PCR tiling of SARS-CoV-2 virus - Rapid Barcoding Kit 96 V14 and Midnight RT PCR Expansion (SQK-RBK114.96 and EXP-MRT001)

Overview

  • This protocol uses extracted RNA samples
  • Includes reverse transcription and tiled PCR amplification
  • For multiplexing 1-96 samples
  • Library preparation time ~315 minutes
  • Fragmentation
  • Compatible with R10.4.1 flow cells

For Research Use Only

This is an Early Access product For more information about our Early Access programmes, please see this article on product release phases.

Document version: MRT_9186_v114_revG_17Aug24

1. Overview of the protocol

IMPORTANT

This protocol is a work in progress and some details are expected to change over time. Please make sure you always use the most recent version of the protocol.

The PCR tiling of SARS-CoV-2 virus with Rapid Barcoding Kit 96 V14 and Midnight RT PCR Expansion (SQK-RBK114.96 and EXP-MRT001) protocol is an updated version of the PCR tiling of SARS-CoV-2 virus with rapid barcoding and Midnight RT PCR Expansion (SQK-RBK110.96 and EXP-MRT001) using our most recent Kit 14 chemistry and an updated downstream analysis.

Introduction to the protocol

To enable support for the rapidly expanding user requests, the team at Oxford Nanopore Technologies have put together an updated workflow based on the ARTIC Network protocols and analysis methods. The protocol uses Oxford Nanopore Technologies' Rapid Barcoding Kit 96 V14 (SQK-RBK114.96) and Midnight RT PCR Expansion (EXP-MRT001) for barcoding and library preparation.

While this protocol is available in the Nanopore Community, we kindly ask users to ensure they are citing the members of the ARTIC network who have been behind the development of these methods.

This protocol is similar to the ARTIC amplicon sequencing protocol for MinION for SARS-CoV-2 v3 (LoCost) by Josh Quick and the method used in Freed et al., 2020. The protocol generates amplicons in a tiled fashion across the whole SARS-CoV-2 genome.

To generate tiled PCR amplicons from the SARS-CoV-2 viral cDNA for use with the Rapid Barcoding Kit 96 V14 (SQK-RBK114.96), primers were designed by Freed et al., 2020 using Primal Scheme. These primers are in the Midnight RT PCR Expansion (EXP-MRT001) and are designed to generate 1.2 kb amplicons. Primer sequences can be found here.

As mutations in SARS-CoV-2 variants emerge amplicon drop out may be observed; for users wishing to design their own primer spike-ins to address this we suggest adding to the appropriate primer pool at a final concentration between 3.33 µM and 6.66 µM.

Steps in the sequencing workflow:

Prepare for your experiment you will need to:

  • Extract your RNA
  • Ensure you have your sequencing kit, the correct equipment and 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

Prepare your library You will need to:

  • Reverse transcribe your RNA samples with random hexamers
  • Amplify the samples by tiled PCR using separate primer pools
  • Combine the primer pools
  • Attach Rapid Barcodes supplied in the kit to the DNA ends, pool the samples and SPRI purify
  • Prime the flow cell and load your DNA library into the flow cell

ARTIC SQK-RBK110.96 96 samples spike-seq (3)

Sequencing and analysis You will need to:

  • Start a sequencing run using the MinKNOW software, selecting SQK-RBK114.96 in kit selection, which will collect raw data from the device and convert it into basecalled reads
  • (Optional): Perform downstream analysis of the data using the wf-artic analysis workflow integrated within the EPI2ME Labs application

Before starting

This protocol outlines how to carry out PCR tiling of SARS-CoV-2 viral RNA samples on a 96-well plate using the Rapid Barcoding Kit 96 V14 (SQK-RBK114.96) with the Midnight RT PCR Expansion (EXP-MRT001).

It is required to use total RNA extracted from samples that have been screened by a suitable qPCR assay.

When processing multiple samples at once, we recommend making master mixes with an additional 10% of the volume. We also recommend using a template-free pre-PCR hood for making up the master mixes, and a separate template pre-PCR hood for handling the samples. It is important to clean and/or UV irradiate these hoods between sample batches. Furthermore, to track and monitor cross-contamination events, it is important to run a negative control reaction at the reverse transcription stage using nuclease-free water instead of sample, and carrying this control through the rest of the prep.

All post-PCR procedures must be carried out in a separate area to the pre-PCR preparation, with dedicated equipment for liquid handling in each area.

