targets in a single pass that are not amenable to long-range PCR (> 30 kb)
   • Optionally wishes to run multiple barcoded samples on a single flow cell.

   * Note: This is known as ‘tiling’ an ROI.

3. Excision/single cut vs tiling approach

   This protocol can be used for any probe design method (details of which can be found in the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet). The recommended ‘excision approach’ and ‘single cut and read out’ method can follow the main flow of this protocol (shown in Figure 1). The ‘tiling’ approach requires the alterations to the protocol described in the Important (orange) boxes in the library preparation section. The main difference with the tiling approach is that both pools of probes need to be prepared separately (RNP complex formation, cleavage and dA-tailing and adapter ligation performed in separate tubes) then pooled during the final AMPure XP bead purification (shown in Figure 2).

   
   Figure 1. Cas9 targeted sequencing protocol using the 'excision approach' or 'single cut and read out'.

   
   Figure 2. Cas9 targeted sequencing protocol using the 'tiling' approach

4. Important Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet

   For more details about the Cas9 targeted sequencing approach, how to design probes, and general expectations and guidance, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet. We strongly recommend that you read it before proceeding with your targeted sequencing experiments.

5. Introduction to the CRISPR/Cas protocol

   This protocol describes how to carry out sequencing of genomic DNA using the Ligation Sequencing Kit (SQK-LSK109) with enrichment of specific genomic regions using CRISPR/Cas. For users with no previous nanopore sequencing experience, we recommend that a Lambda control experiment is completed first to become familiar with the technology.

   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
   Figure 1. shows and explains the biochemical steps used to prepare your DNA library using a SQK-LSK109 kit, plus several third party reagents.

   
   Figure 1. Cas9 targeted library preparation for sequencing. The steps below correspond to the steps in the figure.

   1. After DNA extraction, 5’ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands.
   2. Cas9 ribonucleoprotein particles (RNPs), with bound crRNA and tracrRNA, are added to the genomic DNA, and bind then cleave the Region of Interest (ROI).
   3. dsDNA cleavage by Cas9 reveals blunt ends with ligatable 5’ phosphates.
   4. All of the DNA in the sample is dA-tailed, which prepares the blunt ends for sequencing adapter ligation.
   5. Sequencing adapters are ligated primarily to Cas9 cut sides, which are both 3’ dA-tailed and 5’ phosphorylated. The library preparation is cleaned up to remove excess adapters using AMPure XP beads and resuspended in Sequencing Buffer. Non-target molecules are not removed. The subsequent library preparation is added to the flow cell for sequencing.

   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)

6. Enrichment experiment steps and associated instructions

   Step	Instructions
   1. Extract and prepare DNA	Extraction methods
   2. Design probes	Targeted, amplification-free DNA sequencing using CRISPR/Cas (Probe design)
   3. QC input DNA	Input DNA/RNA QC
   4. Perform enrichment, and prepare sequencing library	Cas9 Sequencing Kit (SQK-CS9109) - this is the protocol using the Oxford Nanopore Technologies Cas9 Sequencing Kit for Cas9 enrichment and library preparation Cas9 targeted sequencing - this is the protocol that uses the Ligation Sequencing Kit (SQK-LSK109) for library preparation after Cas9 enrichment. It requires more 3rd party reagent than if doing library preparation using the Cas9 Sequencing Kit
   5. Sequence on device	Cas9 Sequencing Kit (SQK-CS9109) Cas9 targeted sequencing
   6. Take basecalled FASTQ files into analysis pipeline	Cas9 Targeted Sequencing Tutorial (EPI2ME Labs)
   7. Assess success of experiment and feed back into probe design and quality of input

7. Important Compatibility of this protocol

   This protocol should only be used in combination with:

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

1. For this protocol, you will need 1–10 µg or 0.1–2 pmol high molecular weight genomic DNA (5 µg recommended).
2. 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

3. 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

4. Important Genomic DNA and its quality

   Unsheared, high-molecular weight DNA, as isolated e.g., using the Qiagen Genomic DNA Kit, at ≥210 ng/µl by Qubit, and stored in TE (pH 8.0) or similar, or nuclease-free water.

