cDNA-PCR Sequencing V14 - Barcoding (SQK-PCB114.24)

Oxford Nanopore Technologies
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PromethION: Protocol

cDNA-PCR Sequencing V14 - Barcoding (SQK-PCB114.24) V PCB_9201_v114_revD_06Dec2023

The fastest and simplest protocol for full-length cDNA sequencing

  • Offering highest yield
  • Higher yields than traditional cDNA synthesis
  • Splice variants and fusion transcripts
  • Multiplex up to 24 different samples
  • Compatible with R10.4.1 flow cells only

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

The fastest and simplest protocol for full-length cDNA sequencing

  • Offering highest yield
  • Higher yields than traditional cDNA synthesis
  • Splice variants and fusion transcripts
  • Multiplex up to 24 different samples
  • Compatible with R10.4.1 flow cells only

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: PCB_9201_v114_revD_06Dec2023

1. Overview of the protocol

重要

本试剂盒为早期试用产品

如需有关早期试用计划的更多信息，请参阅 本文了解产品的不同发布阶段。

请确保您始终使用最新版本的实验指南。

Introduction to the cDNA-PCR Barcoding Kit 24 V14 protocol

This protocol describes how to carry out sequencing of multiple cDNA samples using a strand-switching method and the cDNA-PCR Barcoding Kit 24 V14 (SQK-PCB114.24). There are 24 unique barcodes available, allowing the user to pool up to 24 different samples in one sequencing experiment. During the strand-switching step, a UMI is incorporated, before the double-stranded cDNA is amplified by PCR using primers containing 5' tags. The amplified and barcoded samples are then pooled together and the Rapid Sequencing Adapters are added to the pooled mix.

A control experiment can be completed first using RNA Control Sample (RCS) from the RNA Control Expansion (EXP-RCS001) as your input to troubleshoot your library preparation or to become familiar with the protocol.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

  • Extract your RNA, and check its length, quantity and purity using the Input DNA/RNA QC protocol. The quality checks performed during the protocol are essential in ensuring experimental success
  • Ensure you have your sequencing kit, the correct equipment and third-party reagents
  • Download the software for acquiring and analysing your data
  • Check your flow cell to ensure it has enough pores for a good sequencing run

Library preparation

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

Library preparation step Process Time Stop option
Reverse transcription and strand-switching Prepare full-length cDNA from Poly(A)+ RNA (or total RNA) with the incorporation of the UMI 170 minutes -20°C overnight
Selecting for full-length transcripts by PCR Amplify the cDNA by PCR using rapid attachment barcode primers during the PCR step 40 minutes 4°C short-term storage or for repeated use, such as re-loading your flow cell.
-80°C for single-use long-term storage.
Adapter ligation Attach the sequencing adapters to the to the PCR products. 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 cDNA library for sequencing 5 minutes

PCB114.24 workflow

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
  • Optional: Start the EPI2ME software and select a workflow for further analysis
重要

Compatibilities of the protocol

This protocol should only be used in combination with:

  • cDNA-PCR Barcoding Kit 24 V14 (SQK-PCB114.24)
  • R10.4.1 flow cells (FLO-PRO114M)
  • Flow Cell Wash Kit (EXP-WSH004)
  • RNA Control Expansion (EXP-RCS001)
  • Rapid Adapter Auxiliary V14 (EXP-RAA114)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)
  • Flow Cell Priming Kit V14 (EXP-FLP004)
  • PromethION 24/48 - PromethION IT Requirements document
  • PromethION 2 Solo - PromethION 2 Solo IT requirements document