IMPORTANT

Compatibility of this protocol

This protocol should only be used in combination with:

  • Rapid Barcoding Kit 96 V14 (SQK-RBK114.96)
  • Midnight RT PCR Expansion (EXP-MRT001)
  • R10.4.1 flow cells (FLO-MIN114)
  • Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

Materials
  • Input RNA in 10 mM Tris-HCl, pH 8.0
  • Rapid Barcoding Kit 96 V14 (SQK-RBK114.96)
  • Midnight RT PCR Expansion (EXP-MRT001)

Consumables
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Freshly prepared 80% ethanol in nuclease-free water
  • Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 2 ml Eppendorf DNA LoBind tubes
  • 5 ml Eppendorf DNA LoBind tubes
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with PCR seals
  • Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)

Equipment
  • Hula mixer (gentle rotator mixer)
  • Magnetic rack
  • Centrifuge capable of taking 96-well plates
  • Microfuge
  • Vortex mixer
  • Thermal cycler
  • Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • Ice bucket with ice
  • Timer
  • Qubit fluorometer (or equivalent)
Optional equipment
  • Eppendorf 5424 centrifuge (or equivalent)
  • PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
  • PCR-Cooler (Eppendorf)
  • Stepper pipette and tips

For this protocol, you will need your extracted RNA in 8 µl 10 mM Tris-HCl, pH 8.0.

IMPORTANT

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

Rapid Barcoding Kit 96 V14 (SQK-RBK114.96) contents

RBK114.96 tubes (1)

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Rapid Adapter RA Green 2 15
Adapter Buffer ADB Clear 1 100
AMPure XP Beads AXP Amber 3 1,200
Elution Buffer EB Black 1 1,500
Sequencing Buffer SB Red 1 1,700
Library Beads LIB Pink 1 1,800
Library Solution LIS White cap, pink label 1 1,800
Flow Cell Flush FCF Clear 1 15,500
Flow Cell Tether FCT Purple 2 200
Rapid Barcodes RB01-96 - 3 plates 8 µl per well

This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

Midnight RT PCR Expansion (EXP-MRT001) contents

EXP-MRT001 1

Name Acronym Cap colour Number of vials Fill volume per vial (µl)
LunaScript RT SuperMix LS RT Blue 3 500
Q5 HS Master Mix Q5 Orange 6 1,500
Midnight Primer Pool A MP A White 3 15
Midnight Primer Pool B MP B Clear 3 15

Midnight Primer sequences

As mutations in SARS-CoV-2 variants emerge amplicon drop out may be observed; for users wishing to design their own primer spike-ins to address this we suggest adding to the appropriate primer pool at a final concentration between 3.33 µM and 6.66 µM.

Below are the sequences for the V3 primer scheme used in the Midnight RT PCR Expansion.

Pool A

Primer name Primer Sequence
SARSCoV_1200_1_LEFT ACCAACCAACTTTCGATCTCTTGT
SARSCoV_1200_1_RIGHT GGTTGCATTCATTTGGTGACGC
SARSCoV_1200_3_LEFT GGCTTGAAGAGAAGTTTAAGGAAGGT
SARSCoV_1200_3_RIGHT GATTGTCCTCACTGCCGTCTTG
SARSCoV_1200_5_LEFT ACCTACTAAAAAGGCTGGTGGC
SARSCoV_1200_5_RIGHT AGCATCTTGTAGAGCAGGTGGA
SARSCoV_1200_7_LEFT ACCTGGTGTATACGTTGTCTTTGG
SARSCoV_1200_7_RIGHT GCTGAAATCGGGGCCATTTGTA
SARSCoV_1200_9_LEFT AGAAGTTACTGGCGATAGTTGTAATAACT
SARSCoV_1200_9_RIGHT TGCTGATATGTCCAAAGCACCA
SARSCoV_1200_11_LEFT AGACACCTAAGTATAAGTTTGTTCGCA
SARSCoV_1200_11_RIGHT GCCCACATGGAAATGGCTTGAT
SARSCoV_1200_13_LEFT ACCTCTTACAACAGCAGCCAAAC
SARSCoV_1200_13_RIGHT CGTCCTTTTCTTGGAAGCGACA
SARSCoV_1200_15_LEFT TTTTAAGGAATTACTTGTGTATGCTGCT
SARSCoV_1200_15_RIGHT ACACACAACAGCATCGTCAGAG
SARSCoV_1200_17_LEFT TCAAGCTTTTTGCAGCAGAAACG
SARSCoV_1200_17_RIGHT CCAAGCAGGGTTACGTGTAAGG
SARSCoV_1200_19_LEFT GGCACATGGCTTTGAGTTGACA
SARSCoV_1200_19_RIGHT CCTGTTGTCCATCAAAGTGTCCC
SARSCoV_1200_21_LEFT TCTGTAGTTTCTAAGGTTGTCAAAGTGA
SARSCoV_1200_21_RIGHT GCAGGGGGTAATTGAGTTCTGG
21_right_spike GTGTATGATTGAGTTCTGGTTGTAAG
SARSCoV_1200_23_LEFT ACTTTAGAGTCCAACCAACAGAATCT
23_left_spike ACTTTAGAGTTCAACCAACAGAATCT
SARSCoV_1200_23_RIGHT TGACTAGCTACACTACGTGCCC
SARSCoV_1200_25_LEFT TGCTGCTACTAAAATGTCAGAGTGT
SARSCoV_1200_25_RIGHT CATTTCCAGCAAAGCCAAAGCC
SARSCoV_1200_27_LEFT TGGATCACCGGTGGAATTGCTA
SARSCoV_1200_27_RIGHT TGTTCGTTTAGGCGTGACAAGT
SARSCoV_1200_29_LEFT TGAGGGAGCCTTGAATACACCA
SARSCoV_1200_29_RIGHT TAGGCAGCTCTCCCTAGCATTG

