Rapid metagenomic sequencing for surveillance of bacterial, fungal and viral pathogens using SQK-RPB114.24

Oxford Nanopore Technologies
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MinION: Protocol

Rapid metagenomic sequencing for surveillance of bacterial, fungal and viral pathogens using SQK-RPB114.24 V RMS_9215_v114_revA_11Dec2024

This protocol:

  • is a rapid method to perform metagenomic sequencing for identification of bacterial, fungal and viral pathogens.
  • consists of a two-in-one method that splits sample into bacterial/fungal sample preparation and viral/acellular sample preparation.
  • provides a recommended host depletion and extraction method for respiratory samples.
  • uses a shotgun approach to RT-PCR.
  • involves tagmentation, barcoding and PCR amplification.
  • is compatible with R10.4.1 flow cells.

For Research Use Only

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

FOR RESEARCH USE ONLY

概要

This protocol:

  • is a rapid method to perform metagenomic sequencing for identification of bacterial, fungal and viral pathogens.
  • consists of a two-in-one method that splits sample into bacterial/fungal sample preparation and viral/acellular sample preparation.
  • provides a recommended host depletion and extraction method for respiratory samples.
  • uses a shotgun approach to RT-PCR.
  • involves tagmentation, barcoding and PCR amplification.
  • is compatible with R10.4.1 flow cells.

For Research Use Only

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

Document version: RMS_9215_v114_revA_11Dec2024

1. Overview of protocol

Introduction to the rapid metagenomic sequencing protocol

This protocol outlines a method to perform agnostic metagenomic sequencing from extracted nucleic acid.

The method offers two options for sample preparation depending on your target sample and input:

  • The DNA-only bacterial/fungal sample preparation utilises SQK-RPB114.24 reagents to tagment all DNA in the extract for amplification and sequencing.
  • The DNA/RNA viral sample preparation has been optimised by Oxford Nanopore Technologies, and is derived from a method established by Josh Quick and Ingra M. Claro which utilises a shotgun approach using 9N primers to randomly reverse transcribe RNA and subsequently PCR-amplify DNA/RNA present in a sample.

Other areas of note:

  • The performance of this method is reliant on sample type and was optimised for respiratory samples.
  • While effort is made to reduce host background, sample types that are less complex/have less nucleic acid background are likely to perform better.

This protocol uses the Rapid PCR Barcoding Kit V14 (SQK-RPB114.24), which allows the potential to use up to 24 barcodes in one sequencing experiment.

Workflow image ER 11 svg jan2025 final


Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

  • Ensure you have your sequencing kit, the correct equipment and third-party reagents
  • Extract your DNA/RNA if not using the respiratory sample processing workflow in this protocol
  • Download the software for acquiring and analysing your data
  • Check your flow cell to ensure it has enough pores for a good sequencing run

Host depletion and extraction

  • If using the recommended respiratory sample host depletion and extraction, ensure you have the correct third-party reagents
  • Ensure you have fresh samples to use (not recommended for frozen/deactivated samples)

Library preparation

The table below is an overview of the steps required in the library preparation, including timings and optional stopping points.

Step Process Time Stop option
Viral samples: reverse transcription Reverse transcribe your RNA samples with the RLB RT 9N primer mix and the TSOmG template-switching oligo ~90 minutes
Bacterial/fungal samples: DNA tagmentation Tagment your DNA using the Fragmentation Mix from the sequencing kit 10 minutes
PCR amplification PCR your sample using the barcoded primer supplied in the sequencing kit 180 minutes Optional: The PCR amplification can be performed and left at the hold temperature overnight.
Sample quantification and pooling Quantify your barcoded PCR samples and pool them in equimolar ratios 15 minutes
Cleanup and quantification Perform a purification on your pooled samples and quantify 15 minutes 4°C overnight
Adapter attachment and clean-up Attach the sequencing adapters to the DNA ends 5 minutes We strongly recommend sequencing your library as soon as it is adapted.
Priming and loading the flow cell Prime the flow cell and load the prepared library for sequencing 5 minutes

Sequencing and analysis

You will need to:

  • Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
  • Analyse your data using the wf-metagenomics workflow available through EPI2ME or the command line
重要

Compatibility of this protocol

This protocol should only be used in combination with:

2. Equipment and consumables

材料
  • (FOR SAMPLE PREPARATION) Bacterial/fungal or viral/acellular sample inputs (see details below)
  • (FOR LIBRARY PREPARATION) Extracted nucleic acid
  • Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24)
消耗品
  • MinIONとGridIONのFlow Cell
  • Sputasol (Fisher, 11943262)
  • RT-PCR Grade Water (Fisher, 10289774)
  • Phosphate Buffer Saline (PBS) (Fisher, 11593377)
  • 2X HL-SAN Buffer (4.5 M NaCl and 100 mM MgCl2) formulated using the two reagents below:
  • • Invitrogen 5M NaCl (Fisher, 10255984)
  • • Invitrogen 1M MgCl2 (Fisher, 10418464)
  • HL-SAN Triton Free DNase (ArticZymes, 70911-202)
  • Saponin solution (0.2% saponin in PBS) formulated using the reagent below:
  • • Saponin (Sigma-Aldrich, 47036-50G-F)
  • Matrix Lysing E tubes (Fisher, 11452420)
  • MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Fisher, 16346582)
  • 12 µM RLB RT 9N primer (IDT - Sequence: 5’-TTTTTCGTGCGCCGCTTCAACNNNNNNNNN-3’)
  • 12 µM TSOmG template-switching oligo (IDT - Sequence: 5’-GCTAATCATTGCTTTTTCGTGCGCCGCTTCAACATmGmGmG-3’)
  • 10 mM dNTPs (Fisher, 10610851)
  • Maxima H Minus Reverse Transcriptase (Fisher, 13233159)
  • RNaseOUT (Fisher, 10777019)
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • Thermolabile Exonuclease I (NEB, M0568)
  • Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
装置
  • MinIONかGridION のデバイス
  • MinIONとGridIONのFlow Cell ライトシールド
  • Centrifuge e.g. Eppendorf max speed 15,000rpm (Fisher, 15881635)
  • 小型遠心機
  • タイマー
  • ボルテックスミキサー
  • Thermomixer set at 37°C
  • Thermomixer set at 65°C
  • Magnetic rack
  • Bead-beater (e.g. FastPrep-24™ Classic bead beating grinder and lysis system)
  • サーマルサイクラー
  • 96-well PCR block
  • Ice bucket with ice
  • Hula mixer（緩やかに回転するミキサー）
  • Qubit fluorometer (or equivalent)
  • P1000 pipette and tips
  • P200 ピペットとチップ
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 ピペットとチップ
  • P2 pipette and tips
オプション装置
  • Suitable biosafety equipment for your sample(s) (e.g. Microbiological Safety Cabinet II contamination level 3 or equivalent)
重要

Ensure pathogenic samples are handled in appropriate biosafety conditions.