   Carryover of 1 mM EDTA from TE will not significantly affect this protocol; however, care should be taken to reduce other contaminants, such as detergents, phenol, chloroform, and salts.

5. Tip Wide-bore tips

   Use wide-bore tips (or regular pipette tips with the narrow ends cut off) where possible to minimise shearing of long DNA.

6. Important Enzyme buffers

   CutSmart Buffer

   Please note that the RNP formation, dephosphorylation, cleavage and dA-tailing steps are performed in CutSmart buffer (NEB Cat # B7204), and the ligation performed in the LNB buffer from LSK109. CutSmart buffer is provided with the NEB Quick Dephosphorylation Kit, but for scaling up the protocol, additional buffer may be ordered directly from NEB (Cat # B7204). The buffers provided with the other enzymes listed above should not be used.

   Tris-EDTA (TE) Buffer

   We strongly recommend TE at pH 7.5, rather than pH 8.0, for the long-term stability of RNA oligos in storage.

7. Sourcing crRNA and tracrRNA

   We recommend using synthetic crRNA and tracrRNA from IDT, which are of sufficient purity and carry modifications that confer stability and nuclease resistance. For this reason we caution against using single-guide RNAs (sgRNAs).

   • S. pyogenes Cas9 Alt-R™ crRNAs
   • S. pyogenes Cas9 tracrRNA (e.g. IDT Alt-R™, Cat # 1072532, 1072533 or 1072534)

   Individual crRNAs and tracrRNA should be resuspended at 100 µM each in TE, pH 7.5, aliquoted to avoid freeze-thawing, and stored at –20° C for up to two weeks or –80° C if stored long-term. The crRNAs/tracrRNAs can be freeze-thawed a maximum of five times.

   crRNAs may be pooled to make panels for generating multiple cuts in a single reaction. To pool crRNAs, we recommend dispensing equal volumes of each crRNA (up to 100 crRNAs, each at 100 µM) into a separate 1.5 ml Eppendorf DNA LoBind tube to make an equimolar crRNA mix.

1. 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.

2. 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.

3. 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 the EPI2ME Platform protocol.

4. 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

1. Important Here, the Cas9 is loaded with crRNA and tracrRNA to form ribonucleoprotein complexes (RNPs) in preparation for the cleavage reaction.
2. Important If using a 'tiling' approach

   The formula below prepares a pool of RNPs for making multiple excisions in a single reaction. If using a tiling approach for probe design (a method for designing probes in two separate overlapping pools to cover a target region >20 kb), prepare 2x RNP complexes, one for each pool of crRNA probes. For more information about tiling, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet.

3. Important RNP complex stability

   Upon receipt, we recommend aliquoting individual probes or pools of crRNAs for storage, to minimise freeze-thawing.

   Panels of RNPs may be formed ahead of time and stored at 4°C for up to a week, or at -80°C for up to a month with no discernible loss of activity. However, we recommend making a fresh RNP complex for every experiment if possible.

4. Tip crRNA and ribonucleoproteins (RNPs)

   The following protocol applies to a single crRNA. For panels of multiple crRNAs, see “Notes on multiple crRNAs” below.

5. Important Notes on multiple crRNAs

   If you have validated a set of crRNA probes that are always run together, we recommend pooling all crRNA probes and then aliquoting that pool and freezing each aliquot at -80°C. Each time you run a Cas9 experiment and need to make an RNP complex, take out a crRNA pool aliquot and combine with the tracrRNA.

6. Pre-heat a thermal cycler to 95ºC.
7. Thaw an aliquot of NEB CutSmart Buffer, mix by vortexing, and place on ice.
8. In an 1.5 ml Eppendorf DNA LoBind tube, pool the crRNA probes for each cleavage reaction by combining equal volumes of each crRNA probe, resuspended at 100 µM in TE (pH 7.5).
   • A single crRNA or many crRNA probes (up to ~100) may be used in a single cleavage reaction.
   • The crRNA probes may also be pre-mixed as an off-catalogue request from IDT.
   • For example, probes for the HTT gene, found here, can be used as an individual experiment or in addition to other probes as an in-run control.
   • Unused crRNA probe mix may be stored at -80ºC and minimal freeze thaw recommended.