2. Equipment and consumables

材料
  • 10 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 500 ng total RNA per sample
  • cDNA-PCR Barcoding Kit 24 V14 (SQK-PCB114.24)
耗材
  • PromethION 测序芯片
  • NEBNext®快速连接反应缓冲液（NEB，B6058）
  • T4 DNA Ligase 2M U/ml (NEB, M0202M)
  • RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
  • Lambda Exonuclease (NEB, Cat # M0262L)
  • Thermolabile Exonuclease I (NEB, cat # M0568)
  • USER (Uracil-Specific Excision Reagent) Enzyme (NEB, cat # M5505L)
  • 10 mM dNTP solution (e.g. NEB N0447)
  • Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0752)
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • Agencourt RNAClean XP磁珠（Beckman Coulter™，A63987）
  • Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（ThermoFisher，Q32851）
  • Qubit RNA HS Assay（RNA高灵敏度检测）试剂盒（ThermoFisher，Q32852）
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Qubit™ 分析管（Invitrogen, Q32856）
  • 0.2 ml thin-walled PCR tubes
仪器
  • PromethION 测序设备
  • PromethION 测序芯片遮光片
  • Hula混匀仪（低速旋转式混匀仪）
  • 适用于1.5ml Eppendorf 离心管的磁力架
  • 迷你离心机
  • 涡旋混匀仪
  • 热循环仪
  • Qubit荧光计（或用于质控检测的等效仪器）
  • Agilent Bioanalyzer (or equivalent)
  • P1000 移液枪和枪头
  • P200 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
  • P2移液枪和枪头
  • 盛有冰的冰桶
  • 计时器

For this protocol, you will need 10 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 500 ng total RNA per sample.

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.

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。

cDNA-PCR Barcoding Kit 24 V14 (SQK-PCB114.24) contents

SQK PCB114.24 Kit content

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Strand Switching Primer II SSPII Violet 1 350
RT Primer RTP Yellow 1 200
cDNA RT Adapter CRTA Amber 1 200
Annealing Buffer AB Orange 1 200
Rapid Adapter RA Green 1 15
Adapter Buffer ADB Clear 1 100
Elution Buffer EB Black 2 500
Short Fragment Buffer SFB Clear 4 7,500
Sequencing Buffer SB Red 1 700
Library Beads LIB Pink 1 600
Library Solution LIS White cap, pink label 1 600
Barcode Primers 1-24 BP01-24 White 24 10
Flow Cell Tether FCT Purple 1 200
Flow Cell Flush FCF Clear cap, light blue label 1 8,000

3. Reverse transcription and strand-switching

材料
  • 10 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 500 ng total RNA per sample
  • cDNA RT Adapter (CRTA)
  • Annealing Buffer (AB)
  • Short Fragment Buffer (SFB)
  • RT Primer (RTP)
  • Strand Switching Primer II (SSPII)
耗材
  • NEBNext®快速连接反应缓冲液（NEB，B6058）
  • T4 DNA Ligase 2M U/ml (NEB, M0202M)
  • Lambda Exonuclease (NEB, Cat # M0262L)
  • USER (Uracil-Specific Excision Reagent) Enzyme (NEB, cat # M5505L)
  • Agencourt RNAClean XP磁珠（Beckman Coulter™，A63987）
  • 10 mM dNTP solution (e.g. NEB cat # N0447)
  • Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0752)
  • RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
  • Qubit RNA HS Assay（RNA高灵敏度检测）试剂盒（ThermoFisher，Q32852）
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • 1.5 ml Eppendorf DNA LoBind离心管
  • Qubit™ 分析管（Invitrogen, Q32856）
  • 0.2 ml thin-walled PCR tubes
仪器
  • 迷你离心机
  • 热循环仪
  • 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 移液枪和枪头
  • P20 移液枪和枪头
  • P10 移液枪和枪头
  • P2移液枪和枪头
CHECKPOINT

测序芯片质检

我们强烈建议您在开始文库制备前，对测序芯片的活性纳米孔数进行质检，以确保测序实验顺利运行。

详情请参阅 MinKNOW 实验指南中的 测序芯片质检说明。

提示

Preparing the laboratory for handling RNA samples:

For optimal results, we recommend preparing your laboratory space and equipment prior to handling RNA to ensure the presence of RNAse and contaminants is minimal:

  • Clean the lab bench space where you will carry out the work with RNaZap and tech wipes.
  • Clean all equipment such as pippettes, tube racks, centrifuge and vortex with RNaZap and tech wipes.
  • Use fresh tip boxes and reagents to minimise risk of contamination.