Please adhere to the correct health and safety practices in accordance to your laboratory standards and local rules and regulations.

Note: We recommend as a minimum the use of a Class II Biological Safety Cabinet. Please consider that some organisms can survive bead-beating steps during extraction. Ensure you are taking the necessary precautions during your sample and library preparation.

For this protocol you will need the following sample input(s):

For sample preparation:

  • ≥ 250 µl of Sputum/Endotracheal aspirates

OR

  • ≥ 250 µl of Bronchoalveolar lavages/Mini-BALs

OR

  • ≥ 500 µl of VTM/UTM-stored oral/nasal swabs (only suitable for viral samples)

Note: If quantities allow, a sample can be processed for both bacterial/fungal sample preparation and viral sample preparation. Please note, each preparation type will differ in method and should be treated as a separate sample.

For library preparation:

  • Extracted nucleic acid from the sample types above

Input DNA/RNA

How to QC your input

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

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

Chemical contaminants

Depending on how the DNA/RNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA/RNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.

オプショナルステップ

Input controls

Negative internal controls

We recommend a negative control is included and processed as a real sample through the entire process to monitor for contamination events.

The negative control can be PBS or a third-party negative sample matrix.

サードパーティー試薬

このプロトコールで使用されているすべてのサードパーティー試薬は、当社が検証し、使用を推奨しているものです。Oxford Nanopore Technologiesでは、それ以外の試薬を用いたテストは行っていません。

すべてのサードパーティ製試薬については、製造元の指示に従って使用の準備をすることをお勧めします。

Agencourt AMPure XP beads

Additional Agencourt AMPure XP beads may be required alongside the AMPure XP Beads (AXP) provided in the sequencing kit for the clean-up step following PCR amplification.

ヒント

Eppendorf tube orientation in centrifuge

For all centrifugation steps, ensure that tubes are loaded into the centrifuge with the hinge side of the tube facing outwards. This will assist in visual identification of the pellet.

Ensure gentle handling when removing the tubes from the centrifuge to avoid dislodging the pellet.

Eppendorf tube hinge into centrifuge image

フローセルのチェックをしてください

シークエンシング実験を開始する前に、フローセルのポアの数を確認することを強くお勧めします。このフローセルの確認は、MinION/GridION/PromethIONの場合は代理店への到着から１２週間以内に行ってください。またはFlongle Flow Cellの場合は代理店への到着から4週間以内に行う必要があります。Oxford Nanopore Technologiesは、フローセルチェックの実施から2日以内に結果が報告され、推奨される保管方法に従っていた場合に、以下の表に記載されているナノポアの有効数に満たさない場合には、フローセルを交換します。 フローセルのチェックを行うには、Flow Cell Check documentの指示に従ってください。

Flow cell 保証する最小有効ポア数（以下の数未満のフローセルが交換対象となります）
Flongle Flow Cell 50
MinION/GridION Flow Cell 800
PromethION Flow Cell 5000
重要

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

Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24) contents

sqk-rpb114.24 tubes

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Fragmentation Mix FRM Brown 1 160
Rapid Adapter RA Green 1 15
Adapter Buffer ADB Clear 1 100
AMPure XP Beads AXP Amber 3 1,200
Elution Buffer EB Black 2 500
EDTA EDTA Blue 1 700
Sequencing Buffer SB Red 1 700
Library Beads LIB Pink 1 600
Library Solution LS White cap, pink label 1 600
Flow Cell Flush FCF Clear 1 8,000
Flow Cell Tether FCT Purple 1 200
Rapid Barcode Primer 01-24 RLB01-24 Clear 24 (one per barcode) 15

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.

Rapid barcode primers

Component Sequence
RLB01 AAGAAAGTTGTCGGTGTCTTTGTG
RLB02 TCGATTCCGTTTGTAGTCGTCTGT
RLB03 GAGTCTTGTGTCCCAGTTACCAGG
RLB04 TTCGGATTCTATCGTGTTTCCCTA
RLB05 CTTGTCCAGGGTTTGTGTAACCTT
RLB06 TTCTCGCAAAGGCAGAAAGTAGTC
RLB07 GTGTTACCGTGGGAATGAATCCTT
RLB08 TTCAGGGAACAAACCAAGTTACGT
RLB09 AACTAGGCACAGCGAGTCTTGGTT
RLB10 AAGCGTTGAAACCTTTGTCCTCTC
RLB11 GTTTCATCTATCGGAGGGAATGGA
RLB12 GTTGAGTTACAAAGCACCGATCAG
RLB13 AGAACGACTTCCATACTCGTGTGA
RLB14 AACGAGTCTCTTGGGACCCATAGA
RLB15 AGGTCTACCTCGCTAACACCACTG
RLB16 CGTCAACTGACAGTGGTTCGTACT
RLB17 ACCCTCCAGGAAAGTACCTCTGAT
RLB18 CCAAACCCAACAACCTAGATAGGC
RLB19 GTTCCTCGTGCAGTGTCAAGAGAT
RLB20 TTGCGTCCTGTTACGAGAACTCAT
RLB21 GAGCCTCTCATTGTCCGTTCTCTA
RLB22 ACCACTGCCATGTATCAAAGTACG
RLB23 CTTACTACCCAGTGAACCTCCTCG
RLB24 GCATAGTTCTGCATGATGGGTTAG

3. Host depletion and extraction

材料
  • Sputum/Endotracheal aspirates
  • Bronchoalveolar lavages/Mini-BALs
  • VTM/UTM-stored oral/nasal swabs (viral-arm only)
消耗品
  • Sputasol (Fisher, 11943262)
  • RT-PCR Grade Water (Fisher, 10289774)
  • Phosphate Buffer Saline (PBS) (Fisher, 11593377)
  • 2X HL-SAN Buffer (4.5 M NaCl and 100 mM MgCl2) formulated using the two reagents below:
  • • Invitrogen 5M NaCl (Fisher, 10255984)
  • • Invitrogen 1M MgCl2 (Fisher, 10418464)
  • HL-SAN Triton Free DNase (ArticZymes, 70911-202)
  • Saponin solution (0.2% saponin in PBS) formulated using the reagent below:
  • • Saponin (Sigma-Aldrich, 47036-50G-F)
  • Matrix Lysing E tubes (Fisher, 11452420)
  • MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Fisher, 16346582)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 15 ml Falcon tubes
装置
  • Centrifuge e.g. Eppendorf max speed 15,000rpm (Fisher, 15881635)
  • Microfuge
  • タイマー
  • ボルテックスミキサー
  • Thermomixer set at 37°C
  • Thermomixer set at 65°C
  • Magnetic rack
  • Bead-beater (e.g. FastPrep-24™ Classic bead beating grinder and lysis system)
  • P1000 pipette and tips
  • P200 ピペットとチップ
  • P20 pipette and tips
  • P10 ピペットとチップ
オプション装置
  • Suitable biosafety equipment for your sample(s) (e.g. Microbiological Safety Cabinet II contamination level 3 or equivalent)
重要

Ensure you are keeping your bacterial/fungal samples separate from your viral samples.

