PCR tiling of SARS-CoV-2 virus - automated Agilent (SQK-RBK110.96 with EXP-MRT001)

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Overview

For Research Use Only

This protocol uses extracted RNA samples in an automated library preparation using the Agilent Bravo to increase reproducibility and reduce human error. Multiple samples can be prepared simultaneously for high sequencing output.

Document version: MRTA_9157_v110_revG_18May2022

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 and scripts.

The PCR tiling of SARS-CoV-2 virus - automated Agilent Bravo (SQK-RBK110.96 with EXP-MRT001) protocol is an automated 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 the Agilent Bravo liquid handling robot.

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 (SQK-RBK110.96) and Midnight RT PCR Expansion (EXP-MRT001) for barcoding and library preparation.

We have developed this automated protocol on the Agilent Bravo liquid handling robot. The majority of the process is automated with minimal hands-on time which is required for sample quantification and deck re-loading.

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 (SQK-RBK110.96), 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.

Steps in the sequencing workflow:

Prepare for your experiment

you will need to:

Before starting - Manual steps:

  • Extract your RNA
  • Ensure you have your sequencing kit, the correct equipment and reagents
  • Prepare your reagents, samples and labware to load on the Agilent Bravo
  • 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:

Automated steps:

  • 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

__Manual steps:__
  • Quantify your DNA library as a quality control
  • Prime the flow cell and load your DNA library into the flow cell

Overview of library preparation workflow:

The image below is representative of the steps that take place in the automated runs for X96 samples.

ARTIC SQK-RBK110.96 96 samples spike-seq

Note: Timings are dependent on number of samples and include hands on time, such as deck loading and sample quantification

Sequencing and analysis

You will need to:

  • Start a sequencing run using the MinKNOW software, selecting SQK-RBK110.96 and EXP-MRT001 in kit selection, which will collect raw data from the device and convert it into basecalled reads

Timings

Note: Timings are approximate and subject to change with updates.

Process X24 samples X48 samples X96 samples Hands-on time
Deck set-up ~30 minutes
Process 1:
cDNA synthesis
8 minutes 16 minutes 24 minutes
Off-Deck thermocyler 17 minutes 17 minutes 17 minutes
Process 2:
cDNA amplification
10 minutes 16 minutes 21 minutes
Off-Deck thermocyler ~3 hours 30 minutes ~3 hours 30 minutes ~3 hours 30 minutes
Process 3:
Rapid Barcoding
5 minutes 8 minutes 10 minutes
Off-Deck thermocyler 8 minutes 8 minutes 8 minutes
Process 4:
cDNA amplicon pooling/cleanup
40 minutes 50 minutes 60 minutes
Quantification ~10 minutes
Total 4 hours 58 minutes 5 hours 25 minutes 5 hours 50 minutes ~40 minutes

Nomenclature for automation protocol

Throughout this document, 'Protocol' is defined as the assay on the whole and 'Run' refers to the individual scripts for the automated liquid handling robot, which are specific to indicated protocol step(s).

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 (SQK-RBK110.96) with the Midnight RT PCR Expansion (EXP-MRT001) using the Agilent Bravo liquid handling robot.

When processing multiple samples at once, we recommend making master mixes following the indicated volumes to account for the necessary excess. 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 should be carried out in a separate area to the pre-PCR preparation, with dedicated equipment for liquid handling in each area to minimise risk of contamination.

If only one liquid handling robot is available, both pre-PCR and post-PCR sections of the assay can be performed in the same robot. In this scenario, cleaning the equipment thoroughly between runs is required and we recommend validating the process for your unique set-up.