Thaw the following reagents, then spin down briefly using a microfuge and mix as indicated in the table below. Then place the reagents on ice.

Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting
cDNA RT Adapter (CRTA) ✓ ✓ ✓
Annealing Buffer (AB) ✓ ✓ ✓
Short Fragment Buffer (SFB) ✓ ✓ ✓
RT Primer (RTP) ✓ ✓ ✓
Strand Switching Primer II (SSPII) ✓ ✓ ✓
NEBNext® Quick Ligation Reaction Buffer ✓ ✓ Mix by vortexing
T4 DNA Ligase 2M U/ml Not frozen ✓ ✓
RNaseOUT Not frozen ✓ ✓
Lambda Exonuclease Not frozen ✓ ✓
Uracil-Specific Excision Reagent (USER) Not frozen ✓ ✓
10 mM dNTP solution ✓ ✓ ✓
Maxima H Minus Reverse Transcriptase Not frozen ✓ ✓
Maxima H Minus 5x RT Buffer ✓ ✓ Mix by vortexing
重要

It is important that the NEBNext Quick Ligation Reaction Buffer is mixed well by vortexing.

Check for any visible precipitate; vortexing for at least 30 seconds may be required to solubilise all precipitate.

可选操作

To run a control experiment, replace your sample input with 10 μl diluted RNA Control Sample (RCS) from the RNA Control Expansion (EXP-RCS001) as follows:


  • Thaw the RNA Control Sample (RCS) at room temperature, briefly spin down and mix well by pipetting.
  • Dilute the RNA Control Sample (RCS) in a 1.5 ml Eppendorf DNA LoBind tube as follows:

Reagent Volume
RNA Control Sample (RCS) 1 μl
Nuclease-free water 14 μl
Total 15 μl

Note: This will provide enough volume for 1 sample, adjust your volumes accordingly for the number of samples you wish to run in your control experiment.

  • Mix thoroughly by pipetting 10-20 times and briefly spin down.
  • Use the 10 μl of diluted RNA Control Sample (RCS) as your RNA input.

For each sample, prepare the RNA in nuclease-free water:

  • Transfer 10 ng Poly(A)+ RNA, or 500 ng total RNA into a 0.2 ml thin-walled PCR tube
  • Adjust the volume to up to 10 μl with nuclease-free water
  • Mix by flicking the tube to avoid unwanted shearing
  • Spin down briefly in a microfuge

Prepare the following in a 0.2 ml PCR tube per sample:

Reagent Volume
RNA 10 µl
cDNA RT Adapter (CRTA) 1 µl
Annealing Buffer (AB) 1 µl
Total volume 12 µl
提示

The cDNA RT Adapter (CRTA) is a double stranded adapter with a poly(T) overhang which anneals to the very end of the poly(A) tail of the RNA strand. This ensures that the full length of the RNA is reverse transcribed and that the poly(A) length can be estimated accurately. Annealing Buffer (AB) has been included to improve CRTA ligation.

Mix gently by flicking the tubes, and spin down.

Incubate the reactions in the thermal cycler at 60°C for 5 mins, then cool for 5 minutes at room temperature.

To each of the 0.2 ml PCR tubes containing you RNA sample(s), add the following:

Reaction Volume
RNA sample (from previous step) 12 µl
NEBNext® Quick Ligation Reaction Buffer 3.6 µl
T4 DNA Ligase 2M U/ml 1.4 µl
RNaseOUT 1 µl
Total volume (including all reagents) 18 µl

Ensure the components are thoroughly mixed by pipetting the contents of the tubes 10 times and spin down.

Note: Mix gently to minimise introducing air bubbles to the reactions.

Incubate for 10 minutes at room temperature.

To each of the 0.2 ml PCR tubes, add the following:

Reagent Volume
RNA sample (from previous step) 18 µl
Lambda Exonuclease 1 µl
USER (Uracil-Specific Excision Reagent) 1 µl
Total volume (including all reagents) 20 µl
提示

The Lambda Exonuclease and Uracil-Specific Excision Reagent (USER) are third-party reagents used in the preparation of the reverse transcription step. Lambda Exonuclease and USER digest the bottom strand of the ligated CRTA so that the RT Primer (RTP) can bind the CRTA sequence as a primer for the reverse transcription of the RNA.