Pool B

Primer name Primer sequences
SARSCoV_1200_2_LEFT CCATAATCAAGACTATTCAACCAAGGGT
SARSCoV_1200_2_RIGHT ACAGGTGACAATTTGTCCACCG
SARSCoV_1200_4_LEFT GGAATTTGGTGCCACTTCTGCT
SARSCoV_1200_4_RIGHT CCTGACCCGGGTAAGTGGTTAT
SARSCoV_1200_6_LEFT ACTTCTATTAAATGGGCAGATAACAACTG
SARSCoV_1200_6_RIGHT GATTATCCATTCCCTGCGCGTC
SARSCoV_1200_8_LEFT CAATCATGCAATTGTTTTTCAGCTATTTTG
SARSCoV_1200_8_RIGHT TGACTTTTTGCTACCTGCGCAT
SARSCoV_1200_10_LEFT TTTACCAGGAGTTTTCTGTGGTGT
SARSCoV_1200_10_RIGHT TGGGCCTCATAGCACATTGGTA
SARSCoV_1200_12_LEFT ATGGTGCTAGGAGAGTGTGGAC
SARSCoV_1200_12_RIGHT GGATTTCCCACAATGCTGATGC
SARSCoV_1200_14_LEFT ACAGGCACTAGTACTGATGTCGT
SARSCoV_1200_14_RIGHT GTGCAGCTACTGAAAAGCACGT
SARSCoV_1200_16_LEFT ACAACACAGACTTTATGAGTGTCTCT
SARSCoV_1200_16_RIGHT CTCTGTCAGACAGCACTTCACG
SARSCoV_1200_18_LEFT GCACATAAAGACAAATCAGCTCAATGC
SARSCoV_1200_18_RIGHT TGTCTGAAGCAGTGGAAAAGCA
SARSCoV_1200_20_LEFT ACAATTTGATACTTATAACCTCTGGAACAC
SARSCoV_1200_20_RIGHT GATTAGGCATAGCAACACCCGG
SARSCoV_1200_22_LEFT GTGATGTTCTTGTTAACAACTAAACGAACA
SARSCoV_1200_22_RIGHT AACAGATGCAAATCTGGTGGCG
22_right_spike AACAGATGCAAATTTGGTGGCG
SARSCoV_1200_24_LEFT GCTGAACATGTCAACAACTCATATGA
24_left_spike GCTGAATATGTCAACAACTCATATGA
SARSCoV_1200_24_RIGHT ATGAGGTGCTGACTGAGGGAAG
SARSCoV_1200_26_LEFT GCCTTGAAGCCCCTTTTCTCTA
SARSCoV_1200_26_RIGHT AATGACCACATGGAACGCGTAC
SARSCoV_1200_28_LEFT TTTGTGCTTTTTAGCCTTTCTGCT
SARSCoV_1200_28_RIGHT GTTTGGCCTTGTTGTTGTTGGC
SARSCoV_1200_28_LEFT_27837T TTTGTGCTTTTTAGCCTTTCTGTT

