Ligation sequencing influenza whole genome V14 (SQK-NBD114.24 or SQK-NBD114.96)

Oxford Nanopore Technologies
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MinION: Protocol

Ligation sequencing influenza whole genome V14 (SQK-NBD114.24 or SQK-NBD114.96) V INF_9189_v114_revI_20Nov2024

  • This protocol uses extracted RNA samples
  • Includes reverse transcription and PCR amplification using two separate primer schemes for Influenza A and Influenza B
  • Includes quantification and normalisation steps to ensure equal distribution of barcodes for 24, 48 and 96 samples.
  • Compatible with R10.4.1 flow cells

For Research Use Only

FOR RESEARCH USE ONLY

Overview

  • This protocol uses extracted RNA samples
  • Includes reverse transcription and PCR amplification using two separate primer schemes for Influenza A and Influenza B
  • Includes quantification and normalisation steps to ensure equal distribution of barcodes for 24, 48 and 96 samples.
  • Compatible with R10.4.1 flow cells

For Research Use Only

Document version: INF_9189_v114_revI_20Nov2024

1. Overview of the protocol

Introduction to the influenza whole genome sequencing protocol

This protocol has been upgraded to our Kit 14 chemistry and describes how to carry out PCR amplification and native barcoding of influenza amplicons using the Native Barcoding Kit 24 or 96 V14 (SQK-NBD114.24 or SQK-NBD114.96). There are 96 unique barcodes available, allowing the user to pool up to 96 different Influenza A and/or Influenza B samples in one sequencing experiment.

While this protocol is available in the Nanopore Community, we kindly ask users to ensure they are citing the following references, that this protocol is based on.

Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza A viruses by Bin Zhou et al., 2009. and Universal influenza B virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics by Bin Zhou et al., 2014.


Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

  • Ensure you have the sequencing kit, the correct equipment and third-party reagents
  • Download the software for acquiring and analysing your data
  • Check your flow cell to ensure it has enough pores for a good sequencing run

Prepare your library

You will need to:

  • Perform RT-PCR amplification on influenza samples
  • Prepare the DNA ends for adapter attachment
  • Ligate native barcodes supplied in the kit to the DNA ends
  • Ligate sequencing adapters supplied in the kit to the DNA ends
  • Prime the flow cell, and load your DNA library into the flow cell

Sequencing

You will need to:

  • Start a sequencing run using the MinKNOW software, which will collect raw data from the device and basecall the reads
  • Demultiplex barcoded reads in MinKNOW choosing the SQK-NBD114.24 or SQK-NBD114.96 kit option
  • Analyse your data using the Influenza typing workflow (wf-flu) in EPI2ME Labs.
IMPORTANT

We do not recommend mixing barcoded libraries with non-barcoded libraries prior to sequencing.

IMPORTANT

Compatibility of this protocol

This protocol should only be used in combination with:

  • Native Barcoding Kit 24 V14 (SQK-NBD114.24)
  • Native Barcoding Kit 96 V14 (SQK-NBD114.96)
  • R10.4.1 flow cells (FLO-MIN114)
  • Flow Cell Wash Kit (EXP-WSH004)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)
  • Native Barcoding Expansion V14 (EXP-NBA114)

2. Equipment and consumables

Materials
  • Input influenza RNA
  • Influenza A primers
  • Influenza B primers
  • Native Barcoding Kit 24 V14 (SQK-NBD114.24) OR Native Barcoding Kit 96 V14 (SQK-NBD114.96)
  • SFB Expansion (EXP-SFB001)
Consumables
  • SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (ThermoFisher, cat # 12574018 or 12574026)
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
  • Freshly prepared 80% ethanol in nuclease-free water
  • NEB Blunt/TA Ligase Master Mix (NEB, M0367)
  • NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
  • NEBNext Quick Ligation Module (NEB, E6056)
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 2 ml Eppendorf DNA LoBind tubes
  • 5 ml Eppendorf DNA LoBind tubes
  • 15 ml Eppendorf DNA LoBind tubes
  • Reagent reservoirs for multichannel pipetting
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
  • Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
Equipment
  • Magnetic rack, suitable for 1.5 ml Eppendorf tubes
  • Magnetic rack suitable for 96 well plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher CAT#12027)
  • Microfuge
  • Vortex mixer
  • Thermal cycler
  • Hula mixer (gentle rotator mixer)
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Multichannel pipette and tips
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
  • Ice bucket with ice
  • Timer
  • Qubit fluorometer (or equivalent for QC check)
Optional equipment
  • PCR hoods with UV steriliser
  • PCR-Cooler (Eppendorf)
  • Eppendorf 5424 centrifuge (or equivalent)

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

A minimum volume of 1 µl is required per sample input of Influenza A or B.

If performing typing of an unknown sample, 2 µl of input will be required:

  • 1 µl input for the Influenza A primer mix
  • 1 µl for the Influenza B primer mix

Before starting

This protocol outlines how to carry out PCR amplification and native barcoding of influenza amplicons from multiple samples on a 96-well plate using the Native Barcoding Kit 24 or 96 V14 (SQK-NBD114.24 or SQK-NBD114.96).

When processing multiple samples simultaneously, 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.

Third-party reagents

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

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

Influenza primer sequences

Influenza A primer sequences described in the protocol originated from: Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza A viruses by Bin Zhou et al., 2009.

Component Sequence
Tuni 12 ACGCGTGATCAGCAAAAGCAGG
Tuni 12.4 ACGCGTGATCAGCGAAAGCAGG
Tuni 13 ACGCGTGATCAGTAGAAACAAGG

Influenza B primer sequences described in the protocol originated from: Universal influenza B virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics by Bin Zhou et al., 2014.

