cDNA-PCR sequencing - sequence-specific (SQK-PCS111)

Oxford Nanopore Technologies
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Protocol

cDNA-PCR sequencing - sequence-specific (SQK-PCS111) VCPSS_9185_v111_revD_19Apr2023

The fastest and simplest protocol for targeted cDNA sequencing

  • Higher yields than traditional cDNA synthesis
  • Splice variants and fusion transcripts
  • Compatible with R9.4.1 flow cells

For Research Use Only

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 RESEARCH USE ONLY

Overview

The fastest and simplest protocol for targeted cDNA sequencing

  • Higher yields than traditional cDNA synthesis
  • Splice variants and fusion transcripts
  • Compatible with R9.4.1 flow cells

For Research Use Only

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.

Document version: CPSS_9185_v111_revD_19Apr2023

1. Overview of the protocol

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.

cDNA-PCR Sequencing Kit features

This kit is highly recommended for users who:

  • Would like to identify and quantify full-length transcripts
  • Are looking for a faster and simpler method for cDNA synthesis: ~210 minutes library prep + variable time for PCR
  • Want to explore isoforms, splice variants and fusion transcripts using full-length cDNAs
  • Wish to start from total RNA or have a low starting amount of RNA
  • Would like to generate high amounts of cDNA data

Introduction to cDNA-PCR protocol

This protocol outlines a targeted cDNA sequencing method using the cDNA-PCR Sequencing Kit (SQK-PCS111) and a user-defined sequence-specific primer. You can swap out the cDNA RT Adapter (CRTA) and design your own primer to target a specific RNA sequence during reverse transcription. During the strand-switching step, a UMI is incorporated before the double-stranded cDNA is amplified by PCR using primers containing rapid attachment chemistry. The rapid sequencing adapters are then added to the amplified sample.

Steps in the sequencing workflow:

Prepare for your experiment You will need to:

  • Extract your RNA, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
  • Ensure you have your sequencing kit, the correct equipment and third-party reagents.
  • Download the software for acquiring and analysing your data.
  • Check your flow cell to ensure it has enough pores for a good sequencing run.

**Library preparation** You will need to:
  • Perform reverse transcription with sequence-specific primer.
  • Using the strand-switching protocol, prepare full-length cDNAs from your RNA sample.
  • Amplify the samples by PCR, adding rapid attachment primers during the PCR step.
  • Ligate sequencing adapters to the PCR products.
  • Prime the flow cell, and load your cDNA library into the flow cell.

SQK PCS111 seq specific workflow v1

Sequencing and analysis You will need to:

  • Start a sequencing run using the MinKNOW software, which will collect raw data from the device and will basecall the reads.
  • (Optional) Start the EPI2ME software and select a workflow for further analysis, e.g. metagenomic analysis or drug resistance mapping
IMPORTANT

Compatibility of this protocol

This protocol should only be used in combination with:

  • cDNA-PCR Sequencing Kit (SQK-PCS111)
  • R9.4.1 flow cells (FLO-MIN106)
  • Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

Materials
  • 4 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 200 ng total RNA
  • cDNA-PCR Sequencing Kit (SQK-PCS111)
  • Custom-ordered sequence-specific primer
Consumables
  • Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 10 mM dNTP solution (e.g. NEB N0447)
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
  • RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
  • Exonuclease I (NEB, Cat # M0293)
  • Qubit RNA HS Assay Kit (ThermoFisher, cat # Q32852)
  • Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
Equipment
  • Hula mixer (gentle rotator mixer)
  • Magnetic rack, suitable for 1.5 ml Eppendorf tubes
  • Microfuge
  • 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
  • Qubit fluorometer (or equivalent for QC check)
  • Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf cat # 022510509)
Optional equipment
  • Agilent Bioanalyzer (or equivalent)

For this protocol, you will need 4 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 200 ng total RNA.

Sequence-specific primer sequence

The cDNA RT Adapter (CRTA) supplied in the cDNA-PCR Sequencing Kit (SQK-PCS111) is designed to ligate to poly(A) tailed RNAs. However, you can design your own primer to target a specific RNA sequence (for example, to only sequence 16S rRNA) for subsequent cDNA sequencing.