Rapid barcode sequences

Component Sequence
RB01 AAGAAAGTTGTCGGTGTCTTTGTG
RB02 TCGATTCCGTTTGTAGTCGTCTGT
RB03 GAGTCTTGTGTCCCAGTTACCAGG
RB04 TTCGGATTCTATCGTGTTTCCCTA
RB05 CTTGTCCAGGGTTTGTGTAACCTT
RB06 TTCTCGCAAAGGCAGAAAGTAGTC
RB07 GTGTTACCGTGGGAATGAATCCTT
RB08 TTCAGGGAACAAACCAAGTTACGT
RB09 AACTAGGCACAGCGAGTCTTGGTT
RB10 AAGCGTTGAAACCTTTGTCCTCTC
RB11 GTTTCATCTATCGGAGGGAATGGA
RB12 CAGGTAGAAAGAAGCAGAATCGGA
RB13 AGAACGACTTCCATACTCGTGTGA
RB14 AACGAGTCTCTTGGGACCCATAGA
RB15 AGGTCTACCTCGCTAACACCACTG
RB16 CGTCAACTGACAGTGGTTCGTACT
RB17 ACCCTCCAGGAAAGTACCTCTGAT
RB18 CCAAACCCAACAACCTAGATAGGC
RB19 GTTCCTCGTGCAGTGTCAAGAGAT
RB20 TTGCGTCCTGTTACGAGAACTCAT
RB21 GAGCCTCTCATTGTCCGTTCTCTA
RB22 ACCACTGCCATGTATCAAAGTACG
RB23 CTTACTACCCAGTGAACCTCCTCG
RB24 GCATAGTTCTGCATGATGGGTTAG
RB25 GTAAGTTGGGTATGCAACGCAATG
RB26 CATACAGCGACTACGCATTCTCAT
RB27 CGACGGTTAGATTCACCTCTTACA
RB28 TGAAACCTAAGAAGGCACCGTATC
RB29 CTAGACACCTTGGGTTGACAGACC
RB30 TCAGTGAGGATCTACTTCGACCCA
RB31 TGCGTACAGCAATCAGTTACATTG
RB32 CCAGTAGAAGTCCGACAACGTCAT
RB33 CAGACTTGGTACGGTTGGGTAACT
RB34 GGACGAAGAACTCAAGTCAAAGGC
RB35 CTACTTACGAAGCTGAGGGACTGC
RB36 ATGTCCCAGTTAGAGGAGGAAACA
RB37 GCTTGCGATTGATGCTTAGTATCA
RB38 ACCACAGGAGGACGATACAGAGAA
RB39 CCACAGTGTCAACTAGAGCCTCTC
RB40 TAGTTTGGATGACCAAGGATAGCC
RB41 GGAGTTCGTCCAGAGAAGTACACG
RB42 CTACGTGTAAGGCATACCTGCCAG
RB43 CTTTCGTTGTTGACTCGACGGTAG
RB44 AGTAGAAAGGGTTCCTTCCCACTC
RB45 GATCCAACAGAGATGCCTTCAGTG
RB46 GCTGTGTTCCACTTCATTCTCCTG
RB47 GTGCAACTTTCCCACAGGTAGTTC
RB48 CATCTGGAACGTGGTACACCTGTA
RB49 ACTGGTGCAGCTTTGAACATCTAG
RB50 ATGGACTTTGGTAACTTCCTGCGT
RB51 GTTGAATGAGCCTACTGGGTCCTC
RB52 TGAGAGACAAGATTGTTCGTGGAC
RB53 AGATTCAGACCGTCTCATGCAAAG
RB54 CAAGAGCTTTGACTAAGGAGCATG
RB55 TGGAAGATGAGACCCTGATCTACG
RB56 TCACTACTCAACAGGTGGCATGAA
RB57 GCTAGGTCAATCTCCTTCGGAAGT
RB58 CAGGTTACTCCTCCGTGAGTCTGA
RB59 TCAATCAAGAAGGGAAAGCAAGGT
RB60 CATGTTCAACCAAGGCTTCTATGG
RB61 AGAGGGTACTATGTGCCTCAGCAC
RB62 CACCCACACTTACTTCAGGACGTA
RB63 TTCTGAAGTTCCTGGGTCTTGAAC
RB64 GACAGACACCGTTCATCGACTTTC
RB65 TTCTCAGTCTTCCTCCAGACAAGG
RB66 CCGATCCTTGTGGCTTCTAACTTC
RB67 GTTTGTCATACTCGTGTGCTCACC
RB68 GAATCTAAGCAAACACGAAGGTGG
RB69 TACAGTCCGAGCCTCATGTGATCT
RB70 ACCGAGATCCTACGAATGGAGTGT
RB71 CCTGGGAGCATCAGGTAGTAACAG
RB72 TAGCTGACTGTCTTCCATACCGAC
RB73 AAGAAACAGGATGACAGAACCCTC
RB74 TACAAGCATCCCAACACTTCCACT
RB75 GACCATTGTGATGAACCCTGTTGT
RB76 ATGCTTGTTACATCAACCCTGGAC
RB77 CGACCTGTTTCTCAGGGATACAAC
RB78 AACAACCGAACCTTTGAATCAGAA
RB79 TCTCGGAGATAGTTCTCACTGCTG
RB80 CGGATGAACATAGGATAGCGATTC
RB81 CCTCATCTTGTGAAGTTGTTTCGG
RB82 ACGGTATGTCGAGTTCCAGGACTA
RB83 TGGCTTGATCTAGGTAAGGTCGAA
RB84 GTAGTGGACCTAGAACCTGTGCCA
RB85 AACGGAGGAGTTAGTTGGATGATC
RB86 AGGTGATCCCAACAAGCGTAAGTA
RB87 TACATGCTCCTGTTGTTAGGGAGG
RB88 TCTTCTACTACCGATCCGAAGCAG
RB89 ACAGCATCAATGTTTGGCTAGTTG
RB90 GATGTAGAGGGTACGGTTTGAGGC
RB91 GGCTCCATAGGAACTCACGCTACT
RB92 TTGTGAGTGGAAAGATACAGGACC
RB93 AGTTTCCATCACTTCAGACTTGGG
RB94 GATTGTCCTCAAACTGCCACCTAC
RB95 CCTGTCTGGAAGAAGAATGGACTT
RB96 CTGAACGGTCATAGAGTCCACCAT

