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

概览

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)
耗材
  • MinION 和 GridION测序芯片
  • 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)
  • （非必需）牛血清白蛋白（BSA）（50 mg/mL）（例如 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（双链DNA高灵敏度检测）试剂盒（ThermoFisher，Q32851）
  • Qubit™ 分析管（Invitrogen, Q32856）
仪器
  • MinION 或 GridION 测序仪
  • MinION 及GridION 测序芯片遮光片
  • Centrifuge e.g. Eppendorf max speed 15,000rpm (Fisher, 15881635)
  • 迷你离心机
  • 计时器
  • 涡旋混匀仪
  • Thermomixer set at 37°C
  • Thermomixer set at 65°C
  • 磁力架
  • Bead-beater (e.g. FastPrep-24™ Classic bead beating grinder and lysis system)
  • 热循环仪
  • 96-well PCR block
  • Ice bucket with ice
  • Hula混匀仪（低速旋转式混匀仪）
  • Qubit荧光计 （或用于质控检测的等效仪器）
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 移液枪和枪头
  • P2 移液枪和枪头
可选仪器
  • 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

起始DNA (1)

DNA质控

选择符合质量和浓度要求的起始DNA至关重要的。使用过少或过多的DNA，或者质量较差的DNA（如，高度碎片化、含有RNA或化学污染物的DNA）都会影响文库制备。

有关如何对DNA样品进行质控，请参考起始DNA/RNA质控实验指南 。

化学污染物

从原始样本中提取DNA的方法不同，可能会导致经纯化的DNA中所残留的化学污染物不同。这会影响文库的制备效率和测序质量。请在牛津纳米孔社区的 Contaminants（污染物）页面 了解更多信息。

可选操作

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.

第三方试剂

Oxford Nanopore Technologies推荐您使用本实验指南中提及的所有第三方试剂，并已对其加以验证。我们尚未对其它替代试剂进行测试。

我们建议您按制造商说明准备待用的第三方试剂.

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.

提示

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

测序芯片质检

我们强烈建议您在开始测序实验前，对测序芯片的活性纳米孔数进行质检。质检需在您收到MinION /GridION /PremethION测序芯片12周之内进行，或者在您收到Flongle测序芯片四周内进行。Oxford Nanopore Technologies会对活性孔数量少于以下标准的芯片进行替换** ：

测序芯片 芯片上的活性孔数确保不少于
Flongle 测序芯片 50
MinION/GridION 测序芯片 800
PromethION 测序芯片 5000

** 请注意：自收到之日起，芯片须一直贮存于Oxford Nanopore Technologies推荐的条件下。且质检结果须在质检后的两天内递交给我们。请您按照 测序芯片质检文档中的说明进行芯片质检。

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)
耗材
  • 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
仪器
  • Centrifuge e.g. Eppendorf max speed 15,000rpm (Fisher, 15881635)
  • 迷你离心机
  • 计时器
  • 涡旋混匀仪
  • 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移液枪和枪头
  • P200 移液枪和枪头
  • P20 pipette and tips
  • P10 移液枪和枪头
可选仪器
  • 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.
可选操作

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.

提示

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
步骤结束

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)
耗材
  • 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 离心管
  • 0.2 ml thin-walled PCR tubes
仪器
  • Hula混匀仪（低速旋转式混匀仪）
  • 迷你离心机
  • 热循环仪
  • 96-well PCR block
  • Timer
  • 盛有冰的冰桶
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P100 移液枪和枪头
  • P20 pipette and tips
  • P10 移液枪和枪头
  • P2 移液枪和枪头
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.