To perform this type of targeting, order the oligo below, replacing the [sequence-specific] region with ~22 bases complementary to the 3' end of your target RNA sequence. Please note that the exact number of bases may need to be optimised depending on the sequence targeted.

5' - ACTTGCCTGTCGCTCTATCTTC - [sequence-specific] - 3'

We recommend starting with a stock concentration of 2 μM. However, you may find it useful to perform a titration of different primer concentrations. All primers should be HPLC purified.

Input RNA

It is important that the input RNA meets the quantity and quality requirements. Using too little or too much RNA, or RNA of poor quality (e.g. fragmented or containing chemical contaminants) can affect your library preparation.

For instructions on how to perform quality control of your RNA sample, please read the Input DNA/RNA QC protocol.

For further information on using RNA as input, please read the links below.

These documents can also be found in the DNA/RNA Handling page.

Third-party reagents

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

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

IMPORTANT

Rapid Adapter T (RAP T) used in this kit and protocol is not interchangeable with other sequencing adapters.

cDNA-PCR Sequencing Kit (SQK-PCS111) contents

SQK-PCS111 1

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Strand Switching Primer II SSPII Violet 1 20 µl
RT Primer RTP Yellow 1 10 µl
cDNA RT Adapter CRTA Amber 1 10 µl
Rapid Adapter T RAP T Green 1 10 µl
Annealing Buffer AB Orange 1 10 µl
cDNA Primer cPRM White cap, grey label 1 40 µl
Elution Buffer EB Black 1 500 µl
Short Fragment Buffer SFB Clear 1 1,800 µl
Sequencing Buffer II SBII Red 1 500 µl
Loading Beads II LBII Pink 1 360 µl
Loading Solution LS White cap, pink label 1 400 µl
Flush Buffer FB Blue 6 1,170 µl
Flush Tether FLT White cap, purple label 1 200 µl

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 three months 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. Reverse transcription and strand-switching

Materials
  • 4 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 200 ng total RNA
  • Custom-ordered sequence-specific primer
  • Strand Switching Primer II (SSPII)
Consumables
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • 10 mM dNTP solution (e.g. NEB cat # N0447)
  • Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
  • RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Qubit RNA HS Assay Kit (ThermoFisher, cat # Q32852)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
Equipment
  • Microfuge
  • 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
  • Qubit fluorometer (or equivalent for QC check)
  • Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf cat # 022510509)

Thaw the following reagents and spin down briefly using a microfuge, before mixing as indicated in the table below, and place on ice.

Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting
Custom-ordered sequence-specific primer ✓ ✓ ✓
Strand Switching Primer II (SSPII) ✓ ✓ ✓
RNaseOUT Not frozen ✓ ✓
10 mM dNTP solution ✓ ✓ ✓
Maxima H Minus Reverse Transcriptase Not frozen ✓ ✓
Maxima H Minus 5x RT Buffer ✓ ✓ Mix by vortexing

Prepare the RNA in nuclease-free water.

  • Transfer 4 ng Poly(A)+ RNA, or 200 ng total RNA into a 1.5 ml Eppendorf DNA LoBind tube
  • Adjust the volume to up to 9 μl with nuclease-free water
  • Mix by flicking the tube to avoid unwanted shearing
  • Spin down briefly in a microfuge

Prepare the following reaction in a 0.2 ml PCR tube:

Reagent Volume
RNA 9 μl
Custom-ordered sequence-specific primer, diluted to 2 μM 1 μl
10 mM dNTPs 1 μl
Total volume 11 μl

Mix gently by flicking the tube, and spin down.

Incubate at 65°C for 5 minutes and then snap cool on a pre-chilled freezer block for 1 minute.