Ensure the components are thoroughly mixed by flicking the tubes and spin down.

Incubate for 5 minutes at 37°C in the thermal cycler.

Transfer each sample to clean 1.5 ml Eppendorf DNA LoBind tubes.

Resuspend the RNase-free XP beads by vortexing.

Add 36 µl of resuspended RNase-free XP beads to each reaction and mix gently by flicking the tubes.

将离心管置于Hula混匀仪（低速旋转式混匀仪）上室温孵育5分钟。

Spin down the samples and pellet on a magnet. Keep the tubes on the magnet, and pipette off the supernatant.

Keep the tubes on the magnet and wash the beads with 100 µl of Short Fragment Buffer (SFB) as follows:

  1. Wash the beads with 100 µl of Short Fragment Buffer (SFB).
  2. Keeping the magnetic rack on the benchtop, rotate the tube by 180°. Wait for the beads to migrate towards the magnet and to form a pellet.
  3. Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet again.
  4. Without disturbing the pellet, remove the Short Fragment Buffer (SFB) using a pipette and discard.

Repeat the previous step.

Spin down and place the tubes back on the magnet. Pipette off any residual buffer. Briefly allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspend each pellet in 12 µl of nuclease-free water.

Incubate at room temperature for 10 minutes.

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

Remove and retain 12 µl of eluate into a clean 0.2 ml thin-walled PCR tube per sample.

To each of the 0.2 ml PCR tubes, add the following:

Reagents Volume
Eluted sample (from previous step) 12 µl
RT Primer (RTP) 1 µl
dNTPs (10 mM) 1 µl
Total volume (including all reagents) 14 µl
提示

RT Primer (RTP) is a single stranded primer and binds upstream of the poly(A) tail of the RNA transcript to prime for reverse transcription.

Ensure the components are thoroughly mixed by flicking the tubes and spin down.

Incubate the reaction for 5 minutes at room temperature.

To each of the 0.2 ml PCR tubes, add the following:

Reagents Volume
RT primed sample (from previous step) 14 µl
Maxima H Minus 5x RT Buffer 4.5 µl
RNaseOUT 1 µl
Strand Switching Primer II (SSPII) 2 µl
Total (including all reagents) 21.5 µl
提示

Strand Switching Primer II (SSPII) base pairs to the deoxycytidine present at the 5' end of the first cDNA strand synthesised. This allows the reverse transcriptase to "strand-switch" for synthesis of the second cDNA strand.

Mix gently by flicking the tubes, and spin down.

Incubate at 42°C for 2 minutes in the thermal cycler.

Add 1 µl of Maxima H Minus Reverse Transcriptase to each tube. The total volume will be 22.5 µl per tube.

Mix gently by flicking the tubes, and spin down.

Incubate using the following protocol using a thermal cycler:

Cycle step Temperature Time No. of cycles
Reverse transcription and strand-switching 42°C 30 mins 1
Heat inactivation 85°C 5 mins 1
Hold 4°C ∞
步骤结束

Take your samples forward into the next step. However, at this point it is also possible to store the sample at -20°C overnight.

4. Selecting for full-length transcripts by PCR

材料
  • Barcode Primers (BP01-24)
  • Elution Buffer (EB)
耗材
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • Thermolabile Exonuclease I (NEB, cat # M0568)
  • Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
  • Freshly prepared 70% ethanol in nuclease-free water
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • Qubit™ 分析管（Invitrogen, Q32856）
  • 0.2 ml PCR tubes
仪器
  • 热循环仪
  • 涡旋混匀仪
  • Hula混匀仪（低速旋转式混匀仪）
  • 适用于1.5ml Eppendorf 离心管的磁力架
  • 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 移液枪和枪头
  • P10 移液枪和枪头
  • P2移液枪和枪头
  • Qubit荧光计（或用于质控检测的等效仪器）
  • Agilent Bioanalyzer (or equivalent)
重要

This kit enables multiplexing of up to 24 samples. The default method allows you to perform one 25 µl PCR reaction per sample. If multiplexing two or three samples, however, two separate PCR reactions per sample should be performed; if running just one sample, four separate PCR reactions should be performed as per the PCR-cDNA Sequencing Kit V14 (SQK-PCS114) protocol. These recommendations aim to ensure that enough PCR product is generated for optimal flow cell performance.