Component Sequence
B-PBs-UniF GGGGGGAGCAGAAGCGGAGC
B-PBs-UniR CCGGGTTATTAGTAGAAACACGAGC
B-PA-UniF GGGGGGAGCAGAAGCGGTGC
B-PA-UniR CCGGGTTATTAGTAGAAACACGTGC
B-HANA-UniF GGGGGGAGCAGAAGCAGAGC
B-HANA-UniR CCGGGTTATTAGTAGTAACAAGAGC
B-NP-UniF GGGGGGAGCAGAAGCACAGC
B-NP-UniR CCGGGTTATTAGTAGAAACAACAGC
B-M-Uni3F GGGGGGAGCAGAAGCACGCACTT
B-Mg-Uni3F GGGGGGAGCAGAAGCAGGCACTT
B-M-Uni3R CCGGGTTATTAGTAGAAACAACGCACTT
B-NS-Uni3F GGGGGGAGCAGAAGCAGAGGATT
B-NS-Uni3R CCGGGTTATTAGTAGTAACAAGAGGATT
IMPORTANT

Short Fragment Buffer (SFB)

Within the Native Barcoding Kit 24 V14 (SQK-NBD114.24) and Native Barcoding Kit 96 V14 (SQK-NBD114.96), Short Fragment Buffer (SFB) is supplied at the volume needed to complete the "reverse transcription, PCR and clean-up" and "adapter ligation and clean-up" steps of the protocol.

However, extra Short Fragment Buffer (SFB) is required for the "native barcode ligation" step of the protocol. This can be purchased with our SFB Expansion (EXP-SFB001).

IMPORTANT

AMPure XP Beads

Within the Native Barcoding Kits V14 (SQK-NBD114.24 and SQK-NBD114.96), AMPure XP Beads (AXP) are supplied at the volume needed to complete the native barcoding sections of the protocol: "Native barcode ligation" and "Adapter ligation and clean-up".

However, extra AMPure XP Beads are required for the "Reverse transcription, PCR and clean-up" step of the protocol.

IMPORTANT

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

Native Barcoding Kit 24 V14 (SQK-NBD114.24) contents

Note: We are in the process of reformatting the barcodes provided in this kit into a plate format. This will reduce plastic waste and will facilitate automated applications.

Plate format

SQK-NBD114.24 plate format

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
DNA Control Sample DCS Yellow 2 35
Native Adapter NA Green 1 40
Sequencing Buffer SB Red 1 700
Library Beads LIB Pink 1 600
Library Solution LIS White cap, pink label 1 600
Elution Buffer EB Black 2 500
AMPure XP Beads AXP Clear cap, light teal label 1 6,000
Long Fragment Buffer LFB Orange 1 1,800
Short Fragment Buffer SFB Clear 1 1,800
EDTA EDTA Blue 1 700
Flow Cell Flush FCF Clear cap, light blue label 1 8,000
Flow Cell Tether FCT Purple 1 200
Native Barcode plate NB01-24 - 2 plates, 3 sets of barcodes per plate 15 µl per well

Note: 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.

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.


Vial format

SQK-NBD114.24 bottle format

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Native Barcodes NB01-24 Clear 24 (one per barcode) 20
DNA Control Sample DCS Yellow 2 35
Native Adapter NA Green 1 40
Sequencing Buffer SB Red 1 700
Library Beads LIB Pink 1 600
Library Solution LIS White cap, pink label 1 600
Elution Buffer EB Black 2 500
AMPure XP Beads AXP Clear cap, light teal label 1 6,000
Long Fragment Buffer LFB Orange 1 1,800
Short Fragment Buffer SFB Clear 1 1,800
EDTA EDTA Blue 1 700
Flow Cell Flush FCF Clear cap, light blue label 1 8,000
Flow Cell Tether FCT Purple 1 200

Note: 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.

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

Native Barcoding Kit 96 V14 (SQK-NBD114.96) contents

Note: We are in the process of updating our kits with reduced EDTA concentration.

Higher EDTA concentration format:

SQK-NBD114.96 2

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Native Barcode plate NB01-96 - 3 plates 8 µl per well
DNA Control Sample DCS Yellow 3 35
Native Adapter NA Green 2 40
Sequencing Buffer SB Red 2 700
Library Beads LIB Pink 2 600
Library Solution LIS White cap, pink label 2 600
Elution Buffer EB Black 1 1,500
AMPure XP Beads AXP Amber 1 6,000
Long Fragment Buffer LFB Orange 1 7,500
Short Fragment Buffer SFB Clear 1 7,500
EDTA† EDTA Clear 1 700
Flow Cell Flush FCF Blue 1 15,500
Flow Cell Tether FCT Purple 2 200

† Higher concentration of EDTA with a clear cap.


Reduced EDTA concentration format:

SQK-NBD114.96 EDTA

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Native Barcode plate NB01-96 - 3 plates 8 µl per well
DNA Control Sample DCS Yellow 3 35
Native Adapter NA Green 2 40
Sequencing Buffer SB Red 2 700
Library Beads LIB Pink 2 600
Library Solution LIS White cap, pink label 2 600
Elution Buffer EB Black 1 1,500
AMPure XP Beads AXP Clear cap, light teal 1 6,000
Long Fragment Buffer LFB Clear cap, orange label 1 7,500
Short Fragment Buffer SFB Clear cap, dark blue label 1 7,500
EDTA‡ EDTA Blue 1 700
Flow Cell Flush FCF Clear cap, light blue label 1 15,500
Flow Cell Tether FCT Purple 2 200

‡ Reduced concentration of EDTA with a blue cap.

Note: 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.