IMPORTANT

Compatibility of this protocol

This protocol should only be used in combination with:

  • Rapid Barcoding Kit 96 (SQK-RBK110.96)
  • Midnight RT PCR Expansion (EXP-MRT001)
  • R9.4.1 flow cells (FLO-MIN106)
  • 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 (SQK-RBK110.96)
  • 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
  • Bravo Lab Disposable Pipette Tips 250 µl - compatible with Bravo 96LT head (19477-022)
  • Arvensis B-Frame BIOCOMPOSITE 96 Well PCR Plate Fully Skirted Low Profile 0.2 ml wells
  • Hard-Shell® 96-Well PCR Plates, low profile, thin-walled, skirted, white/clear (Bio-Rad, Cat # HSP9601)
  • 96-well 0.8 ml MIDI plate (we recommend Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate: ThermoFisher, Cat # AB0859)
  • PCR plate seals

Equipment
  • Agilent Bravo liquid handling robot
  • Centrifuge capable of taking 96-well plates
  • 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
  • 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

Rapid Barcoding Kit 96 (SQK-RBK110.96) contents

RBK110.96 kit contents

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Rapid Barcode plate RB96 - 3 plates 8 µl per well
AMPure XP Beads AXP Brown 3 1,200
Sequencing Buffer II SBII Red 1 500
Rapid Adapter F RAP-F Green 1 25
Elution Buffer EB Black 1 500
Loading Beads II LBII Pink 1 360
Loading Solution LS White cap, pink label 1 400
Flush Tether FLT Purple 1 400
Flush Buffer FB White 1 bottle 15,500

This product contains AMPure XP reagent manufactured by Beckman Coulter, Inc.

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. Pre-PCR

Materials
  • Input RNA in 10 mM Tris-HCl, pH 8.0
  • Midnight Primer Pool A (MP A)
  • Midnight Primer Pool B (MP B)

Consumables
  • Arvensis B-Frame BIOCOMPOSITE 96 Well PCR Plate Fully Skirted Low Profile 0.2 ml wells
  • Bravo Lab Disposable Pipette Tips 250 µl - compatible with Bravo 96LT head (19477-022)
  • PCR plate seals
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • LunaScript RT SuperMix (LS RT)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Q5 HS Master Mix (Q5)

Equipment
  • Ice bucket with ice
  • PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
  • Agilent Bravo liquid handling robot
  • P1000 pipette and tips
  • P200 pipette and tips
  • P20 pipette and tips
  • P2 pipette and tips
  • Microfuge
  • Thermal cycler
  • Centrifuge capable of taking 96-well plates

Run setup:

Turn on the Agilent Thermocube and set Position 4 to 4°C.

To cool position 4 prior to the run: open the file, under the deck layout, and tick the box near the block you want to cool. Set up the temperature and hit 'Run' to execute.

Agilent Themocube CPAC Temperature

Thaw and keep the samples, LunaScript, Q5 Hot Start Master mix, Midnight Primer Pool A (MPA) and Midnight Primer Pool B (MPB) on ice.

IMPORTANT

To minimise risk of contamination, we recommend handling the Lunascript and primers in a clean template-free PCR hood.

IMPORTANT

Prior to use, ensure all reagents have been thoroughly mixed by performing 20-30 full-volume pipette mixes.

Note: This is especially important with the Q5 Hot start master mix as the contents can precipitate following freeze-thaw cylces.

Take care when mixing and vortexing as this will introduce air bubbles, resulting in loss of volume.

In the template-free pre-PCR hood, prepare the following Primer master mixes in 1.5 ml Eppendorf DNA LoBind tubes and mix thoroughly as follows:

For x24 samples:

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

For x48 samples:

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

For x96 samples:

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

Note: Taking care not to introduce air bubbles, pipette mix 10-15 times between each addition and perform a final full-volume pipette mix 10 times.

Keep on ice until use.

In the template-free pre-PCR hood and using a clean Arvensis plate, prepare the reagent input plate as follows:

For x24 samples:

Reagents Agilent PRE x24

For x48 samples:

Reagents Agilent PRE x48

For x96 samples:

Reagents Agilent PRE x96

Note: Take care to not introduce air bubbles while aliquoting the reagents into the Arvensis plate.

Once complete seal the plate and keep on ice until ready to transfer over to the Agilent Bravo robot.