Reverse transcriptase is a PCR inhibitor and the reverse-transcribed sample must be diluted enough for PCR to take place.

Note: Use one set of Barcode Primers per sample.

Thaw the following reagents, then spin down briefly using a microfuge and mix as indicated in the table below. Then place the reagents on ice.

Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting
Barcode Primers (BP01 - BP24) ✓ ✓ ✓
Elution Buffer (EB) ✓ ✓ ✓
LongAmp Hot Start Taq 2X Master Mix ✓ ✓ ✓
Thermolabile Exonuclease I Not frozen ✓ ✓

Spin down the reverse-transcribed RNA samples.

Prepare a separate 0.2 ml PCR tube for each sample and add 5 μl of reverse-transcribed RNA per tube.

重要

Only 5 µl of the reverse-transcribed sample is to be taken forward. Do NOT use all the 22.5 µl of the reverse transcription reaction in a single PCR reaction.

In each of the 0.2 ml PCR tubes containing reverse-transcribed RNA sample, prepare the following reaction at room temperature:

Reagent Volume
Reverse-transcribed sample (from previous step) 5 μl
Unique Barcode Primer (BP01-24) 0.75 μl
Nuclease-free water 6.75 μl
2x LongAmp Hot Start Taq Master Mix 12.5 μl
Total (including all reagents) 25 μl

Mix gently by pipetting.

Amplify using the following cycling conditions.

Cycle step Temperature Time No. of cycles
Initial denaturation 95°C 30 secs 1
Denaturation 95°C 15 secs 10-18*
Annealing 62°C 15 secs 10-18*
Extension 65°C 60 secs per kb 10-18*
Final extension 65°C 6 mins 1
Hold 4°C ∞

*We recommend 14 cycles as a starting point. However, the number of cycles can be adjusted between the values shown according to experimental needs.

For further information, please read The effect of varying the number of PCR cycles in the PCR-cDNA Sequencing Kit document.

Add 1 μl Thermolabile Exonuclease I directly to each PCR tube. Mix by flicking the tube and briefly spin down.

提示

The Thermolabile Exonuclease I is added to remove any excess primers which have not successfully annealed.

Incubate the reaction at 37°C for 5 minutes, followed by 80°C for 2 minutes in the thermal cycler.

Transfer each sample to a clean 1.5 ml Eppendorf DNA LoBind tube.

Resuspend the AMPure XP beads by vortexing.

Add 18 µl of resuspended AMPure XP beads to each 1.5 ml Eppendorf DNA LoBind tube.

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

Prepare 5 ml of fresh 70% ethanol in nuclease-free water.

Spin down the samples and pellet on a magnet. Keep the tubes on the magnet, and pipette off the supernatant.

Keep the tubes on the magnet and wash the beads with 100 µl of freshly-prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

重复上述步骤。

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

Remove the tubes from the magnetic rack and resuspend each pellet in 12 µl of Elution Buffer (EB).

Incubate at room temperature for 10 minutes.

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

Remove and retain 12 µl of each eluate into a separate clean 1.5 ml Eppendorf DNA LoBind tube.

  • Remove and retain the eluate which contains the cDNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
  • Dispose of the pelleted beads

For each sample, analyse 1 µl of the amplified cDNA for size, quantity and quality using a Qubit fluorometer and Agilent Bioanalyzer (or equivalent) for a QC check.

重要

Sometimes a high-molecular weight product is visible in the wells of the gel when the PCR products are run, instead of the expected smear. These libraries are typically associated with poor sequencing performance. We have found that repeating the PCR with fewer cycles can remedy this.

