Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109)

Oxford Nanopore Technologies
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MinION: Protocol

Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109) V CAS_9106_v109_revH_16Sep2020

  • We advise all customers to read and consider the information in the "Targeted, amplification-free DNA sequencing using CRISPR/Cas" info sheet before starting this protocol
  • This protocol uses genomic DNA
  • Targeted cutting around specific genomic Regions of Interest (ROI) to achieve enrichment
  • Library preparation time is ~110 minutes
  • Fragment lengths are determined by the cut spacing, and not fragmentation
  • No PCR is required

For Research Use Only

This is a Legacy product This kit is soon to be discontinued. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

FOR RESEARCH USE ONLY

Overview

  • We advise all customers to read and consider the information in the "Targeted, amplification-free DNA sequencing using CRISPR/Cas" info sheet before starting this protocol
  • This protocol uses genomic DNA
  • Targeted cutting around specific genomic Regions of Interest (ROI) to achieve enrichment
  • Library preparation time is ~110 minutes
  • Fragment lengths are determined by the cut spacing, and not fragmentation
  • No PCR is required

For Research Use Only

This is a Legacy product This kit is soon to be discontinued. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

Document version: CAS_9106_v109_revH_16Sep2020

1. Overview of the protocol

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.

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).

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

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

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.

Introduction to the CRISPR/Cas protocol

This protocol describes how to carry out sequencing of genomic DNA using the Cas9 Sequencing Kit (SQK-CS9109) 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 barcoded DNA library using a Cas9 Sequencing Kit (SQK-CS9109), plus several third-party reagents.

Cas9 sequencing workflow

Figure 1. Cas-mediated PCR-free enrichment library preparation for sequencing.

  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, then bind and 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 samples are dA-tailed, which prepares the blunt ends for barcode 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)

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 Cas Sequencing Kit protocol
5. Sequence on device Cas Sequencing Kit protocol
6. Take basecalled FASTQ files into analysis pipeline Targeted, amplification-free DNA sequencing using CRISPR/Cas - Evaluation of read-mapping characteristics from a Cas9 targeted sequencing experiment
7. Assess success of experiment and feed back into probe design and quality of input
IMPORTANT

Compatibility of this protocol

This protocol should only be used in combination with:

  • Cas9 Sequencing Kit (SQK-CS9109)
  • R9.4.1 (FLO-MIN106) flow cells
  • Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

Materials
  • 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.
  • Cas9 Sequencing Kit (SQK-CS9109)
  • Flow Cell Priming Kit (EXP-FLP002)
Consumables
  • S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
  • S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
  • Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
  • Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
Equipment
  • Microfuge
  • Magnetic rack
  • Vortex mixer
  • Thermal cycler
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
  • Ice bucket with ice
  • Timer
Optional equipment
  • Agilent Bioanalyzer (or equivalent)
  • Qubit fluorometer (or equivalent for QC check)
  • Eppendorf 5424 centrifuge (or equivalent)

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

Cas9 Sequencing Kit contents (SQK-CS9109)

cs9109 v1

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Adapter Mix AMX Green 2 40
Ligation Buffer LNB Clear 2 200
Elution Buffer EB Black 1 200
Sequencing Buffer SQB Red 2 300
L Fragment Buffer LFB Orange 2 1,800
S Fragment Buffer SFB Grey 2 1,800
Loading Beads LB Pink 1 360
Phosphatase PHOS Brown tube, yellow label 1 50
TAQ Polymerase TAQ Brown tube, green label 1 15
SPRI Dilution Buffer SDB Brown tube, red label 1 1,200
T4 DNA Ligase LIG Brown tube, blue label 1 140
dATP dATP Brown tube, grey label 1 15
Reaction Buffer RB Brown tube, orange label 1 180

Flow Cell Priming Kit contents (EXP-FLP002)

FLP

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
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.

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.

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.

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

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. Read more in 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. Read more in the MinION Mk1C IT requirements document.

Software for nanopore sequencing

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data 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.