If processing a mix of bacterial/fungal samples and viral samples, ensure you keep them separate to avoid following the wrong sample preparation method.

Both methods can be followed simultaneously if processing different sample types prior to library preparation. Please ensure you are following the correct method for each sample type.

Note: A sample can be processed for both bacterial/fungal sample preparation and viral sample preparation. Please follow the instructions below, where the supernatant is used for viral sample preparation and the pellet is used for bacterial/fungal sample preparation. Please note, each preparation type will differ in method and should be treated as a separate sample.

Ensure you have sufficient sample input. You will need:

  • 250 µl of sputum, endotracheal aspirate or other viscous samples (e.g. mucosal BAL).

OR

  • 500 µl of swab sample in transport media or other liquid samples.
オプショナルステップ

Input controls

Negative internal controls

We recommend a negative control is included and processed as a real sample through the entire process to monitor for contamination events.

The negative control can be PBS or a third-party negative sample matrix.

Prepare a working solution of Sputasol in nuclease-free water (1:13.33) as follows:

  • In a fresh 15 ml falcon tube dispense 750 µl Sputasol.
  • Add 9.25 ml nuclease-free water.
  • Throughly mix by vortexing.

* Volumes can be adjusted to meet your experiment requirements.

Prepare a 0.2% saponin-PBS solution:

Reagent Quantity/Volume
Saponin (Sigma-Aldrich, 47036-50G-F) 0.01 g
Phosphate Buffer Saline (PBS) 5,000 µl
Total volume 0.2% saponin-PBS solution 5,000 µl

* Quantity/volumes can be adjusted to meet your experiment requirements.

The 0.2% saponin-PBS solution can be stored in the fridge (~4°C) for up to 30 days.

Prepare the 2X HLSAN Buffer (4.5 M NaCl and 100 mM MgCl2) as follows:

Reagent Volume
Invitrogen 5M NaCl 4,500 µl
Invitrogen 1M MgCl2 500 µl
Total 5,000 µl

* Volumes can be adjusted to meet your experiment requirements.

The formulated 2X HLSAN Buffer can be stored at room temperature (~22°C) long-term.

Ensure your thermomixers are set to 37°C and 65°C.

Prepare your sputum, endotracheal (ETT), or any other mucoid sample(s):

Note: Transport Media and BAL samples do not require this unless they are mucoid.

  • To each sample, add an equal volume (1:1) of Sputasol working solution.
  • Mix by vortexing for 30 seconds.
  • Incubate at room temperature until liquefication (at least 5 minutes).

Tip: If the sample is still viscous or sticky after 5 minutes, repeat the process above. Full liquefication of sputum is important for good depletion and efficient extraction.

For each sample, transfer 500 µl into a separate clean 1.5 ml Eppendorf tube.

Centrifuge for 5 minutes at 10,000 x g.

Carefully remove tubes from centrifuge without disturbing the solution.

ヒント

Remember, your sample(s) can be processed for both viral sample preparation and bacterial/fungal sample preparation.

Please follow the instructions outlined below and treat each preparation as a separate sample.

Perform sample preparation side-by-side depending on your sample type:

Step For viral sample preparation For bacterial/fungal samples
1. For each sample, transfer 300 µl of the supernatant (by aspirating from the top) to a separate new 1.5 ml Eppendorf tube. OPTIONAL: the remaining pellet and supernatant from the sample used in the viral sample preparation can be taken forward and processed in the bacterial/fungal sample preparation as seen below
2. Using a pipette, carefully remove most of the supernatant without disturbing the pellet, leaving enough volume to cover the pellet.

Note: Ensure there is enough supernatant left (~50 µl) above the pellet so as to not disturb it.
3. Thoroughly resuspend the pellet in 200 µl of the prepared 0.2% saponin-PBS solution. Mix by pipetting.
4. Add the depletion reagent to the sample supernatant:

• Add 10 µl of HLSAN enzyme.
• Vortex mix for 3 seconds.
Add the depletion reagents to the resuspended pellet as follows:

• Add 200 µl of the prepared 2X HLSAN Buffer.
• Add 10 µl of HLSAN enzyme.
• Vortex mix for 3 seconds.
5. Incubate the reaction on a thermomixer at 37°C for 10 minutes at 1,000 rpm. Incubate the reaction on a thermomixer at 37°C for 10 minutes at 1,000 rpm.
6. To each tube containing the sample supernatant, prepare the following reaction:

viral arm 1

Ensure the reagents are mixed by inverting the tube multiple times.
Avoid mixing the lysis reagents too vigorously or with any method that would lead to foam (e.g. vortexing).

Note: A mastermix of the extraction reagents can be made and added together instead.
7. Add 900 µl of PBS to your sample(s) and mix by pipetting.
8. Centrifuge your sample(s) at 12,000 x g for 3 minutes.
9. Using a pipette, carefully remove most of the supernatant without disturbing the pellet, leaving enough volume to cover the pellet.

Ensure there is enough supernatant left (~50 µl) above the pellet so as to not disturb it.
10. Resuspend pellet in 500 µl of PBS and mix by pipetting.
11. For each sample, transfer whole volume of sample into a separate new bead-beating tube (matrix lysing E tubes).
12. Insert the bead-beating tubes into the FastPrep-24 Classic bead beating grinder and lysis system and run the program as follows:

Bact arm 2
13. Remove the bead-beating tubes from the Fast-prep device, and centrifuge for 30 seconds at max speed (>20,000 x g).
14. For each sample, transfer 300 µl of the supernatant to a separate new 1.5 ml Eppendorf tube.

Note: Avoid aspirating the beads from the bead-beating tube.
15. To each tube containing the sample supernatant, prepare the following reaction:

Bact arm 1

Ensure the reagents are mixed by inverting the tube multiple times.
Avoid mixing the lysis reagents too vigorously or with any method that would lead to foam (e.g. vortexing).

Note: A mastermix of the extraction reagents can be made and added together instead.

Transfer all sample tubes to a thermomixer and incubate the reactions at 65°C for 5 minutes at 1,000 rpm.

Transfer the sample tube(s) to a hulamixer and incubate/mix at room temperature for 5 min.

Hulamixer settings:

  • Orbital 15 rpm (10 seconds)
  • Reciprocal 70° (15 seconds)
  • Vibro/pause 5° (3 seconds)
  • 5 minutes total.

Note: Tube inversion is crucial during this incubation, as the beads may collect at the bottom of the tube. We highly recommend the use of a hulamixer, however if this is not available, ensure the samples are manually inverted during the incubation.