The barcodes are orientated in columns in the barcode plate.

2021-09-14 Native Barcoding 96 kit contents v2 columns

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

To maximise the use of the Native Barcoding Kits, the Native Barcode Auxiliary V14 (EXP-NBA114) and the Sequencing Auxiliary Vials V14 (EXP-AUX003) expansion packs are available.

These expansions provide extra library preparation and flow cell priming reagents to allow users to utilise any unused barcodes for those running in smaller subsets.

Both expansion packs used together will provide enough reagents for 12 reactions. For customers requiring extra EDTA to maximise the use of barcodes, we recommend using 0.25 M EDTA and adding 4 µl for library preps using the SQK-NBD114.24 kit and 2 µl for preps using the SQK-NBD114.96 kit.

Native Barcode Auxiliary V14 (EXP-NBA114) contents:

EXP-NBA114 tubes

Note: 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.

Sequencing Auxiliary Vials V14 (EXP-AUX003) contents:

EXP-AUX003 bottles

Native barcode sequences

Below is the full list of our native barcode (NB01-96) sequences. The first 24 unique barcodes are available in the Native Barcoding Kit 24 V14 (SQK-NBD114.24). The Native Barcoding Kit 96 V14 (SQK-NBD114.96) include the first 24 native barcodes, with the additional 72 unique barcodes. The native barcodes are shipped at 640 nM.

In addition to the barcodes, there are also flanking sequences which add an extra level of context during analysis.

Barcode flanking sequences:

Forward sequence: 5' - AAGGTTAA - barcode - CAGCACCT - 3' Reverse sequence: 5' - GGTGCTG - barcode - TTAACCTTAGCAAT - 3'