步骤结束

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

5. Clean-up, quantification and adapter attachment

材料
  • AMPure XP 磁珠（AXP）
  • Elution Buffer (EB)
  • 快速测序文库接头（RA）
  • 接头缓冲液（ADB）
耗材
  • Thermolabile Exonuclease I (NEB, M0568)
  • 新制备的80%乙醇（用无核酸酶水配制）
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml thin-walled PCR tubes
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（ThermoFisher，Q32851）
  • Qubit™ 分析管（Invitrogen, Q32856）
仪器
  • 热循环仪
  • Hula混匀仪（低速旋转式混匀仪）
  • 磁力架
  • Qubit荧光计 （或用于质控检测的等效仪器）
  • Timer
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 移液枪和枪头
  • P2 移液枪和枪头

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
可选操作

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.

在一支1.5ml Eppendorf DNA LoBind离心管内，按下表稀释快速测序文库接头（RA），并吹打混匀：

试剂 体积
快速测序文库接头（RA） 1.5 μl
接头缓冲液（ADB） 3.5 μl
总体积 5 μl

向11 μl带条形码的DNA洗脱液中加入1 μl 经过稀释的快速测序文库接头（RA）。

Mix gently by flicking the tube, and spin down.

Incubate the reaction for 5 minutes at room temperature.

步骤结束

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. MinION 及 GridION 测序芯片的预处理及上样 (1)

材料
  • 测序芯片冲洗液（FCF）
  • 测序芯片系绳（FCT）
  • 文库颗粒（LIB）
  • 测序缓冲液（SB）
耗材
  • MinION及GridION测序芯片
  • （非必需）牛血清白蛋白（BSA）（50 mg/mL）（例如 Invitrogen™ UltraPure™ BSA 50 mg/mL， AM2616）
  • 1.5 ml Eppendorf DNA LoBind 离心管
仪器
  • MinION 或 GridION 测序仪
  • MinION 及GridION 测序芯片遮光片
  • P1000 移液枪和枪头
  • P100 移液枪和枪头
  • P20 移液枪和枪头
  • P10 移液枪和枪头
重要

请注意：本试剂盒仅兼容R10.4.1测序芯片（FLO-MIN114）。

提示

测序芯片的预处理及上样

我们建议所有新用户在首次运行测序芯片前，观看视频测序芯片的预处理及上样。

于室温下解冻测序缓冲液（SB）、文库颗粒（LIB）或文库溶液（LIS）、测序芯片系绳（FCT）和一管测序芯片冲洗液（FCF）。完全解冻后，涡旋振荡混匀，然后瞬时离心并置于冰上。 (1)

重要

为在MinION及GridION R10.4.1测序芯片（FLO-MIN114）上获得最优的测序表现并提高测序产出，我们推荐您向测序芯片预处理液中加入终浓度为0.2 mg/ml的牛血清白蛋白（BSA）。

请注意： 我们不推荐使用其它类型的白蛋白（例如重组人血清白蛋白）。

在一支洁净的 1.5 ml Eppendorf DNA LoBind离心管中，按下表制备测序芯片的预处理液，室温下颠倒离心管并吹打混匀：

试剂 体积（每张芯片）
测序芯片冲洗液 (FCF) 1170 µl
50mg/ml的牛血清白蛋白 (BSA) 5 µl
测序芯片系绳 (FCT) 30 µl
总体积 1205 µl

打开MinION或GridION测序仪的盖子，将测序芯片插入金属固定夹的下方。用力向下按压芯片，以确保正确的热、电接触。

中文-测序芯片预处理上样1a

中文-测序芯片预处理上样1b-11Dec24

可选操作

为文库上样前，完成测序芯片检测，查看可用孔数目。

如此前已对测序芯片进行过质检，则此步骤可省略。

更多信息，请查看MinKNOW实验手册的 测序芯片质检 部分。

顺时针转动预处理孔孔盖，使预处理孔显露出来。

中文-测序芯片预处理上样2

重要

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将预处理孔打开后，检查孔周围是否有小气泡。请按照以下方法，从孔中排出少量液体以清除气泡：

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入预处理孔中。
  3. 反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。
    __请注意：__ 肉眼检查，确保从预处理孔到传感器阵列的缓冲液连续且无气泡。