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. Reverse transcription

Materials
  • Input RNA in 10 mM Tris-HCl, pH 8.0
  • LunaScript RT SuperMix (LS RT)

Consumables
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with PCR seals

Equipment
  • Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
  • Thermal cycler
  • Centrifuge capable of taking 96-well plates
  • Ice bucket with ice
Optional equipment
  • PCR-Cooler (Eppendorf)
  • PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
  • Stepper pipette and tips
IMPORTANT

Keep the RNA sample on ice as much as possible to prevent nucleolytic degradation, which may affect sensitivity.

In a clean pre-PCR hood, place a fresh 96-well plate (RT plate) into a PCR Cooler (if using). Using a stepper pipette, or multichannel pipette, add 2 µl of LunaScript RT SuperMix (LS RT) per well.

Depending on the number of samples, fill each well per column as follows:

Plate location X24 samples X48 samples X96 samples
Columns 1-3 1-6 1-12

RT plate prep

To each well containing LunaScript RT SuperMix (LS RT), add 8 µl of sample and gently mix by pipetting. If adding less than 8 µl, make up the rest of the volume with nuclease-free water.

Example for X48 samples: RT plate x48 small

IMPORTANT

We recommend having a negative control and a positive control for every plate of samples.

Seal the RT plate and spin down.

Incubate the samples in the thermal cycler using the following program:

Step Temperature Time Cycles
Primer annealing 25°C 2 min 1
cDNA synthesis 55°C 10 min 1
Heat inactivation 95°C 1 min 1
Hold 4°C
END OF STEP

While the reverse transcription reaction is running, prepare the master mixes as described in the next section.

5. PCR

Materials
  • Q5 HS Master Mix (Q5)
  • Midnight Primer Pool A (MP A)
  • Midnight Primer Pool B (MP B)

Consumables
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with PCR seals

Equipment
  • Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
  • P1000 pipette and tips
  • P200 pipette and tips
  • Thermal cycler
  • Microfuge
  • Centrifuge capable of taking 96-well plates
  • Ice bucket with ice
Optional equipment
  • PCR-Cooler (Eppendorf)
  • PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
  • Stepper pipette and tips

Primer design

To generate tiled PCR amplicons from the SARS-CoV-2 viral cDNA, primers were designed by Freed et al., 2020 using Primal Scheme. These primers are designed to generate 1200 bp amplicons that overlap by approximately 20 bp. These primer sequences can be found here.

IMPORTANT

We recommend handling the primers in a clean template-free PCR hood.

In the template-free pre-PCR hood, prepare the following master mixes in Eppendorf DNA LoBind tubes and mix thoroughly as follows:

Volume per sample:

Reagent Pool A Pool B
Nuclease-free water 3.7 µl 3.7 µl
Midnight Primer Pool A (MP A) 0.05 µl -
Midnight Primer Pool B (MP B) - 0.05 µl
Q5 HS Master Mix (Q5) 6.25 µl 6.25 µl
Total 10 µl 10 µl

For x24 samples:

Reagent Pool A Pool B
Nuclease-free water 102 µl 102 µl
Midnight Primer Pool A (MP A) 2 µl -
Midnight Primer Pool B (MP B) - 2 µl
Q5 HS Master Mix (Q5) 172 µl 172 µl
Total 276 µl 276 µl

For x48 samples:

Reagent Pool A Pool B
Nuclease-free water 203 µl 203 µl
Midnight Primer Pool A (MP A) 3 µl -
Midnight Primer Pool B (MP B) - 3 µl
Q5 HS Master Mix (Q5) 344 µl 344 µl
Total 550 µl 550 µl

For x96 samples:

Reagent Pool A Pool B
Nuclease-free water 407 µl 407 µl
Midnight Primer Pool A (MP A) 6 µl -
Midnight Primer Pool B (MP B) - 6 µl
Q5 HS Master Mix (Q5) 687 µl 687 µl
Total 1,100 µl 1,100 µl

Using a stepper pipette or a multichannel pipette, aliquot 10 µl of Pool A and Pool B into a clean 96-well plate(s) as follows:

Plate location X24 samples X48 samples X96 samples
Columns Pool A: 1-3
Pool B: 4-6
Pool A: 1-6
Pool B: 7-12
Pool A: 1-12
Pool B: 1-12

Note: For X96 samples, Pool A is a separate plate to Pool B.