Native barcode sequences

Component Forward sequence Reverse sequence
NB01 CACAAAGACACCGACAACTTTCTT AAGAAAGTTGTCGGTGTCTTTGTG
NB02 ACAGACGACTACAAACGGAATCGA TCGATTCCGTTTGTAGTCGTCTGT
NB03 CCTGGTAACTGGGACACAAGACTC GAGTCTTGTGTCCCAGTTACCAGG
NB04 TAGGGAAACACGATAGAATCCGAA TTCGGATTCTATCGTGTTTCCCTA
NB05 AAGGTTACACAAACCCTGGACAAG CTTGTCCAGGGTTTGTGTAACCTT
NB06 GACTACTTTCTGCCTTTGCGAGAA TTCTCGCAAAGGCAGAAAGTAGTC
NB07 AAGGATTCATTCCCACGGTAACAC GTGTTACCGTGGGAATGAATCCTT
NB08 ACGTAACTTGGTTTGTTCCCTGAA TTCAGGGAACAAACCAAGTTACGT
NB09 AACCAAGACTCGCTGTGCCTAGTT AACTAGGCACAGCGAGTCTTGGTT
NB10 GAGAGGACAAAGGTTTCAACGCTT AAGCGTTGAAACCTTTGTCCTCTC
NB11 TCCATTCCCTCCGATAGATGAAAC GTTTCATCTATCGGAGGGAATGGA
NB12 TCCGATTCTGCTTCTTTCTACCTG CAGGTAGAAAGAAGCAGAATCGGA
NB13 AGAACGACTTCCATACTCGTGTGA TCACACGAGTATGGAAGTCGTTCT
NB14 AACGAGTCTCTTGGGACCCATAGA TCTATGGGTCCCAAGAGACTCGTT
NB15 AGGTCTACCTCGCTAACACCACTG CAGTGGTGTTAGCGAGGTAGACCT
NB16 CGTCAACTGACAGTGGTTCGTACT AGTACGAACCACTGTCAGTTGACG
NB17 ACCCTCCAGGAAAGTACCTCTGAT ATCAGAGGTACTTTCCTGGAGGGT
NB18 CCAAACCCAACAACCTAGATAGGC GCCTATCTAGGTTGTTGGGTTTGG
NB19 GTTCCTCGTGCAGTGTCAAGAGAT ATCTCTTGACACTGCACGAGGAAC
NB20 TTGCGTCCTGTTACGAGAACTCAT ATGAGTTCTCGTAACAGGACGCAA
NB21 GAGCCTCTCATTGTCCGTTCTCTA TAGAGAACGGACAATGAGAGGCTC
NB22 ACCACTGCCATGTATCAAAGTACG CGTACTTTGATACATGGCAGTGGT
NB23 CTTACTACCCAGTGAACCTCCTCG CGAGGAGGTTCACTGGGTAGTAAG
NB24 GCATAGTTCTGCATGATGGGTTAG CTAACCCATCATGCAGAACTATGC
NB25 GTAAGTTGGGTATGCAACGCAATG CATTGCGTTGCATACCCAACTTAC
NB26 CATACAGCGACTACGCATTCTCAT ATGAGAATGCGTAGTCGCTGTATG
NB27 CGACGGTTAGATTCACCTCTTACA TGTAAGAGGTGAATCTAACCGTCG
NB28 TGAAACCTAAGAAGGCACCGTATC GATACGGTGCCTTCTTAGGTTTCA
NB29 CTAGACACCTTGGGTTGACAGACC GGTCTGTCAACCCAAGGTGTCTAG
NB30 TCAGTGAGGATCTACTTCGACCCA TGGGTCGAAGTAGATCCTCACTGA
NB31 TGCGTACAGCAATCAGTTACATTG CAATGTAACTGATTGCTGTACGCA
NB32 CCAGTAGAAGTCCGACAACGTCAT ATGACGTTGTCGGACTTCTACTGG
NB33 CAGACTTGGTACGGTTGGGTAACT AGTTACCCAACCGTACCAAGTCTG
NB34 GGACGAAGAACTCAAGTCAAAGGC GCCTTTGACTTGAGTTCTTCGTCC
NB35 CTACTTACGAAGCTGAGGGACTGC GCAGTCCCTCAGCTTCGTAAGTAG
NB36 ATGTCCCAGTTAGAGGAGGAAACA TGTTTCCTCCTCTAACTGGGACAT
NB37 GCTTGCGATTGATGCTTAGTATCA TGATACTAAGCATCAATCGCAAGC
NB38 ACCACAGGAGGACGATACAGAGAA TTCTCTGTATCGTCCTCCTGTGGT
NB39 CCACAGTGTCAACTAGAGCCTCTC GAGAGGCTCTAGTTGACACTGTGG
NB40 TAGTTTGGATGACCAAGGATAGCC GGCTATCCTTGGTCATCCAAACTA
NB41 GGAGTTCGTCCAGAGAAGTACACG CGTGTACTTCTCTGGACGAACTCC
NB42 CTACGTGTAAGGCATACCTGCCAG CTGGCAGGTATGCCTTACACGTAG
NB43 CTTTCGTTGTTGACTCGACGGTAG CTACCGTCGAGTCAACAACGAAAG
NB44 AGTAGAAAGGGTTCCTTCCCACTC GAGTGGGAAGGAACCCTTTCTACT
NB45 GATCCAACAGAGATGCCTTCAGTG CACTGAAGGCATCTCTGTTGGATC
NB46 GCTGTGTTCCACTTCATTCTCCTG CAGGAGAATGAAGTGGAACACAGC
NB47 GTGCAACTTTCCCACAGGTAGTTC GAACTACCTGTGGGAAAGTTGCAC
NB48 CATCTGGAACGTGGTACACCTGTA TACAGGTGTACCACGTTCCAGATG
NB49 ACTGGTGCAGCTTTGAACATCTAG CTAGATGTTCAAAGCTGCACCAGT
NB50 ATGGACTTTGGTAACTTCCTGCGT ACGCAGGAAGTTACCAAAGTCCAT
NB51 GTTGAATGAGCCTACTGGGTCCTC GAGGACCCAGTAGGCTCATTCAAC
NB52 TGAGAGACAAGATTGTTCGTGGAC GTCCACGAACAATCTTGTCTCTCA
NB53 AGATTCAGACCGTCTCATGCAAAG CTTTGCATGAGACGGTCTGAATCT
NB54 CAAGAGCTTTGACTAAGGAGCATG CATGCTCCTTAGTCAAAGCTCTTG
NB55 TGGAAGATGAGACCCTGATCTACG CGTAGATCAGGGTCTCATCTTCCA
NB56 TCACTACTCAACAGGTGGCATGAA TTCATGCCACCTGTTGAGTAGTGA
NB57 GCTAGGTCAATCTCCTTCGGAAGT ACTTCCGAAGGAGATTGACCTAGC
NB58 CAGGTTACTCCTCCGTGAGTCTGA TCAGACTCACGGAGGAGTAACCTG
NB59 TCAATCAAGAAGGGAAAGCAAGGT ACCTTGCTTTCCCTTCTTGATTGA
NB60 CATGTTCAACCAAGGCTTCTATGG CCATAGAAGCCTTGGTTGAACATG
NB61 AGAGGGTACTATGTGCCTCAGCAC GTGCTGAGGCACATAGTACCCTCT
NB62 CACCCACACTTACTTCAGGACGTA TACGTCCTGAAGTAAGTGTGGGTG
NB63 TTCTGAAGTTCCTGGGTCTTGAAC GTTCAAGACCCAGGAACTTCAGAA
NB64 GACAGACACCGTTCATCGACTTTC GAAAGTCGATGAACGGTGTCTGTC
NB65 TTCTCAGTCTTCCTCCAGACAAGG CCTTGTCTGGAGGAAGACTGAGAA
NB66 CCGATCCTTGTGGCTTCTAACTTC GAAGTTAGAAGCCACAAGGATCGG
NB67 GTTTGTCATACTCGTGTGCTCACC GGTGAGCACACGAGTATGACAAAC
NB68 GAATCTAAGCAAACACGAAGGTGG CCACCTTCGTGTTTGCTTAGATTC
NB69 TACAGTCCGAGCCTCATGTGATCT AGATCACATGAGGCTCGGACTGTA
NB70 ACCGAGATCCTACGAATGGAGTGT ACACTCCATTCGTAGGATCTCGGT
NB71 CCTGGGAGCATCAGGTAGTAACAG CTGTTACTACCTGATGCTCCCAGG
NB72 TAGCTGACTGTCTTCCATACCGAC GTCGGTATGGAAGACAGTCAGCTA
NB73 AAGAAACAGGATGACAGAACCCTC GAGGGTTCTGTCATCCTGTTTCTT
NB74 TACAAGCATCCCAACACTTCCACT AGTGGAAGTGTTGGGATGCTTGTA
NB75 GACCATTGTGATGAACCCTGTTGT ACAACAGGGTTCATCACAATGGTC
NB76 ATGCTTGTTACATCAACCCTGGAC GTCCAGGGTTGATGTAACAAGCAT
NB77 CGACCTGTTTCTCAGGGATACAAC GTTGTATCCCTGAGAAACAGGTCG
NB78 AACAACCGAACCTTTGAATCAGAA TTCTGATTCAAAGGTTCGGTTGTT
NB79 TCTCGGAGATAGTTCTCACTGCTG CAGCAGTGAGAACTATCTCCGAGA
NB80 CGGATGAACATAGGATAGCGATTC GAATCGCTATCCTATGTTCATCCG
NB81 CCTCATCTTGTGAAGTTGTTTCGG CCGAAACAACTTCACAAGATGAGG
NB82 ACGGTATGTCGAGTTCCAGGACTA TAGTCCTGGAACTCGACATACCGT
NB83 TGGCTTGATCTAGGTAAGGTCGAA TTCGACCTTACCTAGATCAAGCCA
NB84 GTAGTGGACCTAGAACCTGTGCCA TGGCACAGGTTCTAGGTCCACTAC
NB85 AACGGAGGAGTTAGTTGGATGATC GATCATCCAACTAACTCCTCCGTT
NB86 AGGTGATCCCAACAAGCGTAAGTA TACTTACGCTTGTTGGGATCACCT
NB87 TACATGCTCCTGTTGTTAGGGAGG CCTCCCTAACAACAGGAGCATGTA
NB88 TCTTCTACTACCGATCCGAAGCAG CTGCTTCGGATCGGTAGTAGAAGA
NB89 ACAGCATCAATGTTTGGCTAGTTG CAACTAGCCAAACATTGATGCTGT
NB90 GATGTAGAGGGTACGGTTTGAGGC GCCTCAAACCGTACCCTCTACATC
NB91 GGCTCCATAGGAACTCACGCTACT AGTAGCGTGAGTTCCTATGGAGCC
NB92 TTGTGAGTGGAAAGATACAGGACC GGTCCTGTATCTTTCCACTCACAA
NB93 AGTTTCCATCACTTCAGACTTGGG CCCAAGTCTGAAGTGATGGAAACT
NB94 GATTGTCCTCAAACTGCCACCTAC GTAGGTGGCAGTTTGAGGACAATC
NB95 CCTGTCTGGAAGAAGAATGGACTT AAGTCCATTCTTCTTCCAGACAGG
NB96 CTGAACGGTCATAGAGTCCACCAT ATGGTGGACTCTATGACCGTTCAG