Pool together equimolar samples of the amplified cDNA barcoded samples to a total of 50 fmols and make the volume up to 31 µl in Elution Buffer (EB).

Mass Molarity if fragment length = 0.5 kb Molarity if fragment length = 1.5 kb Molarity if fragment length = 3 kb
5 ng 16 fmol 5 fmol 3 fmol
10 ng 32 fmol 11 fmol 5 fmol
15 ng 49 fmol 16 fmol 8 fmol
20 ng 65 fmol 22 fmol 11 fmol
25 ng 81 fmol 27 fmol 13 fmol
50 ng 154 fmol 51 fmol 26 fmol
100 ng 324 fmol 108 fmol 54 fmol

If the quantity of amplified cDNA is above 50 fmol, the remaining cDNA can be frozen and stored for another sequencing experiment (in this case, library preparation would start from the Adapter Addition step). We recommend avoiding multiple freeze-thaw cycles to prevent DNA degradation.

提示

文库保存建议

若为 短期 保存或重复使用（例如在清洗芯片后再次上样），我们建议将文库置于Eppendorf LoBind 离心管中 4℃ 保存。 若为一次性使用且储存时长 __超过3个月__，我们建议将文库置于Eppendorf LoBind 离心管中 -80℃ 保存。

5. Adapter addition

材料
  • 快速测序文库接头（RA）
  • Adapter Buffer (ADB)
  • Elution Buffer (EB)
耗材
  • 1.5 ml Eppendorf DNA LoBind tubes
仪器
  • 迷你离心机
  • 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 移液枪和枪头
  • P10 移液枪和枪头
  • P2移液枪和枪头
重要

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

Thaw the kit components at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated by the table below:

Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting
Rapid Adapter (RA) Not frozen ✓ ✓
Adapter Buffer (ADB) Not frozen ✓ ✓

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

Reagents 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 amplified cDNA library, making the total volume 32 μl.

Mix gently by flicking the tube, and spin down.

Incubate the reaction for 5 minutes at room temperature.

Spin down briefly.

步骤结束

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

6. PromethION 测序芯片的预处理及上样

材料
  • 测序芯片冲洗液（FCF）
  • 测序芯片系绳（FCT）
  • 文库溶液（LIS）
  • 文库颗粒（LIB）
  • 测序缓冲液（SB）
耗材
  • PromethION 测序芯片
  • 1.5 ml Eppendorf DNA LoBind 离心管
仪器
  • PromethION 2 Solo 测序设备
  • PromethION测序设备
  • PromethION 测序芯片遮光片
  • P1000 移液枪和枪头
  • P200 移液枪和枪头
  • P20 移液枪和枪头
重要

本试剂盒仅兼容R10.4.1测序芯片（FLO-PRO114M）。

使用文库溶液

对大多数测序实验，我们建议您使用文库颗粒（LIB）给测序芯片上样。然而，对于粘稠的文库，借助文库颗粒上样可能会比较困难，此时使用文库溶液（LIS）可能更为合适。

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。

按下表制备测序芯片的预处理液，再于室温下短暂涡旋振荡混匀。

试剂 体积（每张芯片）
测序芯片冲洗液 (FCF) 1170 µl
测序芯片系绳 (FCT) 30 µl
总体积 1200 µl
重要

将芯片从冰箱中取出后，请将其置于室温环境孵育20分钟再插入PromethION测序仪。潮湿环境下的测序芯片上可能会形成冷凝水。因此，请检查测序芯片顶部和底部的金色连接器引脚处是否有水凝结。如有，请使用无纤维布擦干。请确保测序芯片底部有热垫（黑色）覆盖。