In a pre-PCR hood and using a clean Arvensis plate, prepare the RNA sample input plate as follows:

For x24 samples:

Sample input x24 MRT

For x48 samples:

Sample input x48 MRT

For x96 samples:

Sample input x96 MRT

Note: Take care to not introduce air bubbles while aliquoting the samples into the Arvensis plate.

Once complete seal the plate and keep on ice until ready to transfer over to the Agilent Bravo robot.

On the Agilent Bravo, select the 'cDNA and Multiplex (X) samples' protocol, where (X) indicates the number of samples to be processed.

Set the number of columns of samples and the PCR plate type.

  • For X24 samples, select 3
  • For X48 samples, select 6
  • For X96 samples, select 12

Select 'Display Deck Layout'.

Add the labware, sample plate and reagent input plate as indicated on the form display.

For X24 samples:

Agilent pre deck layout x24

For X48 samples:

Agilent pre deck layout x48

For X96 samples:

Agilent pre deck layout x96

Select 'Run Protocol'.

To start the run, select 'Ok' from the figure below:

Agilent Bravo start run OK

After the Agilent Bravo has added the 2 µl of LunaScript to the samples, the robot will stop with the the following message:

Agilent Bravo RT PCR Wide

Remove the sample plate containing the LunaScript from the Agilent Bravo, seal it and spin it down.

Place the sample plate in a Thermal cycler and incubate 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

After placing the sample plate in the Thermal cycler, press "Continue" on the Agilent Bravo to start the addition of the Primer master mixes into a clean Arvensis plate.

Once the thermal cycler has completed the cDNA synthesis, remove the sample plate and spin down.

When prompted, place the sample plate back in the Agilent Bravo deck into position 4.

After the Agilent Bravo has added the 2.5 µl of RT to the Primer master mix plate/plates, the robot will stop with the the following message:

Agilent PCR AMP WIDE

Remove the PCR plate (or plates if processing X96 samples) from the Agilent Bravo, seal it and spin it down.

Place in the thermal cycler and 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
END OF STEP

Once the PCR amplification is complete, remove the plate(s) from the thermal cycler and spin down. The plate(s) will be taken forward to the Post-PCR section of the protocol.

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

5. Post-PCR

Materials
  • Rapid Barcode Plate (RB96)
  • AMPure XP Beads (AXP, or SPRI)
  • Elution Buffer from the Oxford Nanopore kit (EB)
  • Rapid Adapter F (RAP F)

Consumables
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly prepared 80% ethanol in nuclease-free water
  • Bravo Lab Disposable Pipette Tips 250 µl - compatible with Bravo 96LT head (19477-022)
  • Arvensis B-Frame BIOCOMPOSITE 96 Well PCR Plate Fully Skirted Low Profile 0.2 ml wells
  • 96-well 0.8 ml MIDI plate (we recommend Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate: ThermoFisher, Cat # AB0859)
  • PCR plate seals
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)

Equipment
  • Agilent Bravo liquid handling robot
  • Thermal cycler
  • Centrifuge capable of taking 96-well plates
  • P1000 pipette and tips
  • P200 pipette and tips
  • P20 pipette and tips
  • P2 pipette and tips
  • Qubit fluorometer (or equivalent for QC check)

Rapid Barcoding:

Select the 'Barcoding and clean-up' program on the Agilent Bravo.

Set the number of columns for your run:

  • For three columns of each primer pool (6 in total), select 3 columns.
  • For six columns of each primer pool (12 in total), select 6 columns.
  • For twelve columns of each primer pool (24 in total), select 12 columns.

Select 'Display Deck Layout'.

Add the Primer plates, the Rapid barcode plate and the labware as indicated on the display.

Select 'Run protocol'.

To start the run, select 'Ok' from the figure below:

Agilent Bravo start run OK

OPTIONAL ACTION

For runs using X24 or X48 samples, you will need to enter the starting index plate column for the RBK plate when prompted.

For example: If your barcode starts at column 4, enter "4" as seen in the figure below.