9. Anneal the pooled crRNAs with tracrRNA in Duplex Buffer by assembling the following in a 0.2 ml thin-walled PCR tube, as follows:

   Reagent	Volume
   Duplex buffer	8 µl
   crRNA pool (100 µM, equimolar)	1 µl
   tracrRNA (100 µM)	1 µl
   Total	10 µl

10. Mix well by pipetting and spin down.
11. Using a thermal cycler heat the above reaction mix at 95ºC for 5 mins, then remove the tube from the thermal cycler and allow it to cool to room temperature, then spin down the tube to collect any liquid in the bottom of the tube.
    • Storage and reuse of the annealed mix is not recommended.

12. To form Cas9 RNPs, assemble the components in the table in a 1.5 ml Eppendorf DNA LoBind tube in the following order:

    Reagent	Volume
    Annealed crRNA•tracrRNA pool (10 µM)	10 µl (Step 4, above)
    10x NEB CutSmart buffer	10 µl
    Nuclease-free water	79.2 µl
    HiFi Cas9 (62 µM)	0.8 µl
    Total	100 µl

    Note: Please refer to the Tip below for scaling down this RNP mix.

13. Mix thoroughly by flicking the tube.
14. Tip The above step yields an excess amount of RNPs, but 10 µl are carried forwards for each reaction into the next target cleavage step. Any excess RNPs may be stored at 4º C for up to a week. The reaction may be scaled, as shown in the below table.

    Number of reactions	3	5	10
    Components	Volume (µl)	Volume (µl)	Volume (µl)
    Annealed crRNA•tracrRNA pool (10 µM) (Step 1)	3	5	10
    10x NEB CutSmart buffer	3	5	10
    Nuclease-free water	23.7	39.6	79.2
    HiFi Cas9 (62 µM)	0.3	0.4	0.8
    Total	30	50	100

15. Form the RNPs by incubating the tube at room temperature for 30 minutes, then return the RNPs on ice until required.
16. Tip Proceed to the next step (gDNA dephosphorylation) during the 30 min RNP incubation step.

1. This step reduces background reads by removing 5’ phosphates from non-target DNA ends.
2. Important If using a 'tiling' approach

   If using a tiling approach for probe design (a method for designing probes in two separate overlapping pools to cover a target region >20 kb), and have just produced 2x separate RNP complexes, users need to perform the dephosphorylation, Cas9 cleavage and adapter ligation step twice (one reaction per pool of probes). For more information about tiling, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet.

3. Prepare the DNA in nuclease-free water
   • Transfer 1-10 μg (with 5 μg recommended) genomic DNA into a 0.2 ml thin-walled PCR tubes
   • Adjust to 24 µl with nuclease-free water
   • Mix thoroughly by flicking the tube avoiding unwanted shearing
   • Spin down briefly in a microfuge

4. Mix the Quick calf intestinal phosphatase (CIP) in the tube by pipetting up and down. Ensure that it is at room temperature before use.
5. Assemble the following components in a clean 0.2 ml thin-walled PCR tube:

   Reagent	Volume
   NEB CutSmart Buffer (10x)	3 µl
   HMW genomic DNA (at ≥ 210 ng/µl)*	24 µl
   Total	27 µl

   • Note: For an initial test, we recommend 5 µg genomic DNA input. Preparing input DNA step yields ~100-2000x target coverage. Target coverage scales linearly with input amount, so the input amount may be reduced accordingly if lower throughput is acceptable.

6. Ensure the components are thoroughly mixed by pipetting, and spin down.
7. Add 3 µl of CIP to the tube.
8. Mix gently by flicking the tube, and spin down.
9. Using a thermal cycler, incubate at 37ºC for 10 minutes, 80ºC for 2 minutes then hold at 20ºC (room temperature).

1. In this step, Cas9 RNPs (see 'Preparing the Cas9 ribonucleoprotein complexes') and Taq polymerase are added to the dephosphorylated genomic DNA sample.

   This process cleaves target and dA-tails all available DNA ends in one step, activating the Cas9 cut sites for ligation.