对 PromethION 2 Solo，请按以下步骤为测序芯片上样：

  1. 将测序芯片平放在金属板上。

  2. 将测序芯片推入对接端口，直至金色引脚或绿色电路板不可见。

J2068 FC-into-P2-animation V5

对PromethION 24/48，将测序芯片插入相应卡槽的对接端口：

  1. 将测序芯片与连接器横竖对齐，以便顺利卡入。

  2. 用力下压芯片至卡槽，并确认卡夹位置归位。

Prom Flowcell Loading 1a 中文

Prom Flowcell Loading 1b 中文

重要

如插入配置测试芯片的角度出现偏差，可能会损坏PromethION上的引脚并影响测序结果。如您发现 PromethION测序仪芯片位置上的引脚损坏，请通过电子邮件（support@nanoporetech.com）或微信公众号在线支持（NanoporeSupport）联系我们的技术支持团队。

Screenshot 2021-04-08 at 12.08.37

顺时针滑动加液孔孔盖，将其打开。

Prom Flowcell Loading 2 中文

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。

在加液孔打开的状态下，按下述步骤吸取少量液体，同时避免引入气泡：

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入加液孔中。
  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。

Prom Flowcell Loading 3 中文

使用P1000移液枪向芯片的加液孔中加入500 µl芯片预处理溶液。加入过程中，请避免引入气泡。等待5分钟，与此同时，您可按以下步骤准备上样文库。

Prom Flowcell Loading 4 中文

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 DNA LoBind离心管内，将所有试剂按以下顺序混合：

试剂 每张测序芯片的上样体积
测序缓冲液 (SB) 100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS) 68 µl
DNA 文库 32 µl
总体积 200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

缓慢向芯片的加液口中加入500 µl预处理液，完成芯片的预处理。

Prom Flowcell Loading 5 中文

临上样前，用移液枪轻轻吹打混匀制备好的文库。

使用 P1000 移液枪向加液孔中加入200 µl 文库。

Prom Flowcell Loading 6 中文

合上加液孔孔盖。

Prom Flowcell Loading 7 中文

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。

如遮光片不在测序芯片上，请您按照以下步骤安装：

  1. 将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
  2. 用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

Prom Flowcell Loading 8a 中文

Prom Flowcell Loading 8b 中文

步骤结束

准备就绪后，合上PromethION设备上盖。

请在为PromethION芯片上样后，等待10分钟再启动实验，以提高芯片产出。

7. Data acquisition and basecalling

如何开始测序

在完成测序芯片的加样后，您即可在MinKNOW中启动测序实验。MinKNOW 软件负责仪器控制、数据采集以及实时碱基识别。有关设置和使用 MinKNOW 的详细信息，请参阅MinKNOW 实验指南。

您可以通过多种方式使用并设置MinKNOW：

  • 在直接或远程连接到测序设备的计算机上。
  • 直接在 GridION、MinION Mk1C 或 PromethION 24/48 测序设备上。

有关在测序设备上使用 MinKNOW 的更多信息，请参阅相应设备的用户手册：

在MinKNOW中启动测序：

1. 在 "开始 "（Start）页面上，选择 __开始测序__（Start Sequencing)。 start

2. 输入实验详情：例如实验名称，测序芯片位置及样本ID。 Grid start seq

3. 在"试剂盒"页面上，选择建库试剂盒。 kit selection

4. 配置测序实验参数，或保持“运行选项”和“分析”页面中的默认设置。

请注意： 如果在设置运行参数时关闭了碱基识别，您可在实验结束后，在MinKNOW中运行线下碱基识别。详情请参阅MinKNOW实验指南。

5. 在“输出”页面中，设置输出参数或保持默认设置。 step5c

6. 单击 "参数确认" 页面上的 开始 启动测序。 Step6

测序后数据分析

当于MinKNOW上完成测序后，您可按照“测序芯片的重复利用及回收”一节中的说明重复使用或返还测序芯片。

完成测序和碱基识别后，即可进行数据分析。有关碱基识别和后续分析选项的详细信息，请参阅数据分析文档。

在下游分析部分，我们将概述更多用于数据分析的选项。

8. 结束实验

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2-8℃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。