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. Preparing the Cas9 ribonucleoprotein complexes (RNPs)

Consumables
  • S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
  • Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
  • S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
  • Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
  • 0.2 ml thin-walled PCR tubes
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
Equipment
  • Thermal cycler
  • Ice bucket with wet ice
  • P200 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
IMPORTANT

Here, the Cas9 is loaded with crRNA and tracrRNA to form ribonucleoprotein complexes (RNPs) in preparation for the cleavage reaction.

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.

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.

TIP

crRNA and ribonucleoproteins (RNPs)

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

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.

Pre-heat a thermal cycler to 95ºC.

Thaw an aliquot of Reaction Buffer (RB), mix by vortexing, and place on ice.

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.

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

Mix well by pipetting and spin down.

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.

To form Cas9 RNPs, assemble the components in the table in an 1.5 ml Eppendorf DNA LoBind tube; this will form the annealed crRNA•tracrRNA, through pooling in the stated order:

Reagent Volume
Nuclease-free water 79.2 µl
Reaction Buffer (RB) 10 µl
Annealed crRNA•tracrRNA pool (10 µM) 10 µl (Step 4, above)
HiFi Cas9 (62 µM) 0.8 µl
Total 100 µl

Note: Refer to the tip below for scaling-down this ternary RNP mix.

Mix thoroughly by flicking the tube.

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 4ºC 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
Reaction Buffer (RB) 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

Form the RNPs by incubating the tube at room temperature for 30 minutes, then return the RNPs on ice until required.

TIP

Proceed to the next step (gDNA dephosphorylation) during the 30 min RNP incubation step.

5. Dephosphorylating genomic DNA

Materials
  • 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.
  • Phosphatase (PHOS)
Consumables
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • 0.2 ml thin-walled PCR tubes
Equipment
  • Thermal cycler
  • P100 pipette and tips
  • P10 pipette and tips

This step reduces background reads by removing 5’ phosphates from non-target DNA ends.

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.

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

Mix the Phosphatase (PHOS) in the tube by pipetting up and down. Ensure that it is at room temperature before use.

Assemble the following components in a clean 0.2 ml thin-walled PCR tube:

Reagent Volume
Reaction Buffer (RB) 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.

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

Add 3 µl of PHOS to the tube.

Mix gently by flicking the tube, and spin down.

Using a thermal cycler, incubate at 37ºC for 10 minutes, 80ºC for 2 minutes then hold at 20ºC (room temperature).

6. Cleaving and dA-tailing target DNA

Materials
  • 5 µg high molecular weight dephosphorylated genomic DNA (recommended); 1 - 10 µg (or 0.1-2 pmol) can be used accordingly.
  • crRNA-tracrRNA-Cas9 ribonucleoprotein complexes (RNPs)
  • Taq Polymerase (TAQ)
  • dATP (dATP)
Consumables
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
Equipment
  • Thermal cycler
  • Ice bucket with wet ice
  • Vortex mixer
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips

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.

Thaw the dATP tube, vortex to mix thoroughly and place on ice.

Spin down and place the tube of Taq Polymerase (TAQ) on ice.

To the PCR tube containing 30 µl dephosphorylated DNA sample, add:

Reagent Volume
Dephosphorylated genomic DNA sample 30 µl
Cas9 RNPs 10 µl
dATP 1 µl
Taq Polymerase (TAQ) 1 µl
Total 42 µl

Carefully mix the contents of the tube by gentle inversion, then spin down and place the tube in the thermal cycler.

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.

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.

7. Adapter ligation

Materials
  • Ligation Buffer (LNB)
  • Adapter Mix (AMX)
  • T4 Ligase (LIG)
Consumables
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
Equipment
  • Ice bucket with wet ice
  • Vortex mixer
  • P1000 pipette and tips
  • P100 pipette
  • P20 pipette and tips

Here, AMX adapters from the Cas Sequencing Kit (SQK-CS9109) are ligated to the ends generated by Cas9 cleavage.

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.

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.

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.

Bring the AMPure XP beads to room temperature.