Prepare 2 ml of fresh 80% ethanol per sample in nuclease-free water.

Briefly spin down the tube(s) and pellet on a magnetic rack until supernatant is clear and colourless (for at least 5 minutes). Keep the tube on the magnetic rack, and pipette off the supernatant.

Take care not to disturb the pelleted beads.

Remove the tube from the magnetic rack and add 1 ml of Wash Buffer from the MagMAX kit. Gently mix by inverting the tube until fully resuspended.

Note: Avoid techniques that will create foam when mixing your tubes. This can negatively impact sample recovery.

Briefly spin down the tube(s) and pellet on a magnetic rack until supernatant is clear and colourless (for at least 2 minutes). Keep the tube on the magnetic rack, and pipette off the supernatant.

Remove the tube from the magnetic rack and add 1 ml of 80% ethanol. Gently mix by inverting the tube until fully resuspended.

Briefly spin down the tube(s) and pellet on a magnetic rack until supernatant is clear and colourless (for at least 2 minutes). Keep the tube on the magnetic rack, and pipette off the supernatant.

Remove the tube from the magnetic rack and add 500 µl of 80% ethanol. Gently mix by inverting the tube until fully resuspended.

Briefly spin down the tube(s) and pellet on a magnetic rack until supernatant is clear and colourless (for at least 2 minutes). Keep the tube on the magnetic rack, and pipette off the supernatant.

Keeping the tube on the magnetic rack, leave the lid open and allow to dry for ~2 minutes, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend the pellet by pipetting in 20 µl of nuclease-free water. Ensure the pellet is fully resuspended by pipette mixing.

To aid with sample elution, transfer tubes to a thermomixer and incubate at 65°C for 5 minutes at 1,000 rpm.

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

Remove and retain the eluate (~15 µl) into a clean 1.5 ml Eppendorf DNA LoBind tube.

  • Dispose of the pelleted beads
最終ステップ

Take your extracted sample forward into the library preparation section of this protocol.

Alternatively, if you are not using your sample immediately, store at -20°C.

4. Reverse transcription and PCR

材料
  • For viral sample preparation: 10 µl of extracted nucleic acid from previous step
  • For bacterial/fungal sample preparation: 3 µl of extracted nucleic acid from previous step
  • Fragmentation Mix (FRM)
  • Rapid Barcode Primers (RLB01-24)
消耗品
  • 12 µM RLB RT 9N primer (IDT - Sequence: 5’-TTTTTCGTGCGCCGCTTCAACNNNNNNNNN-3’)
  • 12 µM TSOmG template-switching oligo (IDT - Sequence: 5’-GCTAATCATTGCTTTTTCGTGCGCCGCTTCAACATmGmGmG-3’)
  • 10 mM dNTPs (Fisher, 10610851)
  • Maxima H Minus Reverse Transcriptase (Fisher, 13233159)
  • RNaseOUT (Fisher, 10777019)
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
装置
  • Hula mixer（緩やかに回転するミキサー）
  • Microfuge
  • サーマルサイクラー
  • 96-well PCR block
  • Timer
  • Ice bucket with ice
  • P1000 pipette and tips
  • P200 ピペットとチップ
  • P100 ピペットとチップ
  • P20 pipette and tips
  • P10 ピペットとチップ
  • P2 pipette and tips
重要

Ensure you are following the correct steps in the method for your sample type.

The method differs for bacterial/fungal sample processing and for viral sample processing. Please ensure you keep track of your samples to ensure you are following the correct method.

Note: If a sample has been processed for both bacterial/fungal sample preparation and viral sample preparation, you will have two "separate" samples to process and barcode in library preparation.

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:

Reagents 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting
Fragmentation Mix (FRM) Not frozen ✓ ✓
Rapid Barcode Primer 1-24 (RLB01-24) ✓ ✓ ✓
RLB RT 9N primer (12 µM) ✓ ✓ ✓
TSOmG template-switching oligo (2 μM) ✓ ✓ ✓
10 mM dNTP solution ✓ ✓ ✓
Maxima H Minus Reverse Transcriptase Not frozen ✓ ✓
Maxima H Minus 5x RT Buffer ✓ ✓ ✓
LongAmp Taq 2X Master Mix ✓ ✓ ✓
RNaseOUT Not frozen ✓ ✓

Prepare each of your extracted nucleic acid samples from the previous step in a separate clean 0.2 ml tube:

For viral sample preparation:

  • Take forward 10 µl of each viral nucleic acid extract.

Note: Quantifying viral samples is not recommended, as these will likely be below the limit of detection for Qubit HS or similar fluorescence-based quantification methods. Please proceed with the recommended volume of viral nucleic acid extract.


For bacterial/fungal samples:

  • Quantify 1 µl of each bacterial/fungal extraction using Qubit High Sensitivity.
    • If the extract is more than 1 ng/µl: dilute to 1 ng/µl using nuclease-free water.
    • If the extract is less than 1 ng/µl (or unquantifiable): no dilution is necessary, and extract is used directly.
  • Take forward 3 µl of each bacterial/fungal DNA extract.

Perform the RT reaction / tagmentation reaction side-by-side depending on your sample type:

Note: If processing both sample types we recommend preparing the viral samples first as they have a longer incubation time. The bacterial/fungal samples can be processed during the viral sample incubation.

Step For viral sample preparation (RT reaction) For bacterial/fungal samples (tagmentation)
1. In a clean 0.2 ml thin-walled PCR tube, prepare the following master mix.

Tip: Generate sufficient volume for each viral sample preparation + 1 extra volume for excess:

RT viral 1
2. Mix the master mix prepared above by vortexing and spin down briefly.
3. Add 2 µl of the prepared mastermix to each of your 10 µl of viral nucleic acid extract.
4. Incubate the reaction at 65°C for 5 minutes, then transfer the samples to ice immediately.
5. Keep your samples on ice for 2 minutes.
6. In a clean 0.2 ml thin-walled PCR tube, prepare the Maxima H master mix.

Tip: Generate sufficient volume for each viral sample preparation + 1 extra volume for excess:

RT viral 2
Note: Add the Maxima H Enzyme last. Keep the master mix on ice.

The master mix should be aliquoted to your samples and incubated relatively quickly, as the enzymes are not hot-start, and the reaction will start as soon as it comes in contact with your sample.
7. Add 8 µl of the Maxima H master mix to each of your viral samples for a total combined volume of 20 µl.
8. Pre-heat a PCR block to 42°C, then add your sample tubes and incubate using the following conditions:

RT viral 3 inc
During the RT reaction (viral samples), the bacterial/fungal samples can be prepared.
9. To each of the 3 µl of bacterial/fungal DNA extract, add 1 µl of FRM, and mix gently by flicking the tube.
10. Pre-heat a PCR block to 30°C, then add your sample tubes and incubate using the following conditions:

Frag bact fung 1 inc

.