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, PCR and clean-up

Materials
  • Input influenza RNA
  • Influenza A primers
  • Influenza B primers
Consumables
  • SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (ThermoFisher, cat # 12574018 or 12574026)
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with heat seals
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 5 ml Eppendorf DNA LoBind tubes
  • 15 ml Eppendorf DNA LoBind tubes
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • Freshly prepared 80% ethanol in nuclease-free water
  • Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
Equipment
  • Thermal cycler
  • Qubit fluorometer (or equivalent for QC check)
  • Multichannel pipettes suitable for dispensing 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
  • P2 pipette and tips
  • Magnetic rack suitable for 96 well plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher CAT#12027)
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Microfuge
Optional equipment
  • PCR hoods with UV steriliser
  • PCR-Cooler (Eppendorf) or ice bucket with ice
IMPORTANT

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

To reduce risk of contamination, we recommend the use of PCR hoods with a UV steriliser when setting up the PCR plates.

  • When handling the primer stocks and derivatives, use a clean template-free PCR hood.
  • When handling the samples and/or a positive control, use a clean template-addition PCR hood.

In a clean template-free pre-PCR hood, prepare the primer mixes for influenza A and influenza B as follows in 1.5 ml Eppendorf DNA LoBind tubes:

Note: The volume requirements can be adjusted according to stock concentrations and experiment needs.

Influenza A primer mix

Primer Concentration Volume
Nuclease-free water - 378 µl
Tuni 12 100 µM 16.8 µl
Tuni 12.4 100 µM 4.2 µl
Tuni 13 100 µM 21 µl
Total 420 µl

Influenza B primer mix

Primer Concentration Volume
Nuclease-free water - 378 µl
B-PBs-UniF 100 µM 5 µl
B-PBs-UniR 100 µM 5 µl
B-PA-UniF 100 µM 2.5 µl
B-PA-UniR 100 µM 2.5 µl
B-HANA-UniF 100 µM 5 µl
B-HANA-UniR 100 µM 5 µl
B-NP-UniF 100 µM 3 µl
B-NP-UniR 100 µM 3 µl
B-M-Uni3F 100 µM 1.5 µl
B-Mg-Uni3F 100 µM 1.5 µl
B-M-Uni3R 100 µM 3 µl
B-NS-Uni3F 100 µM 2.5 µl
B-NS-Uni3R 100 µM 2.5 µl
Total 420 µl

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

For X12 samples, use 1.5 ml Eppendorf DNA LoBind tubes:

Component Influenza A RT-PCR Master Mix Influenza B RT-PCR Master Mix
Nuclease free water 280 µl 280 µl
Influenza A primer mix 28 µl -
Influenza B primer mix - 28 µl
2X Reaction Mix 350 µl 350 µl
SuperScript™ III RT/Platinum™ Taq Mix 28 µl 28 µl
Total volume 686 µl 686 µl

For X24 samples, use 1.5 ml Eppendorf DNA LoBind tubes:

Component Influenza A RT-PCR Master Mix Influenza B RT-PCR Master Mix
Nuclease free water 560 µl 560 µl
Influenza A primer mix 56 µl -
Influenza B primer mix - 56 µl
2X Reaction Mix 700 µl 700 µl
SuperScript™ III RT/Platinum™ Taq Mix 56 µl 56 µl
Total volume 1372 µl 1372 µl

For X48 samples, use 5 ml Eppendorf DNA LoBind tubes:

Component Influenza A RT-PCR Master Mix Influenza B RT-PCR Master Mix
Nuclease free water 1120 µl 1120 µl
Influenza A primer mix 112 µl -
Influenza B primer mix - 112 µl
2X Reaction Mix 1400 µl 1400 µl
SuperScript™ III RT/Platinum™ Taq Mix 112 µl 112 µl
Total volume 2744 µl 2744 µl

For X96 samples, use 15 ml Eppendorf DNA LoBind tubes:

Component Influenza A RT-PCR Master Mix Influenza B RT-PCR Master Mix
Nuclease free water 2240 µl 2240 µl
Influenza A primer mix 224 µl -
Influenza B primer mix - 224 µl
2X Reaction Mix 2800 µl 2800 µl
SuperScript™ III RT/Platinum™ Taq Mix 224 µl 224 µl
Total volume 5488 µl 5488 µl

For each influenza type, place a clean 96-well RT-PCR plate into a PCR-cooler or ice bucket with ice (if using).