中文-测序芯片预处理上样3

通过预处理孔向芯片中加入800µl预处理液，避免引入气泡。等待5分钟。在此期间，请按照以下步骤准备用于上样的DNA文库。

中文-测序芯片预处理上样4

将含有文库颗粒的LIB管用移液枪吹打混匀。

重要

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

在一支新的1.5ml Eppendorf LoBind离心管中，按下表所示准备上样文库： (1)

试剂 体积（每张测序芯片）
测序缓冲液（SB） 37.5 µl
文库颗粒（LIB），使用前即时混匀；或文库溶液（LIS） 25.5 µl
DNA文库 12 µl
总体积 75 µl

完成测序芯片的预处理：

  1. 轻轻地翻起SpotON上样孔盖，使SpotON上样孔显露出来。 中文-测序芯片预处理上样5
  2. 通过预处理孔（而 非 SpotON加样孔）向芯片中加入200µl预处理液，避免引入气泡。 中文-测序芯片预处理上样6

临上样前，用移液枪轻轻吹打混匀制备好的文库。

通过SpotON加样孔向芯片中逐滴加入75µl样品。确保液滴流入孔内后，再加下一滴。

中文-测序芯片预处理上样7

轻轻合上SpotON加样孔孔盖，确保塞头塞入加样孔内。逆时针转动预处理孔孔盖，盖上预处理孔。

中文-测序芯片预处理上样8

中文-测序芯片预处理上样9

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

按下述步骤安装测序芯片遮光片：

  1. 小心将遮光片的前沿（平端）与金属固定夹的边沿对齐。 请注意： 请勿将遮光片强行压到固定夹下方。

  2. 将遮光片轻轻盖在测序芯片上。遮光片的SpotON加样孔孔盖缺口应与芯片上的SpotON加样孔孔盖接合，遮盖住整个测序芯片的前部。

MinION加装遮光片

注意

MinION测序芯片的遮光片并非固定在测序芯片上，因此当为芯片加装遮光片后，请小心操作。

步骤结束

小心合上测序设备上盖并在MinKNOW上设置测序实验。

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.

可选操作

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. 测序芯片的重复利用及回收

材料
  • 测序芯片清洗剂盒（EXP-WSH004）

完成测序实验后，如您希望再次使用测序芯片，请按照测序芯片清洗试剂盒的说明进行操作，并将清洗后的芯片置于+2至+8℃保存。

您可在纳米孔社区获取 测序芯片清洗试剂盒实验指南。

提示

我们建议您在停止测序实验后尽快清洗测序芯片。如若无法实现，请将芯片留在测序设备上，于下一日清洗。

或者，请按照回收程序将测序芯片返还至Oxford Nanopore。

您可在此处找到回收测序芯片的说明。

重要

如果您遇到问题或对测序实验有疑问，请参阅本实验指南在线版本中的“疑难解答指南”一节。

10. DNA/RNA提取和文库制备过程中可能出现的问题

以下表格列出了常见问题，以及可能的原因和解决方法。

我们还在 Nanopore 社区的“Support”板块 提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。

低质量样本

现象 可能原因 措施及备注
低纯度DNA（Nanodrop测定的DNA吸光度比值260/280<1.8，260/230 <2.0-2.2） 用户所使用的DNA提取方法未能达到所需纯度 您可在 污染物专题技术文档 中查看污染物对后续文库制备和测序实验的影响。请尝试其它不会导致污染物残留的 提取方法。

请考虑将样品再次用磁珠纯化。
RNA完整度低（RNA完整值（RIN）<9.5，或rRNA在电泳凝胶上的条带呈弥散状） RNA在提取过程中降解 请尝试其它 RNA 提取方法。您可在 RNA完整值专题技术文档 中查看更多有关RNA完整值（RIN）的介绍。更多信息，请参阅 DNA/RNA 操作 页面。
RNA的片段长度短于预期 RNA在提取过程中降解 请尝试其它 RNA 提取方法。 您可在 RNA完整值专题技术文档中查看更多有关RNA完整值（RIN）的介绍。更多信息，请参阅DNA/RNA 操作 页面。

我们建议用户在无RNA酶污染的环境中操作，并确保实验设备没有受RNA酶污染.