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
T4 Ligase (LIG) 10 µl
Adapter Mix (AMX)* 5 µl
Total 38 µl

Mix by pipetting the above ligation mix thoroughly. Ligation Buffer (LNB) is very viscous, so the adapter ligation mix needs to be well-mixed.

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.

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

Incubate the reaction for 10 minutes at room temperature.

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.

8. AMPure XP bead purification

Materials
  • Long Fragment Buffer (LFB)
  • Short Fragment Buffer (SFB)
  • Elution Buffer (EB)
  • SPRI Dilution Buffer (SDB)
Consumables
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • 1.5 ml Eppendorf DNA LoBind tubes
Equipment
  • P1000 pipette and tips
  • P200 pipette and tips
  • P20 pipette and tips
  • Magnetic rack, suitable for 1.5 ml Eppendorf tubes
  • Eppendorf 5424 centrifuge (or equivalent)

This step removes excess unligated adapters and other short DNA fragments, and concentrates and buffer-exchanges the library in preparation for sequencing.

IMPORTANT

If using a 'tiling' approach

Complete steps 1 and either 2 or 3 depending on the DNA fragment lengths you wish to retain. Then pool the samples together into a single tube, before performing steps 4, 5, and 6 with the modified volumes for your pooled sample. This includes 1 volume (160 µl) of SPRI Dilution Buffer (SDB) and 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.

Thaw the Elution Buffer (EB) and SPRI Dilution Buffer (SDB) at room temperature, mix by vortexing, spin down and place on ice.

To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down and place on ice.

To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.

Add 1 volume (80 µl) of the SPRI Dilution Buffer (SDB) to the ligation mix. Mix gently by flicking the tube.

Resuspend the AMPure XP beads by vortexing.

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 SDB. Mix gently by inversion. If any sample ends up in the lid, spin down the tube very gently, keeping the beads suspended in liquid.

Incubate the sample for 10 minutes at room temperature. Do not agitate or pipette the sample.

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

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.

Repeat the previous step.

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.

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.

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

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
END OF STEP

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

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.

TIP

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.

9. Priming and loading the SpotON flow cell

Materials
  • Flow Cell Priming Kit (EXP-FLP002)
  • Loading Beads (LB)
  • Sequencing Buffer (SQB)
Consumables
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
Equipment
  • MinION device
  • MinION and GridION Flow Cell Light Shield
  • P1000 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • SpotON Flow Cell

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.

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.

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.

Flow Cell Loading Diagrams Step 1a

Flow Cell Loading Diagrams Step 1b

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 priming port cover clockwise to open the priming port.

Flow Cell Loading Diagrams Step 2

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:

  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.

Flow Cell Loading Diagrams Step 03 V5

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.

Flow Cell Loading Diagrams Step 04 V5

Thoroughly mix the contents of the Loading Beads (LB) by pipetting.

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.

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.

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.

Flow Cell Loading Diagrams Step 5

Flow Cell Loading Diagrams Step 06 V5

Mix the prepared library gently by pipetting up and down just prior to loading.

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.

Flow Cell Loading Diagrams Step 07 V5

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

Step 8 update

Flow Cell Loading Diagrams Step 9

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:

  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.

J2264 - Light shield animation Flow Cell FAW optimised

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.

10. Data acquisition and basecalling

Overview of nanopore data analysis

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

How to start sequencing

The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software. 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.

IMPORTANT

When selecting the sequencing kit in MinKNOW, please choose SQK-CAS109 instead of SQK-LSK109.

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.

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.

Throughput duty time

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.

11. Downstream analysis

Post-basecalling analysis

There are several options for further analysing your basecalled data:

1. Bioinformatics tutorials

For more in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials, which are available in the Bioinformatics resource section of the Community. The tutorials take the user through installing and running pre-built analysis pipelines, which generate a report with the results. The tutorials are aimed at biologists who would like to analyse data without the help of a dedicated bioinformatician, and who are comfortable using the command line.

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 Community-developed data analysis tool library. 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.

12. 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.

13. 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. SPRI cleanup
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.

14. 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. DNA gel2 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)

image2022-3-25 10-43-25 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 FAQ 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.

Last updated: 11/1/2023

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