Note: Enusre the RT-PCR plates for each influenza type are separate:

  • One plate for Influenza A (Influenza A RT-PCR plate)
  • One plate for Influenza B (Influenza B RT-PCR plate)

Using a stepper pipette or a multichannel pipette, aliquot 49 µl of influenza A RT-PCR Master Mix into the influenza A RT-PCR plate.

Using a stepper pipette or a multichannel pipette, aliquot 49 µl of influenza B RT-PCR Master Mix into the influenza B RT-PCR plate.

IMPORTANT

We recommend having a negative control for every plate of samples to monitor for contamination events.

Use 1 µl of nuclease-free water as your negative control input into a single well of each Influenza RT-PCR plate.

Seal the RT-PCR plate(s) and transfer to a template-addition pre-PCR hood.

Transfer 1 µl of influenza A samples to the wells containing influenza A RT-PCR Master Mix in the influenza A RT-PCR plate and mix thoroughly by pipetting the contents of each well up and down.

Transfer 1 µl of influenza B samples to the wells containing influenza B RT-PCR Master Mix in the influenza B RT-PCR plate and mix thoroughly by pipetting the contents of each well up and down.

Seal the RT-PCR plate(s) and spin down in a centrifuge.

IMPORTANT

Please note the thermal cycler programs are different for the Influenza A RT-PCR and Influenza B RT-PCR reactions.

Ensure you are using the correct program for your reaction plate.

Incubate the influenza A RT-PCR plate using the following program, with the heated lid set to 105°C:

Step Temperature Time Cycles
cDNA synthesis 42°C 60 min 1
Initial denaturation 94°C 2 min 1
Denaturation

Annealing and extension		 94°C

45°C
68°C		 30 sec

30 sec
3 min
5
Denaturation

Annealing and extension		 94°C

57°C
68°C		 30 sec

30 sec
3 min
31
Hold 4°C ∞

Incubate the influenza B RT-PCR plate using the following program, with the heated lid set to 105°C:

Step Temperature Time Cycles
cDNA synthesis 45°C 60 min 1
cDNA synthesis 55°C 30 min 1
Initial denaturation 94°C 2 min 1
Denaturation

Annealing and extension		 94°C

40°C
68°C		 20 sec

30 sec
3 min 30 sec
5
Denaturation

Annealing and extension		 94°C

58°C
68°C		 20 sec

30 sec
3 min 30 sec
30
Final extension 68°C 10 min 1
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.

IMPORTANT

From this point onwards, a clean post-PCR hood can be used if available. Decontamination with UV and or DNAzap between sample batches is recommended.

Resuspend the AMPure XP beads by vortexing.

Add 50 µl of resuspended AMPure XP beads to each well of the RT-PCR plate(s) and mix by gently pipetting.

Incubate the PT-PCR plate(s) at room temperature for 10 minutes.

Prepare at least 500 µl 80% ethanol in nuclease-free water per sample.

Spin down the RT-PCR plate(s) and pellet the beads on a magnet for 5 minutes. Keep the plate on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Keep the plate on the magnet and wash the beads in each well with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

Spin down and place the plate 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 plate from the magnetic rack and resuspend each pellet in 15 µl nuclease-free water. Incubate for 2 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 per well, into a clean 96-well plate(s).

Dispose of the pelleted beads.

CHECKPOINT

Quantify 1 µl of each eluted sample using a Qubit fluorometer.

END OF STEP

Take forward your quantified samples to the end-prep step.

However, at this point it is also possible to store the samples at 4°C overnight.

For long-term storage or to store any unused amplified material for use in later experiments, store your samples at -20°C.

5. End-prep

Consumables
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with heat seals
Equipment
  • Multichannel pipette capable of dispensing 0.5 – 10 µL, and tips
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • Thermal cycler
  • Microfuge
  • Ice bucket with ice
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Vortex mixer
IMPORTANT

We recommended carrying the negative control through this step until sequencing.

Prepare the NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer's instructions, and place on ice:

For optimal performance, NEB recommend the following:

  1. Thaw all reagents on ice.

  2. Ensure the reagents are well mixed.
    Note: Do not vortex the Ultra II End Prep Enzyme Mix.

  3. Always spin down tubes before opening for the first time each day.

  4. The NEBNext Ultra II End Prep Reaction Buffer may contain a white precipitate. If this occurs, allow the mixture(s) to come to room temperature and pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix.

Determine the volume of the cleaned-up PCR reaction that yields 200 fmol of DNA per sample and aliquot into a clean 96-well plate (End-prep plate).

Make up each sample per well to 12.5 µl using nuclease-free water.

Prepare the following end-prep master mix in 1.5 ml Eppendorf DNA LoBind tube and mix thoroughly by pipetting:

Reagent Volume per reaction For X24 samples For X48 samples For X96 samples
Ultra II End-prep reaction buffer 1.75 µl 52.5 µl 105 µl 210 µl
Ultra II End-prep enzyme mix 0.75 µl 22.5 µl 45 µl 90 µl
Total 2.5 µl 75 µl 150 µl 300 µl

Using a stepper pipette or multi-channel pipette, add 2.5 µl of the end-prep master mix to each well containing 12.5 µl sample.