In a clean 1.5 Eppendorf tube, prepare the following master mix:

Tip: Generate sufficient volume for each sample preparation + 1 extra volume for excess:

Reagent Volume
LongAmp Hot Start Taq 2X Master Mix 25 µl
Nuclease-free water 20 µl
Total volume 45 µl

Mix by pipetting, taking care not to generate bubbles or foam.


Note: The non-hot start product, LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287) is also compatible with this method. If using this alternative, please ensure you follow the manufacturers instructions and prepare this reaction on ice.

Aliquot out the master mix into a new separate 0.2 ml thin-walled PCR tube for each sample preparation.

To each tube containing 45 µl of master mix, add 1 µl of RLB barcode (1-24). Each tube should have a different barcode.

Note: To make sample tracking easier, we recommend that the two types of sample preparation are given different blocks of barcodes:

e.g. Barcodes 1-4 for the viral samples and barcodes 5-8 for the bacterial/fungal samples.

To each tube containing master mix and a RLB barcode, add your RT (viral) sample or tagmented (bacterial/fungal) sample:

For viral samples:

  • Add 5 µl RT sample to the reaction tube. Do not deviate from the recommended volume as this can lead to sub-optimal results.
Reagent Volume
Master mix + barcode 46 µl
RT viral sample 5 µl
Total 51 µl

Note: If you are concerned that you will not reach the minimum molarity, we recommend assembling two separate reactions for your viral sample using the volumes above, and pooling following PCR. Do not alter the PCR volumes as this will result in poor performance of the PCR and lead to sub-optimal results.


For bacterial/fungal samples:

  • Add the full volume of tagmented sample to the reaction tube:
Reagent Volume
Master mix + barcode 46 µl
Tagmented bacterial/fungal DNA sample ~4 µl
Total 50 µl

Mix gently by flicking the tube, and spin down.

Amplify using the following cycling conditions:

Cycle step Temperature Time No. of cycles
Initial denaturation 95°C 45 seconds 1
Denaturation

Annealing

Extension		 95°C

56°C

65°C		 15 sec

15 sec

4 minutes
30
Final extension 65°C 6 minutes 1
Hold 10°C ∞

Note: Total PCR time 2 hours 43 minutes* (Timing may differ depending on equipment and ambient conditions).

Optional: The PCR amplification can be performed and left at the hold temperature overnight.

最終ステップ

Take forward your sample into the clean up, quantification and adapter attachment step.

5. Clean-up, quantification and adapter attachment

材料
  • AMPure XP Beads (AXP)
  • Elution Buffer (EB)
  • Rapid Adapter (RA)
  • Adapter Buffer (ADB)
消耗品
  • Thermolabile Exonuclease I (NEB, M0568)
  • ヌクレアーゼフリー水で用事調整した 80% エタノール溶液
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
装置
  • サーマルサイクラー
  • Hula mixer（緩やかに回転するミキサー）
  • Magnetic rack
  • Qubit fluorometer (or equivalent)
  • Timer
  • P1000 pipette and tips
  • P200 ピペットとチップ
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 ピペットとチップ
  • P2 pipette and tips

Quantify the sample tubes (PCR products from the previous step) using the Qubit dsDNA HS Assay Kit.

Make a note of each samples concentration.

Note: Sample concentration may vary depending on input. Some of your samples might be below the limit of detection for the Qubit dsDNA HS Assay Kit. This is not an uncommon occurance.

Please proceed with all of your samples, following the recommendations in the instructions below.

Add 1 µl of Thermolabile Exo I to each of the sample tubes (PCR product) and mix by pipetting.

Incubate the reactions using the following conditions:

Temperature Time
37°C 10 minutes
80°C 1 minute
10°C Hold

In a new 1.5 ml Eppendorf DNA LoBind tube, pool all barcoded samples in equimolar ratios to a combined final concentration 800 ng.

For example: if 10 barcodes were used, take forward 80 ng of each sample.


Note: In cases where the concentration of your sample PCR product is too low, take forward the maximum available volume.

Resuspend the AMPure XP beads (AXP) by vortexing.

Add 0.6X volume of AMPure XP beads (AXP) to the pooled samples.

Ensure you have accurately measured the volume of your pooled samples to maintain the 0.6X ratio of the AMPure XP beads (AXP).

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

Prepare at least 1 ml of fresh 80% ethanol in nuclease-free water.

Briefly spin down the sample and pellet on a magnetic rack until supernatant is clear and colourless. Keep the tube on the magnetic rack, and pipette off the supernatant.

Keep the tube on the magnet and wash the beads with 500 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 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 15 µl Elution Buffer (EB).

Incubate for 2 minutes at room temperature.

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

Remove and retain 14 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

  • Remove and retain the eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
  • Dispose of the pelleted beads
オプショナルステップ

Quantify 1 µl of eluted sample using a Qubit fluorometer and the Qubit dsDNA HS Assay Kit.

Expected yield of 10-40 ng/µl.

Take forward 11 µl of your eluted samples into a clean 1.5 ml Eppendorf DNA LoBind tube.

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

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

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

Mix gently by flicking the tube, and spin down.

Incubate the reaction for 5 minutes at room temperature.

最終ステップ

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

6. Priming and loading the MinION and GridION Flow Cell

材料
  • Flow Cell Flush (FCF)
  • Flow Cell Tether (FCT)
  • Library Beads (LIB)
  • Sequencing Buffer (SB)
消耗品
  • MinionとGridIONのFlow Cell
  • Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
  • 1.5 ml Eppendorf DNA LoBind tubes
装置
  • MinIONかGridION のデバイス
  • MinIONとGridIONのFlow Cell ライトシールド
  • P1000 ピペット及びチップ
  • P100 ピペットとチップ
  • P20 ピペットとチップ
  • P10 ピペットとチップ
重要

注意：本キットはR10.4.1フローセル（FLO-MIN114）のみに対応しています。

ヒント

フローセルのプライミングとローディング

新規ユーザーは、 初回使用前に'Priming and loading your flow cell' のビデオをご覧いただくことをお勧めします。

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

重要

MinION R10.4.1フローセル（FLO-MIN114）での最適なシークエンス性能と出力向上のために、フローセルのプライミングミックスに最終濃度0.2 mg/mlでBovine Serum Albumin (BSA) を添加することを推奨します。

(注： その他のアルブミンの種類（組換えヒト血清アルブミンなど）の使用は推奨しません。

To prepare the flow cell priming mix with BSA, combine the following reagents in a fresh 1.5 ml Eppendorf DNA LoBind tube. Mix by inverting the tube and pipette mix at room temperature:

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

MinIONまたはGridIONデバイスの蓋を開け、フローセルをクリップの下にスライドさせます。 フローセルをしっかりと押さえ、サーマルプレートと電気接触が密着しているかを確認してください。