Set initial value variables WIDE

After the Agilent Bravo has completed the Rapid Barcoding plate, the robot will stop with the following message:

Agilent Bravo POST RBK Wide

Remove the barcoded sample plate from the Agilent Bravo, seal it and spin it down.

Place the barcoded sample plate in the thermal cycler and incubate at 30°C for 2 minutes and then at 80°C for 2 minutes.

Sample pooling and clean-up:

Follow the instructions for the Agilent Bravo deck set-up on the screen prompt.

Agilent Bravo POST RBK Wide

Resuspend the AMPure XP beads by vortexing.

While the barcoding plate is in the thermal cycler, using a clean MIDI 96 deep-well plate prepare the reagents as follows:

For X24 samples:

Reagents Agilent POST x24

For X48 samples:

Reagents Agilent POST x48

For X96 samples:

Reagents Agilent POST x96

Note: Take care to not introduce air bubbles while aliquoting the reagents into the deep-well plate.

Ensure all reagents are properly mixed prior to use.

When preparing the plates, well A12 will be empty. This is where the final elution will be found at the end of the Agilent Bravo run.

Prepare the deck of the Agilent Bravo with the reagent plate and labware as follows:

For X24 or X48 samples:

Agilent POST deck layout x24 48

For X96 samples:

Agilent POST deck layout x96

Once the thermal cycler has finished, remove the plate and spin it down.

Remove the seal on the rapid barcoding plate and return it to the allocated position on the Agilent Bravo deck.

Select "Continue" on the Agilent Bravo screen prompt to continue the run.

Ensure all of the labware and reagent plates are in the correct positions, you have removed all of the lids and the plates are unsealed before continuing.

After the run ends, the final library can be collected from position A12 in the deep-well plate. Remove and retain the final elution into a clean 1.5 ml Eppendorf DNA LoBind tube

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

END OF STEP

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

6. 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

Equipment
  • MinION device
  • SpotON Flow Cell
  • MinION and GridION Flow Cell Light Shield
  • P1000 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
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 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 Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down the SBII and FLT at room temperature.

Prepare the flow cell priming mix in a suitable vial for the number of flow cells to flush. Once combined, mix well by briefly vortexing.

Reagent Volume per flow cell
Flush Tether (FLT) 30 µl
Flush Buffer (FB) 1,170 µl

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 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) because the fuel in the buffer will start to be consumed by the adapter.

Complete the flow cell priming:

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

Flow Cell Loading Diagrams Step 5

Flow Cell Loading Diagrams Step 06 V5

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

Add 75 μl of 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.

7. 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.

8. 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 software is natively supported on the GridION device and can be simply installed on most Linux computers and servers. The installation is outlined later in the document.

The Midnight analysis uses the ARTIC bioinformatics workflow.

Demultiplexed sequence reads are processed using the ARTIC FieldBioinformatics software that has been subtly 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.

On GridION devices, the wf-artic workflow will start automatically after sequencing. However, on other devices, this will have to be started manually as outlined further on this page under 'Running a Midnight analysis'.

Software set up and installation

The wf-artic workflow requires the Nextflow and Docker software to have been installed. The EPI2ME quickstart guide provides instructions for the installation of these requirements for GridION, PromethION and general Ubuntu Linux users and provides a little more introduction to the Nextflow software.

Automatic start on GridION:

To set up the Midnight analysis to start automatically after sequencing on GridION, select the Rapid Barcoding Kit 96 (SQK-RBK110.96) kit with the Midnight RT PCR Expansion (EXP-MRT001) pack on MinKNOW when setting up a sequencing run.

When the workflow has finished, the relevant analysis files will be available in the following output folder:

processing/artic/artic_DATE_TIME_67195e17

Post-run analysis on GridION:

The Midnight analysis can also be started post-run on GridION:

  1. On the start page, click 'Analysis'
  2. Click 'Workflow'
  3. From the dropdown menu, select 'post_processing/artic/artic'
  4. Select your input folder with the sequencing data and the location for the output folder

Midnight gridion workflow

Using Linux command line:

The wf-artic workflow can be run from the Linux command line. The workflow can be installed or updated with the command:

$ nextflow pull epi2me-labs/wf-artic

Demultiplexing of multiple barcoded samples

The wf-artic requires FASTQ format sequence data that has already been demultiplexed. Sequences can either be demultiplexed directly in the MinKNOW software or as a post-sequencing step by the guppy_barcoder software provided by the Guppy software.