Primer pools 1

Using a multichannel pipette, transfer 2.5 μl of each RT reaction from the RT plate to the corresponding well for both Pool A and Pool B in the PCR plate(s), taking care not to cross-contaminate different wells. Mix by pipetting the contents of each well up and down.

There should be two PCR reactions per sample.

Example for X48 samples: PCR x48 small

Mix by pipetting the contents of each well up and down.

IMPORTANT

Carry forward the negative control from the reverse transcription reaction to monitor cross-contamination events.

We recommend having a negative control and a positive control for every plate of samples.

Seal the plate(s) and spin down briefly.

Incubate using the following program, with the heated lid set to 105°C:

Step Temperature Time Cycles
Initial denaturation 98°C 30 sec 1
Denaturation

Annealing and extension
98°C

61°C
65°C
15 sec

2 min
3 min

35
Hold 4°C
OPTIONAL ACTION

If necessary, the protocol can be paused at this point. The samples should be kept at 4°C and can be stored overnight.

6. Addition of rapid barcodes

Materials
  • Rapid Barcode Plate (RB01-96)

Consumables
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with PCR seals

Equipment
  • Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl, and tips
  • Thermal cycler
  • Centrifuge capable of taking 96-well plates

Spin down the Rapid Barcode Plate and PCR reactions prior to opening to collect material in the bottom of the wells.

Using a multichannel pipette or stepper pipette, transfer 2.5 μl nuclease-free water to the wells of a fresh 96-well plate (Barcode Attachment Plate).

Depending on the number of samples, aliquot into each well of the columns as follows:

Plate location X24 samples X48 samples X96 samples
Columns 1-3 1-6 1-12

Barcode attachment plate prep

Using a multichannel pipette, transfer the entire contents of each well of PCR Pool B to the corresponding well of PCR Pool A and mix by pipetting.

Depending on the number of samples, Pool B columns will correspond to different Pool A columns.

No. of samples Pool B column Corresponding Pool A column
X24 4
5
6
1
2
3
X48 7
8
9
10
11
12
1
2
3
4
5
6
X96 1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12

Example for X48 samples: PCR pools x48 small

Using a multichannel pipette, transfer 5 µl from each well of PCR Pool A (now containing pooled PCR products) to the corresponding well of the Barcode Attachment Plate and mix by pipetting.

Depending on the number of samples, PCR Pool A will be in each well of the following columns:

Plate location X24 samples X48 samples X96 samples
Columns 1-3 1-6 1-12

Example for X48 samples: Barcode attachment plate x48 small

Using a multichannel pipette, transfer 2.5 μl from the Rapid Barcode Plate to the corresponding well of the Barcode Attachment Plate, taking care not to cross-contaminate different wells. Mix by pipetting.

Depending on the number of samples, aliquot into each well of the columns as follows:

Plate location X24 samples X48 samples X96 samples
Columns 1-3 1-6 1-12

Example for X48 samples: Rapid barcode plate x48 small

IMPORTANT

Samples must be thoroughly mixed.

Seal the Barcode Attachment Plate and spin down.

Incubate the plate in a thermal cycler at 30°C for 2 minutes and then at 80°C for 2 minutes.

7. Pooling samples and clean-up

Materials
  • AMPure XP Beads (AXP)
  • Elution Buffer from the Oxford Nanopore kit (EB)
  • Rapid Adapter (RA)
  • Adapter Buffer (ADB)

Consumables
  • Freshly prepared 80% ethanol in nuclease-free water
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 5 ml Eppendorf DNA LoBind tubes
  • Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)

Equipment
  • Microfuge
  • Centrifuge capable of taking 96-well plates
  • Hula mixer (gentle rotator mixer)
  • Magnetic rack
  • Ice bucket with ice
  • P1000 pipette and tips
  • P200 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • Qubit fluorometer plate reader (or equivalent for QC check)

Briefly spin down the Barcode Attachment Plate to collect the liquid at the bottom of the wells prior to opening.

Pool the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.

We expect to have about ~10 µl per sample.

X24 samples X48 samples X96 samples
Total volume ~240 µl ~480 µl ~960 µl

Mix pooled samples by vortexing.

IMPORTANT

Pooled barcoded samples must be thoroughly mixed.

Transfer half of the barcoded pooled sample to a clean 1.5 ml Eppendorf DNA LoBind tube.

Per sample, we expect to take forward ~5 µl.

X24 samples X48 samples X96 samples
Example volume 120 µl 240 µl 480 µl

Resuspend the AMPure XP Beads (AXP) by vortexing.

To the pooled barcoded sample, add an equal volume of resuspended AMPure XP Beads (AXP, or SPRI) and mix by pipetting.

Example volume X24 samples X48 samples X96 samples
Volume of 1X AXP 120 µl 240 µl 480 µl

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

Prepare at least 3 ml of fresh 80% ethanol in nuclease-free water.