To the same 0.2 ml PCR tube, add the following:

Reagent Volume
Maxima H Minus 5x RT Buffer 4 μl
RNaseOUT 1 μl
Nuclease-free water 1 μl
Strand Switching Primer II (SSPII) 2 μl
Total (including all reagents) 19 μl
TIP

Strand Switching Primer II (SSPII) base pairs to the deoxycytidine present at the 5' end of the first cDNA strand synthesised. This allows the reverse transcriptase to "strand-switch" for synthesis of the second cDNA strand.

Mix gently by flicking the tube, and spin down.

Incubate at 42°C for 2 minutes in the thermal cycler.

Add 1 µl of Maxima H Minus Reverse Transcriptase. The total volume is now 20 µl.

Mix gently by flicking the tube, and spin down.

Incubate using the following protocol using a thermal cycler:

Cycle step Temperature Time No. of cycles
Reverse transcription and strand-switching 42°C 90 mins 1
Heat inactivation 85°C 5 mins 1
Hold 4°C ∞
END OF STEP

Take your samples forward into the next step. However, at this point it is also possible to store the sample at -20°C overnight.

5. Selecting for full-length transcripts by PCR

Materials
  • cDNA Primer (cPRM)
  • Elution Buffer (EB)
Consumables
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • Exonuclease I (NEB, Cat # M0293)
  • Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
Equipment
  • Thermal cycler
  • Vortex mixer
  • Hula mixer (gentle rotator mixer)
  • Magnetic rack, suitable for 1.5 ml Eppendorf tubes
  • Ice bucket with ice
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
  • Qubit fluorometer (or equivalent for QC check)
  • Agilent Bioanalyzer (or equivalent)
IMPORTANT

The 22.5 μl of reverse-transcribed sample is used to make 4x 50 μl PCR reactions which will be pooled at a later stage, with 5 μl of reverse-transcribed sample in each PCR reaction. Do NOT use all 22.5 μl of the reverse transcription reaction in a single PCR reaction.

Reverse transcriptase is a PCR inhibitor and the RT material must be diluted enough for PCR to take place.

Thaw the cDNA Primer (cPRM), Elution Buffer (EB). LongAmp Hot Start Taq 2X Master Mix and Exonuclease I at room temperature, spin down and pipette mix. Store the reagents on ice.

Spin down the reverse-transcribed RNA sample.

Prepare four fresh 0.2 ml PCR tubes and add 5 μl of reverse-transcribed sample per tube.

In each of the 0.2 ml PCR tubes containing the reverse-transcribed sample, prepare the following reaction at room temperature:

Reagent Volume
Reverse-transcribed sample (from previous step) 5 μl
cDNA Primer (cPRM) 1.5 μl
Nuclease-free water 18.5 μl
2x LongAmp Hot Start Taq Master Mix 25 μl
Total (including all reagents) 50 μl

Mix gently by pipetting.

Amplify using the following cycling conditions.

Cycle step Temperature Time No. of cycles
Initial denaturation 95°C 30 secs 1
Denaturation 95°C 15 secs 10-18*
Annealing 62°C 15 secs 10-18*
Extension 65°C 60 secs per kb 10-18*
Final extension 65°C 6 mins 1
Hold 4°C ∞

*We recommend 14 cycles as a starting point. However, the number of cycles can be adjusted between the values shown according to experimental needs.

For further information, please read The effect of varying the number of PCR cycles in the PCR-cDNA Sequencing Kit document.

Add 1 μl Exonuclease I directly to each PCR tube. Mix by pipetting.

TIP

Exonuclease I is added to remove any excess primers which have not successfully annealed.

Incubate the reaction at 37°C for 15 minutes, followed by 80°C for 15 minutes in the thermal cycler.

Pool the four PCR reactions (total 204 μl) in a clean 1.5 ml Eppendorf DNA LoBind tube.

Resuspend the AMPure XP beads by vortexing.

Add 160 µl of resuspended AMPure XP beads to the reaction.

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

Prepare 1 ml of fresh 70% ethanol in nuclease-free water.

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

Keep the tube on the magnet and wash the beads with 500 µl of freshly-prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

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

Remove the tube from the magnetic rack and resuspend pellet in 12 µl of Elution Buffer (EB).