经AMPure磁珠纯化后的DNA回收率低

现象 可能原因 措施及备注
低回收率 AMPure磁珠量与样品量的比例低于预期，导致DNA因未被捕获而丢失 1. AMPure磁珠的沉降速度很快。因此临加入磁珠至样品前，请确保将磁珠重悬充分混匀。

2. 当AMPure磁珠量与样品量的比值低于0.4:1时，所有的DNA片段都会在纯化过程中丢失。
低回收率 DNA片段短于预期 AMPure磁珠量与样品量的比值越低，针对短片段的筛选就越严格。每次实验时，请先使用琼脂糖凝胶（或其他凝胶电泳方法）确定起始DNA的长度，并据此计算出合适的AMPure磁珠用量。 SPRI cleanup
末端修复后的DNA回收率低 清洗步骤所用乙醇的浓度低于70% 当乙醇浓度低于70%时，DNA会从磁珠上洗脱下来。请确保使用正确浓度的乙醇。

11. 在使用快速测序试剂盒测序的过程中，可能产生的问题

以下表格列出了常见问题，以及可能的原因和解决方法。

我们还在 Nanopore 社区的“Support”板块 提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。

Mux扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数

现象 可能原因 措施及备注
MinKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数 纳米孔阵列中引入了气泡 在对通过质控的芯片进行预处理之前，请务必排出预处理孔附近的气泡。否则，气泡会进入纳米孔阵列对其造成不可逆转地损害。 视频中演示了避免引入气泡的最佳操作方法。
MinKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数 测序芯片没有正确插入测序仪 停止测序，将芯片从测序仪中取出，再重新插入测序仪内。请确保测序芯片被牢固地嵌入测序仪中，且达到目标温度。如用户使用的是GridION/PromethION测序仪，也可尝试将芯片插入仪器的其它位置进行测序。
inKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数 文库中残留的污染物对纳米孔造成损害或堵塞 在测序芯片质检阶段，我们用芯片储存缓冲液中的质控DNA分子来评估活性纳米孔的数量。而在测序开始时，我们使用DNA文库本身来评估活性纳米孔的数量。因此，活性纳米孔的数量在这两次评估中会有约10%的浮动。

如测序开始时报告的孔数明显降低，则可能是由于文库中的污染物对膜结构造成了损坏或将纳米孔堵塞。用户可能需要使用其它的DNA/RNA提取或纯化方法，以提高起始核酸的纯度。您可在 污染物专题技术文档中查看污染物对测序实验的影响。请尝试其它不会导致污染物残留的 提取方法 。

MinKNOW脚本失败

现象 可能原因 措施及备注
MinKNOW显示 "Script failed”（脚本失败）
重启计算机及MinKNOW。如问题仍未得到解决，请收集 MinKNOW 日志文件 并联系我们的技术支持。 如您没有其他可用的测序设备，我们建议您先将装有文库的测序芯片置于4°C 储存，并联系我们的技术支持团队获取进一步储存上的建议。

纳米孔利用率低于40%

现象 可能原因 措施及备注
纳米孔利用率<40% 测序芯片中的文库量不足 请确保您按照相应实验指南，向MinION Mk1B/GridION测序芯片中加入正确浓度的优质测序文库。请在上样前对文库进行定量，并使用 Promega Biomath Calculator 等工具中的“ dsDNA：µg to pmol”功能来计算DNA分子的摩尔量。
纳米孔利用率接近0 尽管您使用了快速建库测序试剂盒 V14/快速条形码测序试剂盒V14，但测序接头并未与DNA连接 请务必严格遵照实验指南的操作步骤，并确保使用正确的试剂体积和孵育温度。您可制备Lambda对照文库来检验试剂的质量和有效性。
纳米孔利用率接近0 测序芯片中无系绳 系绳（FCT管）随预处理液加入芯片。因此在制备预处理液时，请确保将FCT加入测序芯片冲洗液（FCF）中。