Flow Cell Loading Diagrams Step 1a_JP

Flow Cell Loading Diagrams Step 1b_JP

オプショナルステップ

ライブラリーをロードする前にフローセルチェックを行い、使用可能なポアの数を把握して下さい。

フローセルが以前にチェックされている場合は、このステップを省略できます。

詳細については、MinKNOWプロトコルのフローセルチェックの手順 flow cell check instructionsを参照してください。

フローセルのプライミングポートカバーを時計方向にスライドさせ、プライミングポートを開きます。

Flow Cell Loading Diagrams Step 2_JP

重要

フローセルからバッファーを引き上げる際には注意してください。20～30μl以上は除去せず、ポアのアレイ全体が常にバッファーで覆われていることを確認して下さい。アレイに気泡が入ると、ポアに不可逆的なダメージを与える可能性があります。

プライミングポートを開けた後に、カバーの下に小さな気泡がないかを確認して下さい。気泡を取り除くために少量の液を引き上げます。

  1. P1000ピペットを200 µ Lに設定して下さい。
  2. ピペットの先端をプライミングポートに差し込みます。
  3. 目盛りが220-230 ulと表示されるまでダイヤルを回して、20-30 ulを吸い上げるか、少量のバッファーがピペットの先端に入るのが見えるまでダイヤルを回します。

(注： プライミングポートからセンサーアレイ全体にバッファーがあることを確認してください。

Flow Cell Loading Diagrams Step 03 V5_JP

気泡が混入しないように、プライミングポートからフローセルにプライミングミックスを800µl注入し、 5分間待ちます。この５分間の間に、以下の手順でライブラリーをロードする準備をして下さい。

Flow Cell Loading Diagrams Step 04 V5_JP

Library Beads(LIB)の液をピペッティングすることで十分に混合して下さい。

重要

Library Beads（LIB）チューブにはビーズの懸濁液が入っています。これらのビーズはすぐに沈殿するので、使用直前に混合することが重要です。

ほとんどのシーケンス実験にはLibrary Beads （LIB）の使用を推奨します。しかし、より粘性の高いライブラリーにはLibrary Solution（LIS）を使ってください。

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 25.5 µl
DNA library 12 µl
Total 75 µl

フローセルのプライミングを完了させます。

  1. SpotON サンプルポートカバーをゆっくりと持ち上げ、SpotON サンプルポートにアクセスできるようにします。
  2. 200μlのプライミングミックスをフローセルのプライミングポート（SpotONサンプルポートではありません）に気泡が入らないように注入します。

Flow Cell Loading Diagrams Step 5_JP

Flow Cell Loading Diagrams Step 06 V5_JP

調製したライブラリーは、ロードする直前にピペッティング混合して下さい。

調製したライブラリー75μlをSpotONサンプルポートからフローセルに滴下します。次の一滴を追加する前に各一滴がポートに入っていることを確認して下さい。

Flow Cell Loading Diagrams Step 07 V5_JP

SpotONサンプルポートカバーをゆっくりと元に戻し、バング（カバーの先）がSpotONポートに入ることを確認し、プライミングポートを閉じます。

Step 8 update_JP

Flow Cell Loading Diagrams Step 9_JP

重要

最適なシークエンス出力を得るために、ライブラリーがロードされたすぐにライトシールドをフローセルに取り付けてください。

ライブラリーがフローセル上にある状態では（ウォッシングやリロードのステップを含める）、フローセルにライトシールドを付けたままにしておくことを推奨します。ライトシールドは、ライブラリーがフローセルから除去された時点で取り外すことができます。

ライトシールドを以下のようにフローセルに設置して下さい。

  1. ライトシールドの先端を慎重にクリップに当てます。 (注： ライトシールドをクリップの下に無理に押し込まないでください。

  2. ライトシールドをフローセルにゆっくりと下ろします。ライトシールドは、フローセルの上部全体を覆うようにSpotONカバーの周囲に取り付けます。

J2264 - Light shield animation Flow Cell FAW optimised-Japanese step10

注意

MinIONフローセルライトシールドは、フローセルに固定されていないため、取り付け後の取り扱いには注意が必要です。

最終ステップ

デバイスの蓋を閉め、MinKNOWでシークエンスランをセットします。

7. Data acquisition and basecalling

重要

We do not recommend sequencing and performing data analysis simultaneously on your device.

To ensure the compute on your device can keep up with the requirements for sequencing and/or analysis, we strongly recommend against running both processes at the same time.

Ensure your sequencing run has completed before setting off data analysis. Data analysis will be performed post-sequencing.

Equally, we do not recommend starting a sequencing run if you are currently performing data analysis on your device.

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. Further instructions for setting up a sequencing run can be found in the MinKNOW protocol.

We recommend setting up your sequencing run using the basecalling and barcoding recommendations outlined below. All other parameters can be left to their default settings.

MinKNOW settings for rapid metagenomic sequencing

For fastest turnaround time and easiest analysis, basecalling should be performed live during the sequencing run using the High Accuracy (HAC) basecaller.

Below are the recommendations for MinKNOW settings:

Positions

Flow cell position: [user defined]

Experiment name: [user defined]

Flow cell type: FLO-MIN114

Sample ID: [user defined]

Kit

Kit selection: Rapid PCR Barcoding Kit (SQK-RPB114.24)

Run configuration

Sequencing and analysis

Basecalling: On [default]
Modified bases: Off
Model: High-accuracy basecalling (HAC) [default]

Barcoding: On [default]
Trim barcodes: Off [default]
Barcode both ends: Off [default]
Custom barcodes selection: Off [default]

Alignment: Off [default]

Adaptive sampling: Off [default]

Advanced options
Active channel selection: On [default]
Time between pore scans: 1.5 [default]
Reserve pores: On [default]

Data targets

Run limit: [user defined]*

*Sequencing time will depend on data requirements. For rapid information, data can be analysed after as little as 30 minutes of sequencing. Or to maximise data generation, you can sequence for up to 72 hours.

Output

Output format
.POD5: On [default]
.FASTQ: On [default]
.BAM: On

Filtering: On [default]
Qscore: 9 [default]
Minimum read length: 200 bp [default]

8. Downstream analysis

重要

We do not recommend sequencing and performing data analysis simultaneously on your device.

To ensure the compute on your device can keep up with the requirements for sequencing and/or analysis, we strongly recommend against running both processes at the same time.

Ensure your sequencing run has completed before setting off data analysis. Data analysis will be performed post-sequencing.

Equally, we do not recommend starting a sequencing run if you are currently performing data analysis on your device.

Post-basecalling analysis

We recommend performing downstream analysis using EPI2ME which facilitates bioinformatic analyses by allowing users to run Nextflow workflows in a desktop application. EPI2ME maintains a collection of bioinformatic workflows which are curated and actively maintained by experts in long-read sequence 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.