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

Keep the tube on the magnet and wash the beads with 1 ml of freshly-prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

Briefly 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 the pellet by pipetting in 15 µl Elution Buffer (EB). Incubate for 10 minutes at room temperature.

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

Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

CHECKPOINT

Quantify DNA concentration by using the Qubit dsDNA HS Assay Kit.

Take forward 11 µl of your eluted DNA library.

In a fresh 1.5 ml Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows and pipette mix:

Reagent Volume
Rapid Adapter (RA) 1.5 μl
Adapter Buffer (ADB) 3.5 μl
Total 5 μl

Add 1 µl of the diluted Rapid Adapter (RA) to the barcoded DNA.

Mix gently by flicking the tubes, and spin down.

Incubate the reaction for 5 minutes at room temperature.

END OF STEP

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

8. Priming and loading the SpotON flow cell

Materials
  • Flow Cell Flush (FCF)
  • Flow Cell Tether (FCT)
  • Library Solution (LIS)
  • Library Beads (LIB)
  • Sequencing Buffer (SB)

Consumables
  • 1.5 ml Eppendorf DNA LoBind tubes
  • MinION and GridION Flow Cell
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)

Equipment
  • MinION or GridION device
  • MinION and GridION Flow Cell Light Shield
  • P1000 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
IMPORTANT

Please note, this kit is only compatible with R10.4.1 flow cells (FLO-MIN114).

TIP

Priming and loading a flow cell

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

Using the Library Solution

For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the Library Solution (LIS).

Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.

IMPORTANT

For optimal sequencing performance and improved output on MinION R10.4.1 flow cells (FLO-MIN114), we recommend adding Bovine Serum Albumin (BSA) to the flow cell priming mix at a final concentration of 0.2 mg/ml.

Note: We do not recommend using any other albumin type (e.g. recombinant human serum albumin).

Prepare the flow cell priming mix with BSA in a suitable tube for the number of flow cells to flush. Once combined, mix well by pipette mixing.

Reagents Volume per flow cell
Flow Cell Flush (FCF) 1,170 µl
Bovine Serum Albumin (BSA) at 50 mg/ml 5 µl
Flow Cell Tether (FCT) 30 µl
Total volume 1,205 µl

Open the MinION or GridION device lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.

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 Library Beads (LIB) by pipetting.

IMPORTANT

The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

We recommend using the Library Beads (LIB) for most sequencing experiments. However, the Library Solution (LIS) is available for more viscous libraries.

In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:

Reagent Volume per flow cell
Sequencing Buffer (SB) 37.5 µl
Library Beads (LIB) mixed immediately before use, or Library Solution (LIS), if using 25.5 µl
DNA library 12 µl
Total 75 µl

Complete the flow cell priming:

  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.

9. 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.

IMPORTANT

Required settings in MinKNOW

The correct barcoding parameters must be set up on MinKNOW prior to the sequencing run. During the run setup, in the Analysis tab:

  1. Enable Barcoding.
  2. Select Edit options.
  3. Enable Mid-read barcode filtering.
  4. Enable Override minimum barcoding score and set the value to 60.
  5. Enable Override minimum mid-read barcoding score and set the value to 50.

MRT Run setup analysis - Barcoding highlights

MRT Run setup Barcoding options

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.

10. Downstream analysis

Recommended pipeline analysis

The wf-artic is a bioinformatics workflow for the analysis of ARTIC sequencing data prepared using the Midnight protocol. The bioinformatics workflow is orchestrated by the Nextflow software. Nextflow is a publicly available and open-source project that enables the execution of scientific workflows in a scalable and reproducible way. The use of the Nextflow software has been integrated into the EPI2ME Labs software that we recommend for running our downstream analysis methods.

Alternative methods for downstream analysis are available using your device terminal or command line, however we only suggest this for experienced users.

Demultiplexed sequence reads are processed using the ARTIC Field Bioinformatics software that has been modified for the analysis of FASTQ sequences prepared using Oxford Nanopore Rapid Sequencing kits. The other modification to the ARTIC workflow is the use of a primer scheme that defines the sequencing primers used by the Midnight protocol and their genomic locations on the SARS-CoV-2 genome.

The wf-artic workflow includes other analytical steps that include cladistic analysis using Nextclade and strain assignment using Pangolin. The data facets included in the report are parameterised and additional information such as plots of depth-of-coverage across the reference genome is optional.

The complete source for wf-artic is linked, and the Nextflow software will download the scripts and logic flow from this location.

The wf-artic workflow needs to be started manually as outlined below in 'Running a Midnight analysis using EPI2ME Labs'.

Software set-up and installation

The EPI2ME application provides a clean interface to accessing bioinformatics workflows, and is our recommended method in performing your post-sequencing analysis.

Follow the instructions in the EPI2ME Installation guide to install the application on your device.