The Midnight protocol uses a rapid barcoding kit; it is therefore important to note that the demultiplexing step must not require barcodes at both ends of the sequence.

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

Running a Midnight analysis

The reference command for running a Midnight analysis is as follows. The parameters are explained further on in the document.

nextflow run epi2me-labs/wf-artic \

--scheme_name SARS-CoV-2 \

--scheme_version V1200 \

--min_len 200 \

--max_len 1100 \

--out_dir PATH_TO_OUTPUT \

--fastq PATH_TO_FASTQ_PASS \

-work-dir PATH_TO_INTERMEDIATE_FILES

Type the command into you linux terminal and press enter.

Picture1Midnight

Nextflow will describe the analysis as it progresses; the figure above shows an example run from a 48-plex analysis. We can see which processes have completed and the processes that are still running and or queued.

Parameter definitions

  • nextflow run epi2me-labs/wf-artic An instruction to use the Nextflow software to run a workflow, which is further explained here.

  • --scheme_name SARS-CoV-2 An instruction for the ARTIC software to use the primer scheme that corresponds to the amplicons tiled across the whole SARS-CoV-2 genome.

  • --scheme_version V1200 This defines the version of the ARTIC primers to use. The Midnight protocol uses the primer set refered to as V1200.

  • –-min_len 200 This sets the minimum allowed sequence length as 200 nucleotides.

  • –-max_len 1100 This sets the maximum allowed sequence length as 1100 nucleotides.

  • --out_dir PATH_TO_OUTPUT This instructs the Nextflow software where the results should be stored; please change PATH_TO_OUTPUT to the location on your computer where files should be stored.

  • --fastq PATH_TO_FASTQ_PASS This instructs Nextflow which sequences should be used in the analysis. Please change PATH_TO_FASTQ_PASS to an existing fastq_pass folder from a Midnight run.

  • -work_dir PATH TO WORK DIRECTORY Please note the single hyphen; this is a Nextflow parameter. This defines where the intermediate files are stored. This folder may contain a significant amount of information; please see the section on housekeeping.

Other command line parameters

Other commands and options can be provided to the Nextflow command:

  • --samples This describes a sample file that links barcode identifier with sample names. These sample names will be reported in the HTML format report and in the CSV file of genotypes. The sample file should be a comma-delimited file and must contain the column names barcode and sample_name.

  • --help This will display the help-file which describes the available parameters and other information on default values and their meanings.

  • --medaka_model This defines the model that should be used by the Medaka software for variant calling (and thus consensus preparation).

IMPORTANT

Basecalling model

If you are basecalling using a FAST model, then this should be changed to reflect the appropriate model and version of Guppy used.

  • Default model used: r941_min_hac_variant_g507.
  • If you have used FAST basecalling, please use: r941_min_fast_variant_g507.
  • If HAC basecalling was performed using an earlier version of MinKNOW, please use: r941_min_high_g360.

Result files

Results will be written to the location specified by the --out_dir parameter. These output results 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 nextflow parameter, -work-dir, was introduced as a parameter to define where the workflow intermediate files are stored. This folder will accumulate a significant number of files that correspond to raw BAM files and other larger intermediates. We recommend this folder to be routinely cleared.

Updating the wf-artic software

Updated versions of the wf-artic software may be released and an alert to the availability of newer workflow versions will be noted by the Nextflow software at run-time.

To update the software:

nextflow pull epi2me-labs/wf-artic

It may be necessary to first delete the cached workflow files. This can be achieved with the command:

nextflow drop -f epi2me-labs/wf-artic

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. 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.

11. 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: 6/29/2023