2. Thaw the dATP tube, vortex to mix thoroughly and place on ice.
3. Spin down and place the tube of Taq polymerase on ice.
4. In a fresh 1.5 ml DNA Eppendorf LoBind tube, make a 10 mM dATP solution by adding 1 µl of the 100 mM dATP stock to 9 µl of nuclease-free water. Vortex to mix, then spin down.
5. To the PCR tube containing 30 µl dephosphorylated DNA sample, add:

   Reagent	Volume
   Dephosphorylated genomic DNA sample (Step 2)	30 µl
   Cas9 RNPs (Step 1)	10 µl
   10 mM dATP	1 µl
   NEB Taq polymerase	1 µl
   Total	42 µl

6. Carefully mix the contents of the tube by gentle inversion, then spin down and place the tube in the thermal cycler.
7. Using the thermal cycler, incubate at 37ºC for 15-60 minutes*, then 72ºC for 5 minutes and hold at 4ºC or return to tube to ice.
8. Important *The Cas9 enzyme is active at 37ºC, and denatured at 72ºC. We recommend a 15 minute cut time by default. Longer 37ºC incubations may increase the amount of off-target reads without increasing the yield of on-target reads, while shorter incubations may result in incomplete target cleavage. However, some regions may benefit from a longer incubation at 37ºC.

1. Here, AMX adapters from the Ligation Sequencing Kit (SQK-LSK109), are ligated to the ends generated by Cas9 cleavage.
2. 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.
3. Thaw the Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
4. Carefully transfer the contents of the 0.2 ml thin-walled PCR tube to a fresh 1.5 ml Eppendorf DNA LoBind Tube using a wide-bore pipette tip.
5. Thaw an aliquot of Adapter Mix (AMX), mix by flicking the tube, pulse-spin to collect the liquid in the bottom of the vial, then return the vial to ice.
6. Bring the AMPure XP beads to room temperature.
7. Assemble the following at room temperature in a separate 1.5 ml Eppendorf DNA LoBind Tube, adding Adapter Mix (AMX) last, before you are ready to begin the ligation:

   Reagent	Volume
   Ligation Buffer (LNB)	20 µl
   Nuclease-free water	3 µl
   NEBNext Quick T4 DNA Ligase	10 µl
   Adapter Mix (AMX)*	5 µl
   Total	38 µl

   Note: The Adapter Mix (AMX) must be added last and immediately before the ligation step.

8. Mix by pipetting the above ligation mix thoroughly. Ligation Buffer (LNB) is very viscous, so the adapter ligation mix needs to be well-mixed.
9. IMPORTANT: Add 20 µl of the adapter ligation mix to the cleaved and dA-tailed sample. Mix gently by flicking the tube. Do not centrifuge the sample at this stage. Immediately after mixing, add the remainder of the adapter ligation mix to the cleaved and dA-tailed sample, to yield an 80 µl ligation mix.
10. Ensure the components are thoroughly mixed by pipetting, and spin down.
11. Incubate the reaction for 10 minutes at room temperature.
12. Important DNA precipitation

    A white precipitate may form upon addition of the adapter ligation mix to the dA-tailed DNA. Adding the ligation mixture in two parts helps to reduce precipitation. However, the presence of a precipitate does not necessarily indicate failure of ligation of the sequencing adapter to target molecule ends.

1. This step removes excess unligated adapters and other short DNA fragments, and concentrates and buffer-exchanges the library in preparation for sequencing.
2. Important If using a 'tiling' approach

   Complete steps 1 and 2 below, then pool the samples together into a single tube, then add 0.3x (96 µl) of AMPure XP beads. For more information about tiling, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet.

3. Resuspend the AMPure XP beads by vortexing and ensure they are at room temperature before use. Thaw an aliquot of LFB (Long Fragment Buffer) or SFB (Short Fragment Buffer) from SQK-LSK109, as required, and an aliquot of EB (Elution Buffer).

   Note: To retain DNA fragments shorter than 3 kb (by purifying fragments of all sizes), use one tube of use Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down, and place on ice. To enrich for DNA fragments of 3 kb or longer, use one tube of Long Fragment Buffer (LFB) at room temperature mix by vortexing, spin down and place on ice.