Ensure the reactions are thoroughly mixed by pipetting. Seal the End-prep plate and spin down briefly.

Using a thermal cycler, incubate the plate at 20°C for 5 minutes and 65°C for 5 minutes.

END OF STEP

Take forward the end-prepped DNA into the native barcode ligation step.

If users want to pause the library preparation here, we recommend cleaning up your sample with 1X Agencourt AMPure XP beads and eluting in nuclease-free water before storing at 4°C.

6. Native barcode ligation

Materials
  • Native Barcodes (NB01-24) OR Native Barcodes (NB01-NB96)
  • AMPure XP Beads (AXP)
  • EDTA (EDTA)
  • Short Fragment Buffer (SFB)
Consumables
  • Freshly prepared 80% ethanol in nuclease-free water
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • NEB Blunt/TA Ligase Master Mix (NEB, M0367)
Equipment
  • Magnetic rack suitable for 96-well plates
  • Thermal cycler
  • Hula mixer (gentle rotator mixer)
  • Vortex mixer
  • Ice bucket with ice
  • Microfuge
  • P1000 pipette and tips
  • P100 pipette and tips
  • P10 pipette and tips
Optional equipment
  • Qubit fluorometer (or equivalent for QC check)
IMPORTANT

To monitor cross-contamination events, we recommend that the negative control is carried through this process and a barcode is used to sequence this control.

Prepare the NEB Blunt/TA Ligase Master Mix according to the manufacturer's instructions, and place on ice:

  1. Thaw the reagents at room temperature.

  2. Spin down the reagent tubes for 5 seconds.

  3. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.

Thaw kit components at room temperature, then spin down briefly using a microfuge and mix as indicated by the table below. Then place on ice:

Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix by pipetting or vortexing 4. Place on ice
EDTA (EDTA) ✓ ✓ Vortexing ✓
Native Barcodes (NB01-24) or (NB01-96) ✓ ✓ Only pipette mix immediately before use ✓
Short Fragment Buffer (SFB) ✓ ✓ Vortexing ✓

Select a unique barcode for each sample to be run together on the same flow cell.

Please note: Only use one barcode per sample.

Using a stepper pipette, or a multichannel pipette, make up the Native Barcode Ligation Plate in a clean 96-well plate. Add the reagents in the following order per well:

Reagent Volume per well for up to x24 samples Volume per well for x25—x96 samples
Nuclease-free water 6 µl 3 µl
End-prepped DNA
Note: Transfer to the corresponding well		 1.5 µl 0.75 µl
Native barcode
Note: Transfer to the corresponding well		 2.5 µl 1.25 µl
Blunt/TA Ligation Master Mix 10 µl 5 µl
Total 20 µl 10 µl

Depending on the number of samples, aliquot to each well of the columns:

Plate location x24 samples x48 samples x96 samples
Columns 1-3 1-6 1-12

NB Ligation Plate prep

Mix the contents thoroughly by pipetting.

Seal the plate(s) and spin down briefly.

Incubate for 20 minutes at room temperature.

Add EDTA to each well and mix thoroughly by pipetting and spin down briefly.

Note: Ensure you follow the instructions for the cap colour of your EDTA tube.

Up to x24 samples x25 — x96 samples
Volume of clear capped EDTA per sample 2 µl 1 µl
Volume of blue capped EDTA per sample 4 µl 2 µl
TIP

EDTA is added at this step to stop the reaction.

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

Note: Ensure you follow the instructions for the cap colour of your EDTA tube.

x24 samples x48 samples x96 samples
Total volume for preps using clear cap EDTA ~528 µl ~528 µl ~1,056 µl
Total volume for preps using blue cap EDTA ~576 µl ~576 µl ~1,152 µl
TIP

We recommend checking the base of your tubes/plate are all the same volume before pooling and after to ensure all the liquid has been taken forward.

Resuspend the AMPure XP Beads (AXP) by vortexing.

Add AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting for a 0.4X clean.

Volume for 10 µl of sample For 265 µl of samples For 528 µl of samples For 1,056 µl of samples
Volume of AXP 4 µl 106 µl 211 µl 422 µl

Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.

Prepare 500 µl of fresh 80% ethanol in nuclease-free water.

Spin down the sample and pellet the beads on a magnet for 5 minutes. Keep the tube on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Wash the beads by adding 700 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Repeat the previous step.

Keep the tube on the magnet and wash the beads with 100 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

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 in 35 µl nuclease-free water by gently flicking.

Incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.

Note: If stuggling to obtain the necessary yield, increasing the incubation period up to 30 minutes may improve elution efficacy.

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

Remove and retain 35 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

CHECKPOINT

Quantify 1 µl of eluted sample using a Qubit fluorometer.

END OF STEP

Take forward the barcoded DNA library to the adapter ligation and clean-up step. However, at this point it is also possible to store the sample at 4°C overnight.

7. Adapter ligation and clean-up

Materials
  • Short Fragment Buffer (SFB)
  • Elution Buffer (EB)
  • Native Adapter (NA)
  • AMPure XP Beads (AXP)
Consumables
  • NEBNext® Quick Ligation Module (NEB, E6056)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
Equipment
  • Microfuge
  • Magnetic rack
  • Vortex mixer
  • Hula mixer (gentle rotator 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
  • Qubit fluorometer (or equivalent for QC check)
IMPORTANT

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

Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice:

  1. Thaw the reagents at room temperature.

  2. Spin down the reagent tubes for 5 seconds.

  3. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes. Note: Do NOT vortex the Quick T4 DNA Ligase.