请按照“回收程序”清洗好芯片，以便送回Oxford Nanopore。

您可在 此处找到回收测序芯片的说明。

请注意： 在将测序芯片寄回之前，请使用去离子水对每张芯片进行冲洗。

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。

9. Downstream analysis

下游分析

您可以选择以下几个途径来进一步分析经过碱基识别的数据：

1. EPI2ME 工作流程

Oxford Nanopore Technologies通过EPI2ME Labs平台提供了一系列针对高阶数据分析的生物信息学教程和工作流程。上述资源汇总于纳米孔社区的 EPI2ME Labs 板块。该平台通过描述性文字、生物信息学代码和示例数据，具象化地展示出我们的研究和应用团队发布在 GitHub 上的工作流程。

2. 科研分析工具

Oxford Nanopore Technologies的研发部门开发了许多分析工具，您可在Oxford Nanopore的 GitHub 资料库中找到。这些工具面向有一定经验的用户，并包含如何安装和运行软件的说明。工具以源代码形式提供，因此我们仅提供有限的技术支持。

3. 纳米孔社区用户开发的分析工具

如果以上工具仍无法为您提供解决研究问题的分析方法，请参考资源中心的生物信息学板块。该板块汇总了许多由纳米孔社区成员开发、且在Github上开源的、针对纳米孔数据的生信分析工具。请注意，Oxford Nanopore Technologies不为这些工具提供支持，也不能保证它们与测序所用的最新的化学试剂/软件配置兼容。

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. Issues during the sequencing run

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。

Pore occupancy below 40%

Observation Possible cause Comments and actions
Pore occupancy <40% Not enough library was loaded on the flow cell Ensure you load the recommended amount of good quality library in the relevant library prep protocol onto your flow cell. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol"
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and sequencing adapters did not ligate to the DNA Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford Nanopore Technologies Ligation Buffer (LNB, provided in the sequencing kit) at the sequencing adapter ligation step, and use the correct amount of each reagent. A Lambda control library can be prepared to test the integrity of the third-party reagents.
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and ethanol was used instead of LFB or SFB at the wash step after sequencing adapter ligation Ethanol can denature the motor protein on the sequencing adapters. Make sure the LFB or SFB buffer was used after ligation of sequencing adapters.
Pore occupancy close to 0 No tether on the flow cell Tethers are adding during flow cell priming (FLT/FCT tube). Make sure FLT/FCT was added to FB/FCF before priming.

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。

运行过程中过孔速度和数据质量（Q值）降低

现象 可能原因 措施及备注
运行过程中过孔速度和数据质量（Q值）降低 对试剂盒9系列试剂（如SQK-LSK109），当测序芯片的上样量过多时（请参阅相应实验指南获取推荐文库用量），能量消耗通常会加快。 请按照MinKNOW 实验指南中的说明为测序芯片补充能量。请在后续实验中减少测序芯片的上样量。

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 MK1B温度控制的更多信息，请参考此 FAQ （常见问题）文档。

Guppy – no input .fast5 was found or basecalled

Observation Possible cause Comments and actions
No input .fast5 was found or basecalled input_path did not point to the .fast5 file location The --input_path has to be followed by the full file path to the .fast5 files to be basecalled, and the location has to be accessible either locally or remotely through SSH.
No input .fast5 was found or basecalled The .fast5 files were in a subfolder at the input_path location To allow Guppy to look into subfolders, add the --recursive flag to the command

Guppy – no Pass or Fail folders were generated after basecalling

Observation Possible cause Comments and actions
No Pass or Fail folders were generated after basecalling The --qscore_filtering flag was not included in the command The --qscore_filtering flag enables filtering of reads into Pass and Fail folders inside the output folder, based on their strand q-score. When performing live basecalling in MinKNOW, a q-score of 7 (corresponding to a basecall accuracy of ~80%) is used to separate reads into Pass and Fail folders.

Guppy – unusually slow processing on a GPU computer

Observation Possible cause Comments and actions
Unusually slow processing on a GPU computer The --device flag wasn't included in the command The --device flag specifies a GPU device to use for accelerate basecalling. If not included in the command, GPU will not be used. GPUs are counted from zero. An example is --device cuda:0 cuda:1, when 2 GPUs are specified to use by the Guppy command.

Last updated: 4/19/2024

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