4. Add 1 volume (80 µl) of TE (pH 8.0) to the ligation mix. Mix gently by flicking the tube.
5. Add 0.3x volume (48 µl) of AMPure XP Beads to the ligation sample. The volume of beads is calculated based on the volume after the addition of TE. Mix gently by inversion. If any sample ends up in the lid, spin down the tube very gently, keeping the beads suspended in liquid.
6. Incubate the sample for 10 minutes at room temperature. Do not agitate or pipette the sample.
7. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
8. Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl Short Fragment Buffer (SFB), depending on the size of your target molecule. Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
9. Repeat the previous step.
10. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
11. Remove the tube from the magnetic rack and resuspend pellet in 13 µl Elution Buffer (EB). Incubate for 10 minutes at room temperature.

    Note: For targets >30 kb, we recommend increasing the elution time to 30 minutes.

12. Pellet the beads on a magnet until the eluate is clear and colourless.
13. Remove and retain 12 µl of eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube.
    • Dispose of the pelleted beads

14. End of Step The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.
15. Optional Action

    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.

1. Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down at room temperature.
2. To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.
3. Open the MinION Mk1B lid and slide the flow cell under the clip.

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

   

   

4. 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.

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

   

6. 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.
7. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
   1. Set a P1000 pipette to 200 µl
   2. Insert the tip into the priming port
   3. Turn the wheel until the dial shows 220-230 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.

   

8. 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.

   

9. Thoroughly mix the contents of the Loading Beads (LB) by pipetting.
10. Important The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
11. In a new tube, prepare the library for loading as follows:

    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 II (SBII) and Loading Beads II (LBII) because the fuel in the buffer will start to be consumed by the adapter.

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

    

    

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

    

15. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.

    

    

16. 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.

17. Place the light shield onto the flow cell, as follows:

    1. Carefully place the leading edge of the light shield against the clip.
       Note: Do not force the light shield underneath the clip.

    2. 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.

    

18. Caution The MinION Flow Cell Light Shield is not secured to the flow cell and careful handling is required after installation.
19. End of Step Close the device lid and set up a sequencing run on MinKNOW.

1. Overview of nanopore data analysis

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

2. How to start sequencing

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

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

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

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

   Follow the instructions in the GridION user manual.

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

   Follow the instructions in the MinION Mk1C user manual.

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

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

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

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

3. Important When selecting the sequencing kit in MinKNOW, please choose SQK-CAS109 instead of SQK-LSK109.
4. Understanding Cas enrichment

   The Duty Time feature in the MinKNOW software can be used to judge the quality of your experiment. The duty time plot shows the distribution of channel states over time, grouped by time chunks, or 'buckets'. The basic view shows the five main channel states: Sequencing, Pore, Recovering, Inactive, and Unclassified. Clicking the "More" button shows a more detailed breakdown of channel states.

   It is recommended to observe the duty time plot populating over the first 30 min-1 hr of the sequencing run. By this time, the channel state distribution will give an indication whether the DNA library is of a good quality, and whether the flow cell is performing well.

   Note: The Duty Time plots will be noticeably different to a conventional SQK-LSK109 run. A much smaller percentage of pores will be observed as Sequencing/Strand.

   If Active Channel Selection is enabled during the run, the software instantly switches to a new channel in the group if a channel is in the “Saturated” or “Multiple” state, or after ~5 minutes if a channel is “Recovering”. This feature maximises the number of channels sequencing at the start of the experiment, however this may also result in an artificially high number of "Sequencing" or "Pore" channels in the duty time plot. For this reason, we recommend referring to the Mux Scan Results plot, which shows the true distribution of channel states at the point of the most recent mux scan.

5. Understanding Duty Time plots during a Cas9 targeted sequencing run

   As discussed above, the user should expect a lower proportion of pores in Sequencing compared to a standard SQK-LSK109 run, while the total number of available pores should be roughly consistent between a Cas9 targeted sequencing experiment and SQK-LSK109 experiment.

   

   FLO-MIN106 Duty time plot for a Cas9 targeted sequencing experiment using a human gene. From the Duty Time plot, there is an equivalent number of active pores between a SQK-LSK109 run and Cas9 taregeted sequencing run. In a Cas9 experiment, the sequencing pore is roughly 5-15% (light green) of the total number of pores.

1. 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.

1. 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.

2. 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.
3. 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.

4. 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.

1. 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.

2. 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.

3. 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.

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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

12. 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.

13. 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.