Your basecalled data generated by the sequencing software can be easily analysed using the wf-metagenomics workflow, a bioinformatic pipeline written in NextfFlow. This workflow provides identification and abundance estimation of taxa present in your sample.

Using the recommended database, human, fungal, bacterial, viral, archaea and protozoal sequences can be accurately identified using the workflow.

We recommend you always use the latest available version of the workflow. Further information about the usage and results provided by the workflow can be found in the wf-metagenomics EPI2ME documentation page.


Note: You can also run this workflow through command line. However, we only recommend this option for experienced users. For more information and the latest version of the workflow, please visit the wf-metagenomics page on GitHub. If using the workflow through commandline, the settings recommend for optimal workflow performance for this protocol are: --kraken2_confidence 0.01 --database_set PlusPF-8 --store_dir /path/to/database/download/directory/

*/path/to/database/download/directory/ is a placeholder, the exact location used will depend on the user system.

オプショナルステップ

Test data is available on Github to test the wf-metagenomics workflow.

The test data can be found in the following repository: wf-metagenomics test data.

Running wf-metagenomics with EPI2ME

Within the EPI2ME app, new workflows can be installed by selecting the appropriate workflow under the "Available Workflows" tab in the Workflows section.

Already installed workflows can be found under the "Installed" tab.

You can test your installation using a small test dataset that is provided with the workflow:

1. Launch the EPI2ME app

2. Select View workflows

3. Select the wf-metagenomics workflow

4. Select Use demo data

5. Launch the workflow

6. Once the run has completed, results can be viewed under the "Report" tab. The workflow outputs an interactive html report.


Running the workflow

To run wf-metagenomics with the settings recommended for the dual arm metagenomics protocol:

1. Launch the EPI2ME app

2. Select View workflows

3. Select the wf-metagenomics workflow

4. Select Run this workflow.

5. Choose local or cloud option (if available).

6. In "Input Options", specify the input data for the workflow (either FASTQ or BAM).

7. In "Sample Options", provide a sample sheet in comma-separated values (csv) format.
We suggest you provide an alias for each barcode that describes both the sample and prep type (e.g. sample_A_viral, sample_A_bacterial).

8. In "Reference Options", choose PlusPF-8 database to ensure archaea, bacterial, viral, human, and fungal taxa can be identified by the workflow.

9. In "Kraken2 Options", set Confidence score threshold to 0.01

10. Click Launch workflow

11. Once the run has completed, results can be viewed under the "Report" tab. The workflow outputs an interactive html report.


Note: All options not specified should be left to their default values.

Optional: If you need to adjust the computational resource available to your workflow, you can change local CPU and memory allocation in the workflow setup.

9. フローセルの再利用と返却

材料
  • Flow Cell Wash Kit (EXP-WSH004)

シークエンス実験終了後、フローセルを再利用する場合は、Flow Cell Wash Kitのプロトコールに従い、洗浄したフローセルを2～8℃で保管してください。

Flow Cell Wash Kit protocolは、Nanoporeコミュニティーで入手できます。

ヒント

運転を停止したらできるだけ早くフローセルをウォッシュすることをお勧めします。しかし、これが不可能な場合はフローセルをデバイスに入れたまま、翌日にウォッシュをして下さい。

または、返送手順に従って、オックスフォード・ナノポアに返送してください。

フローセルの返却方法は hereをご覧ください。

(注： 製品を返却する前に、すべてのフローセルを脱イオン水で洗浄する必要があります。

重要

シークエンシング実験に関して問題が発生した場合や質問がある場合には、このプロトコルのオンライン版にあるトラブルシューティングガイドを参照してください。

10. DNA/RNA抽出、およびライブラリ調製時の問題点

以下は、最もよく起こる問題のリストであり、いくつかの原因と解決策が提案されています。

Nanopore Community Support セクションにFAQをご用意しています。

ご提案された解決策を試しても問題が解決しない場合は、テクニカルサポートに電子メール (support@nanoporetech.com)または LiveChat in the Nanopore Communityでご連絡ください。

サンプルの品質が低い

問題点 この問題が生じた可能性のある原因 解決策とコメント
DNAの純度が低い（DNAのOD 260/280のナノドロップ測定値が1.8未満およびOD 260/230が2.0～2.2未満） DNA抽出で必要な純度が得られていない 夾雑物の影響は、 Contaminants に示されています。コンタミネーションをもたらさないために別の抽出方法extraction method をお試しください。.

追加のSPRIクリーンアップステップの実施を検討して下さい。
低いRNA インテグリティー（RNA Integrity Number: <9.5 RIN、またはrRNAバンドがゲル上でスメアになっている） 抽出中にRNAが分解された 別のRNA抽出方法 RNA extraction methodを試してください。RINの詳細については、 RNA Integrity Number の資料を参照してください。詳細については、 DNA/RNA Handling のページをご覧ください。
RNAのフラグメントが予想より短い 抽出中にRNAが分解された 別のRNA抽出方法 RNA extraction methodを試してください。 RINの詳細については、 RNA Integrity Number の資料を参照してください。詳細については、DNA/RNA Handling のページをご覧ください。

RNAを扱う際には、RNaseフリーの環境で作業し、実験器具もRNaseフリーにしておくことをお勧めします。

AMPureビーズクリーンアップ後のDNA回収率が低い

問題点 この問題が生じた可能性のある原因 解決策とコメント
低回収率 AMPureビーズとサンプルの比率が予想していたのよりも低いことによるDNAの損失 1. AMPureビーズはすぐに沈降するため、サンプルに添加する前によく再懸濁させてください。

2. AMPureビーズ対サンプル比が0.4:1未満の場合、どのようなサイズのDNA断片でもクリーンアップ中に失われます。
低回収率 DNA断片が予想よりも短い サンプルに対するAMPureビーズの比率が低いほど、短い断片に対する選択が厳しくなります。 アガロースゲル(または他のゲル電気泳動法)上でインプットDNAの長さを設定してから、使用するAMPureビーズの適切な量を計算してください。 SPRI cleanup
エンドプレップ後の収率が低い 洗浄ステップで使用したエタノール濃度が低い（70%未満）。 エタノールが70%未満の場合、DNAは洗浄中にビーズから溶出されます。必ず正しい濃度（％）のエタノールを使用してください。

11. Issues during the sequencing run using a Rapid-based sequencing kit

以下は、最もよく起こる問題のリストであり、いくつかの原因と解決策が提案されています。

Nanopore Community Support セクションにFAQをご用意しています。

ご提案された解決策を試しても問題が解決しない場合は、テクニカルサポートに電子メール (support@nanoporetech.com)または LiveChat in the Nanopore Communityでご連絡ください。