读长短于预期

现象 可能原因 措施及备注
读长短于预期 DNA样本降解 读长反映了起始DNA片段的长度。起始DNA在提取和文库制备过程中均有可能被打断。

1. 1. 请查阅纳米孔社区中的 提取方法 以获得最佳DNA提取方案。

2. 在进行文库制备之前，请先跑电泳，查看起始DNA片段的长度分布。DNA gel2 在上图中，样本1为高分子量DNA，而样本2为降解样本。

3. 在制备文库的过程中，请避免使用吹打或/和涡旋振荡的方式来混合试剂。轻弹或上下颠倒离心管即可。

大量纳米孔处于不可用状态

现象 可能原因 Comments and actions
大量纳米孔处于不可用状态 (在通道面板和纳米孔活动状态图上以蓝色表示)

image2022-3-25 10-43-25 上方的纳米孔活动状态图显示：状态为不可用的纳米孔的比例随着测序进程而不断增加。		 样本中含有污染物 使用MinKNOW中的“Unblocking”（疏通）功能，可对一些污染物进行清除。 如疏通成功，纳米孔的状态会变为"测序孔". 若疏通后，状态为不可用的纳米孔的比例仍然很高甚至增加：

1. 用户可使用 测序芯片冲洗试剂盒（EXP-WSH004）进行核酸酶冲洗 can be performed, 操作，或
2. 使用PCR扩增目标片段，以稀释可能导致问题的污染物。

大量纳米孔处于失活状态

现象 可能原因 措施及备注
大量纳米孔处于失活状态（在通道面板和纳米孔活动状态图上以浅蓝色表示。膜结构或纳米孔遭受不可逆转地损伤） 测序芯片中引入了气泡 在芯片预处理和文库上样过程中引入的气泡会对纳米孔带来不可逆转地损害。请观看 测序芯片的预处理及上样 视频了解最佳操作方法。
大量纳米孔处于失活/不可用状态 文库中存在与DNA共纯化的化合物 与植物基因组DNA相关的多糖通常能与DNA一同纯化出来。

1. 请参考 植物叶片DNA提取方法。
2. 使用QIAGEN PowerClean Pro试剂盒进行纯化。
3. 利用QIAGEN REPLI-g试剂盒对原始gDNA样本进行全基因组扩增。
大量纳米孔处于失活/不可用状态 样本中含有污染物 您可在 污染物专题技术文档 中查看污染物对测序实验的影响。请尝试其它不会导致污染物残留的提取方法。

温度波动

现象 可能原因 措施及备注
温度波动 测序芯片和仪器接触不良 检查芯片背面的金属板是否有热垫覆盖。重新插入测序芯片，用力向下按压，以确保芯片的连接器引脚与测序仪牢固接触。如问题仍未得到解决，请联系我们的技术支持。

未能达到目标温度

现象 可能原因 措施及备注
MinKNOW显示“未能达到目标温度” 测序仪所处环境低于标准室温，或通风不良（以致芯片过热） MinKNOW会限定测序芯片达到目标温度的时间。当超过限定时间后，系统会显示出错信息，但测序实验仍会继续。值得注意的是，在错误温度下测序可能会导致通量和数据质量（Q值）降低。请调整测序仪的摆放位置，确保其置于室温下、通风良好的环境中后，再在MinKNOW中继续实验。有关MinION温度控制的更多信息，请参考此 FAQ （常见问题）文档。

Last updated: 1/8/2025

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