The NEBNext Quick Ligation Reaction Buffer (5x) may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for several seconds to ensure the reagent is thoroughly mixed.

IMPORTANT

Do not vortex the Quick T4 DNA Ligase.

Spin down the Native Adapter (NA) and Quick T4 DNA Ligase, pipette mix and place on ice.

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

Thaw the Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and place on ice.

In a 1.5 ml Eppendorf LoBind tube, mix in the following order:

Between each addition, pipette mix 10 - 20 times.

Reagent Volume
Pooled barcoded sample 30 µl
Native Adapter (NA) 5 µl
NEBNext Quick Ligation Reaction Buffer (5X) 10 µl
Quick T4 DNA Ligase 5 µl
Total 50 µl

Thoroughly mix the reaction by gently pipetting and briefly spinning down.

Incubate the reaction for 20 minutes at room temperature.

IMPORTANT

The next clean-up step uses Short Fragment Buffer (SFB) and not 80% ethanol to wash the beads. The use of ethanol will be detrimental to the sequencing reaction.

Resuspend the AMPure XP Beads (AXP) by vortexing.

Add 20 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.

Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.

Spin down the sample and pellet the beads on a magnet for 5 minutes. Keep the tube on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Wash the beads by adding 125 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Keep the tube on the magnet until the eluate is clear and colourless. Remove the supernatant using a pipette and discard.

Repeat the previous step.

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

Remove the tube from the magnetic rack and resuspend pellet in 15 µl Elution Buffer (EB).

Spin down and incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.

Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.

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

Dispose of the pelleted beads

CHECKPOINT

Quantify 1 µl of eluted sample using a Qubit fluorometer.

Depending on your DNA library fragment size, prepare your final library in 12 µl of Elution Buffer (EB).

Fragment library length Flow cell loading amount
Very short (<1 kb) 100 fmol
Short (1-10 kb) 35–50 fmol
Long (>10 kb) 300 ng

Note: If the library yields are below the input recommendations, load the entire library.

If required, we recommend using a mass to mol calculator such as the NEB calculator.

END OF STEP

The prepared library is used for loading onto the flow cell. Store the library on ice or at 4°C until ready to load.

TIP

Library storage recommendations

We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short-term storage or repeated use, for example, re-loading flow cells between washes. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes.

OPTIONAL ACTION

If quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.

Depending on how many flow cells the library will be split across, more Elution Buffer (EB) than what is supplied in the kit will be required.

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
  • 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
  • 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
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).

To prepare the flow cell priming mix with BSA, combine Flow Cell Flush (FCF) and Flow Cell Tether (FCT), as directed below. Mix by pipetting at room temperature.

Note: We are in the process of reformatting our kits with single-use tubes into a bottle format. Please follow the instructions for your kit format.

Single-use tubes format: Add 5 µl Bovine Serum Albumin (BSA) at 50 mg/ml and 30 µl Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).

Bottle format: In a suitable tube for the number of flow cells, combine the following reagents:

Reagent 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, enable Barcoding. Click Edit options and enable Barcode both ends and Mid-read barcodes. Optional: basecalling and/or demultiplexing of sequences can be performed using the stand-alone Guppy software.

Edit options barcoding FLU v14

Barcoding options FLU V14

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 analysis pipeline

The analysis of the FASTQ format sequence data is performed using a Nextflow workflow called Influenza Typing Workflow (wf-flu). 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.

The workflow processes the basecalled and demultiplexed DNA sequence data output generated by MinKNOW:

  • The sequences are filtered for a minimum length and quality thresholds (200 nucleotides and Q9 respectively) prior to sequence alignment to the CDC multi-fasta Influenza reference.
  • The alignment is performed using the Minimap2 software.
  • Depth of coverage across the mapped sequences is measured using Samtools before genetic variants are called using Medaka.
  • A coverage-masked consensus sequence is prepared for each sample using bcftools.
  • The influenza strain typing is then performed using the abricate software with an insaflu database.

The influenza strains included in the database are listed in the project documentation pages for the Influenza Typing Workflow.

The workflow returns a per-run HTML-format summary report along with a CSV file of typing results. Additional files that include mapping BAM files and VCF files of Medaka variants are also included in the workflow output.

For more information, please refer to the Influenza workflow blog.

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-flu workflow in EPI2ME Labs:

Ensure you have installed the wf-flu 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-flu:

EPI2ME FLU 1

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

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

EPI2ME FLU 2

Demultiplexing of multiple barcoded samples

The wf-flu 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-flu 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 FluFastq/

FluFastq/

├── barcode01

├── barcode02

├── barcode03

├── barcode04

├── barcode05

├── barcode06

└── unclassified

IMPORTANT

Basecalling model

The basecalling model should be specified when setting up the wf-flu 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 Flu 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-flu" workflow:

EPI2ME FLU 3

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

EPI2ME FLU 4

Complete the wf-flu run set-up:

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

Set the basecaller cgf to the basecalling model used in your sequencing run.

EPI2ME FLU 5

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

EPI2ME FLU 7

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

Completed analysis and result files

The wf-flu 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-flu 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:

EPI2ME FLU 8

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 for Kit 14

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 for Kit 14

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 10–20 fmol of good quality library can be loaded on to a MinION/GridION 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 Native Barcoding 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 (FCT tube). Make sure FCT was added to 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 Fast fuel consumption is typically seen in Kit 9 chemistry (e.g. SQK-LSK109) when the flow cell is overloaded with library. Please see the appropriate protocol for your DNA library to find 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.

Last updated: 11/15/2024

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