For more information on how to use EPI2ME, refer to the EPI2ME Quick Start guide.

Installing and updating the wf-artic workflow in EPI2ME Labs:

Ensure you have installed the wf-artic workflow prior to the first analysis set-up.

In the EPI2ME Labs home page, scroll down to the "Install workflows" section and click on epi2me-labs/wf-artic:

EPI2ME labs install wfartic

If you have already installed the wf-artic workflow, ensure you are using the latest version.

Updating the workflow can be done directly through EPI2ME Labs by navigating to the wf-artic workflow page and clicking Update Workflow:

12 EPI2ME labs wfartic analysis updating the workflow

Demultiplexing of multiple barcoded samples

The wf-artic analysis requires FASTQ sequence data that has already been demultiplexed.

Reads will be demultiplexed during sequencing if you are following the recommended "Required settings in MinKNOW". However, demultiplexing can also be done post-sequencing using the MinKNOW software.

For more information and guides on demultiplexing using MinKNOW, refer to the "Post-run analysis" section in our MinKNOW Protocol.

The expected input for wf-artic is a folder of folders as shown below. Each of the barcode folders should contain the FASTQ sequence data and files may either be uncompressed or gzipped.

$ tree -d MidnightFastq/

MidnightFastq/

├── barcode01

├── barcode02

├── barcode03

├── barcode04

├── barcode05

├── barcode06

└── unclassified

IMPORTANT

Basecalling model

The basecalling model should be specified when setting up the wf-artic analysis. This should reflect the basecalling model selected during your run set-up as follows:

  • If using the default model, High-accuracy basecalling (HAC): r1041_e82_400bps_hac_variant_g615
  • If you have used Super accurate basecalling (SUP), please use: r1041_e82_400bps_sup_variant_g615
  • If you have used FAST basecalling, please use: r1041_e82_400bps_fast_variant_g615

Running a Midnight analysis using EPI2ME Labs

Open the EPI2ME Labs application on your device.

EPI2ME labs application logo

Open the "Workflows" tab in the EPI2ME Labs application and click on the "wf-artic" workflow:

3 EPI2ME labs wf-artic workflow

In the "wf-artic" workflow page, select "Run this workflow" to open analysis set-up:

4 EPI2ME labs wfartic workflow run

Complete the wf-artic run set-up:

Select your data input file location. Please note, this folder must contain the demultiplexed FASTQ files of your sequencing run.

5 EPI2ME labs wfartic run setup fastqs

Expand the Primer Scheme Selection tab and set the Scheme version to Midnight-ONT/V3.

6 EPI2ME labs wfartic run setup primer scheme selection

Expand the Advanced Options tab and set the Medaka model to the basecalling model used in your sequencing run.

7 EPI2ME labs wfartic run setup advanced options

8 EPI2ME labs wfartic run setup medaka model

Expand the Extra configuration tab and set the Run name for your wf-artic analysis.

9 EPI2ME labs wfartic run setup run name

Click Launch workflow at the bottom of the page to begin your analysis.

Navigate to the "Analysis" tab in the EPI2ME Labs application to monitor your run:

10 EPI2ME labs wfartic analysis run monitoring

Completed analysis and result files

The wf-artic analysis outputs will be written to the Working Directory folder specified in the EPI2ME Labs Settings tab. The location of this folder is specified in the wf-artic run Instance parameters preceeded by out_dir.

However, these files can also be accessed directly in the EPI2ME Labs application from the completed analysis page for your run:

11 EPI2ME labs wfartic analysis completed run

These outputs include:

  • all_consensus.fasta A multi-FASTA format sequence file containing the consensus sequence for each of the samples investigated. This consensus sequence has been prepared for the whole SARS-CoV-2 genome, not just the spike protein region. The consensus sequence masks the non-spike regions and regions of low sequence coverage with N residues.

  • all_variants.vcf.gz A gzipped VCF file that describes all high-quality genetic variants called by medaka from the sequenced samples.

  • all_variants.vcf.gz.tbi An index file for the gzipped VCF file.

  • consensus_status.txt A tab delimited file that reports whether a consensus sequence has been successfully prepared for a sample, or not.

  • wf-artic-report.html A report summarising these data. This HTML format report also includes the output of the Nextclade software that can be used for a visual inspection of, for example, primer drop out or other qualitative consensus sequence aspects.

Other files are included in the work-directory. This includes per sample VCF files of all genetic variants prior to filtering and other sequences.

Housekeeping and disk usage

The "Working Directory" can be specified in the EPI2ME Labs "Settings" tab and defines where the workflow intermediate files and outputs are stored.

This folder will accumulate a significant number of files that correspond to raw BAM files, other larger intermediates and analysis results files. We recommend this folder to be routinely cleared.

11. 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.

12. 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.

13. 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: 9/17/2024

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