シークエンス開始時のポアがフローセルチェック後よりも少ない場合

問題点 予想される原因 解決策とコメント
MinKNOWのフローセルチェックで確認されたポアの数より、シークエンシング開始時のポア数が少なく表示された。 ナノポアアレイに気泡が入ってしまった。 フローセルチェックをした後、フローセルをプライミングする前に、プライミングポート付近の気泡を取り除くことが必要です。 気泡を取り除かないと、気泡がナノポアアレイに移動し、空気に触れたたナノポアが不可逆的なダメージを負った可能性がある。これを防ぐための最適な方法が、 this videoで紹介されています。
MinKNOWのフローセルチェックで確認されたポアの数より、シークエンシング開始時のポア数が少なく表示された。 フローセルがデバイスに正しく挿入されていない。 シークエンスランを停止し、フローセルをシークエンス装置から取り出します。次に再度フローセルを挿入し、装置にしっかりと固定され、目標温度に達していることを確認します。GridIONやPromethIONの場合は別のフローセルの位置をお試しください。
MinKNOWのフローセルチェックで確認されたポアの数より、シークエンシング開始時のポア数が少なく表示された。 ライブラリー内の汚染物質がポアを失活させたり塞いだりしている。 フローセルチェックの際のポア数は、フローセル保存バッファー中のQC用のDNA分子を用いて計測されます。シークエンシングの開始時は、ライブラリ自体を使用してアクティブなポア数を推定します。このため、フローセルチェックとRun開始時のポア数は、約10％程度の変動が起こります。シークエンシング開始時に報告されたポアの数が大幅に減少している場合は、ライブラリー中の汚染物質がメンブレンを損傷していたり、ポアをブロックしている可能性があります。インプット材料の純度を向上させるために、別のDNA/RNA抽出または精製方法が必要となる場合があります。コンタミネーションの影響は、Contaminants Know-how pieceを参照にして下さい。夾雑物を除去するために別の抽出方法extraction method をお試しください。

MinKNOWのスクリプトに問題

問題点 この問題が生じた可能性のある原因 解決策とコメント
MinKNOW に 「Script failed」と表示されている"
コンピューターを再起動し、MinKNOWを再起動します。問題が解決しない場合は MinKNOW log files MinKNOWログファイルを収集し 、テクニカルサポートにご連絡ください。他のシークエンシングデバイスをお持ちでない場合は、 フローセルとロードしたライブラリーを4℃で保管することをお勧めします。詳細な保管方法については、テクニカルサポートにお問い合わせください。

Pore occupancy below 40%

Observation Possible cause Comments and actions
Pore occupancy <40% Not enough library was loaded on the flow cell Ensure the correct concentration of good quality library is loaded on to a MinION/GridION flow cell. To check the concentration, please refer to the library preparation protocol. 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 Rapid Sequencing Kit V14/Rapid Barcoding Kit V14 was used, and sequencing adapters did not attach to the DNA Make sure to closely follow the protocol and use the correct volumes and incubation temperatures. A Lambda control library can be prepared to test the integrity of reagents.
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.

予想より短いリード長

問題点 予想される原因 解決策とコメント
予想より短いリード長 DNAサンプルの不要な断片化 読み取り長はサンプルDNA断片の長さを反映します。サンプルDNAは、抽出およびライブラリー調製中の操作で断片化した可能性があります。

1. 抽出の最適な方法については、Extraction Methods の抽出方法を参照してください。

2. ライブラリー調製に進む前に、アガロースゲル電気泳動で、サンプルDNAのフラグメント長の分布を確認してください。 DNA gel2 上の画像では、サンプル1は高分子量ですが、サンプル2は断片化されています。

3. ライブラリー調製中は、試薬を混合するためのピペッティングやボルテックス操作は、プロトコルで指示がないかぎり行わないでください。

利用できないポアの割合が多い場合

問題点 予想される原因 解決策とコメント
利用できないポアの割合が大きい（チャンネルパネルとポアのアクティブポートで青く表示されています）

image2022-3-25 10-43-25 上のアクティブなポアの図は、時間の経過とともに「利用できない」ポアの割合が増加していることを示しています。		 サンプル内に不純物が含まれている 一部のポアに吸着する不純物は、MinKNOWに組み込まれたポアのブロック解除機能によって、ポアから除去することができます。 このステップが完了すると、ポアの状態が「sequencing pore」に戻ります。利用できないポアの部分が多いか、増加した場合:

1.Flow Cell Wash Kit nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) を用いて、ヌクレアーゼ洗浄を 行うことができます。又は
2. PCRを数サイクル実行してサンプルDNAの量を増やし、サンプルDNAに含まれる問題の不純物が相対的に減る（希釈される）ようにします。

Inactiveのポアの割合が高い

問題点 予想される原因 解決策とコメント
利用できない(inactive/unavailable)ポアの割合が高い（チャネルパネルとポアアクティブポートでは水色で表示されています）ポアまたは膜に損傷が起きてしまった。 気泡がフローセルに混入した。 フローセルのプライミングやライブラリーのロードで気泡が入ると、ポアに不可逆的なダメージを与える可能性があります。 推奨の操作方法については、Priming and loading your flow cell のビデオをご覧ください。
利用できないポアの割合が多い場合 サンプルDNAに含まれる不純物 既知の化合物問題で、サンプルDNAに多糖類が含まれた事で、植物のゲノムDNAと結合しポアをブロックした。

1. 植物葉DNA抽出法 Plant leaf DNA extraction methodをご参照ください。
2. QIAGEN PowerClean Pro キットを使用してクリーンアップして下さい。
3. QIAGEN REPLI-g kit.キットを使用して、元のgDNAサンプルで全ゲノム増幅を実行します。
利用できないポアの割合が多い場合 サンプル内に不純物が含まれている 不純物の影響は、 Contaminants の ノウハウを参照して下さい。 サンプルDNAに不純物を残留させないために別の抽出方法をお試しください。

温度変動

問題点 予想される原因 解決策とコメント
温度変動 フローセルとデバイスの接続が途切れている。 フローセルの背面にある金属プレートを覆っているヒートパッドがあることを確認してください。 フローセルを再度挿入し、コネクターピンがデバイスにしっかりと接触していることを確認するために軽く押してください。問題が解決しない場合は、テクニカルサービスにご連絡してください。

目標温度に到達しない場合

問題点 予想される原因 解決策とコメント
MinKNOWが "Failed to reach target temperature "(目標温度に達しなかった)と表示する。" 装置が通常の室温より低い場所、または風通しの悪い場所（排気が出来ない場所）に置かれた時にフローセルが過熱してします。 MinKNOWでは、フローセルが目標温度に到達するまでの既定の時間枠があります。時間枠を超えると、エラーメッセージが表示され、シークエンシング実験が続行されます。しかし、不適切な温度でシークエンスを行うと、スループットが低下し、qスコアが低下する可能性があります。シークエンシングデバイスが風通しの良い室温に置かれていることを確認して、MinKNOW再スタートしてください。MinION Mk 1Bの温度制御の詳細については、FAQ を参照してください。

Last updated: 1/8/2025

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