Incubate at room temperature for 10 minutes.

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

Remove and retain 12 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

  • Remove and retain the eluate which contains the cDNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
  • Dispose of the pelleted beads

For each sample, analyse 1 µl of the amplified cDNA for size, quantity and quality using a Qubit fluorometer and Agilent Bioanalyzer (or equivalent) for a QC check.

IMPORTANT

Sometimes a high-molecular weight product is visible in the wells of the gel when the PCR products are run, instead of the expected smear. These libraries are typically associated with poor sequencing performance. We have found that repeating the PCR with fewer cycles can remedy this.

Take forward 15-25 fmol of amplified cDNA and make the volume up to 11 μl in Elution Buffer (EB).

Mass Molarity if fragment length = 0.5 kb Molarity if fragment length = 1.5 kb Molarity if fragment length = 3 kb
5 ng 16 fmol 5 fmol 3 fmol
10 ng 32 fmol 11 fmol 5 fmol
15 ng 49 fmol 16 fmol 8 fmol
20 ng 65 fmol 22 fmol 11 fmol
25 ng 81 fmol 27 fmol 13 fmol
50 ng 154 fmol 51 fmol 26 fmol

If the quantity of amplified cDNA is above 25 fmol, the remaining cDNA can be frozen and stored for another sequencing experiment (in this case, library preparation would start from the Adapter Addition step). We recommend avoiding multiple freeze-thaw cycles to prevent DNA degradation.

The new sequencing adapter used in Kit 11 chemistry has a higher capture rate, enabling lower flow cell loading amounts to give optimal pore occupancy.

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, re-loading 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.

6. Adapter addition

Materials
  • Rapid Adapter T (RAP T)
  • Elution Buffer (EB)
Consumables
  • 1.5 ml Eppendorf DNA LoBind tubes
Equipment
  • Microfuge
  • Ice bucket with ice
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
IMPORTANT

Rapid Adapter T (RAP T) used in this kit and protocol is not interchangeable with other sequencing adapters.

Spin down the Rapid Adapter T (RAP T) and place on ice.

Add 1 μl of Rapid Adapter T (RAP T) to the amplified cDNA library.

Mix well by pipetting and spin down.

Incubate the reaction for 5 minutes at room temperature.

Spin down briefly.

END OF STEP

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

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, re-loading 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.

7. Priming and loading the SpotON flow cell

Materials
  • Flush Buffer (FB)
  • Flush Tether (FLT)
  • Loading Beads II (LBII)
  • Sequencing Buffer II (SBII)
  • Loading Solution (LS)
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
  • SpotON Flow Cell
  • P1000 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
TIP

Priming and loading a MinION flow cell

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

Using the Loading Solution

We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water. Note: some customers have noticed that viscous libraries can be loaded more easily when not using Loading Beads II.

Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), 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 device 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 flow cell 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 II (LBII) by pipetting.

IMPORTANT

The Loading Beads II (LBII) 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 II (SBII) 37.5 µl
Loading Beads II (LBII) mixed immediately before use, or Loading Solution (LS), if using 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).

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 the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

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.

8. Data acquisition and basecalling

How to start sequencing

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

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

  • On a computer either directly or remotely connected to a sequencing device.
  • Directly on a GridION, MinION Mk1C or PromethION 24/48 sequencing device.

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

To start a sequencing run on MinKNOW:

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

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

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

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

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

5. Click Start on the Review page to start the sequencing run.

Data analysis after sequencing

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

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

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

9. 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.

10. Downstream analysis

Post-basecalling analysis

There are several options for further analysing your basecalled data:

1. EPI2ME workflows

For in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in EPI2ME. The platform provides a vehicle where workflows deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code and example data.

2. Research analysis tools

Oxford Nanopore Technologies' Research division has created a number of analysis tools, which are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.

3. Community-developed analysis tools

If a data analysis method for your research question is not provided in any of the resources above, please refer to the Bioinformatics section of the Resource centre. 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.

11. 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.

12. 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 Mk 1B 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: 12/6/2023

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