Single-cell transcriptomics with cDNA prepared using 10X Genomics

Oxford Nanopore Technologies
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MinION: Protocol

Single-cell transcriptomics with cDNA prepared using 10X Genomics V SST_v9148_v111_revH_12Jan2022

Single-cell transcriptomics method:

  • Requires cDNA amplicons produced using the 10X Genomics system
  • High output
  • PCR required

For Research Use Only

This is a Legacy product This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

FOR RESEARCH USE ONLY

概览

Single-cell transcriptomics method:

  • Requires cDNA amplicons produced using the 10X Genomics system
  • High output
  • PCR required

For Research Use Only

This is a Legacy product This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

Document version: SST_v9148_v111_revH_12Jan2022

1. Overview of the protocol

重要

This is a Legacy product

This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com. For further information on please see the product update page.

PCR-cDNA Sequencing Kit features

This kit is highly recommended for users who:

  • would like to identify and quantify full-length transcripts
  • want to explore isoforms, splice variants and fusion transcripts using full-length cDNAs
  • would like to generate a large number of cDNA reads

Introduction to the single-cell transcriptomics protocol

This protocol describes how to carry out sequencing of cDNA from single cells using the PCR-cDNA Sequencing Kit (SQK-PCS111). You will need to have reverse-transcribed single cell mRNA into cDNA using the 10X Genomics Next GEM Single Cell 3' Kit (V3.1).

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

  • Have previously-prepared single-cell barcoded cDNA using the 10X Genomics Next GEM Single Cell 3' Kit (V3.1). 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

You will need to:

  • Biotin tag your cDNAs and amplify by PCR
  • Pull down the amplicons on streptavidin beads, and amplify again by PCR
  • Attach sequencing adapters to the PCR products
  • Prime the flow cell, and load your cDNA library into the flow cell

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 the data further using a pipeline of your choice
重要

Compatibility of this protocol

This protocol should only be used in combination with:

  • PCR-cDNA Sequencing Kit (SQK-PCS111)
  • R9.4.1 flow cells (FLO-MIN106)
  • Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

材料
  • 10 ng of cDNA amplicons prepared using 10X Genomics Next GEM Single Cell 3' Kits (V3.1)
  • cDNA-PCR Sequencing Kit (SQK-PCS111)
  • Custom-ordered oligo at 10 μM: [Btn]Fwd_3580_partial_read1_defined (sequence provided below)
  • Custom-ordered oligo at 10 μM: Rev_PR2_partial_TSO_defined_for_5'_cDNA (sequence provided below)
耗材
  • M280 streptavidin, 10 μg/μl (Invitrogen, 11205D)
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • 1 M Tris-HCl, pH 7.5
  • 5 M NaCl (Sigma, 71386)
  • 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • Agilent Technologies DNA 12000 Kit
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • 15 ml Falcon tubes
仪器
  • Hula混匀仪（低速旋转式混匀仪）
  • Magnetic rack (e.g. Invitrogen DynaMag-2 Magnet, Cat # 12321D)
  • 迷你离心机
  • 涡旋混匀仪
  • 热循环仪
  • 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
  • Qubit荧光计（或用于质控检测的等效仪器）
  • Agilent Bioanalyzer (or equivalent)

For this protocol, you will need 10 ng amplified cDNA amplicons prepared using 10X Genomics Next GEM Single Cell 3' Kits (V3.1).

重要

10X Genomics kits

Note: This protocol is compatible and fully supported with 10X Genomics Next GEM Single Cell 3' Kit (V3.1) and the Visium Spatial Gene Expression Kit (V1). Other versions of the kits are not supported.

The 10X Genomics Next GEM Single Cell 5' Kit (V2) is compatible with our Ligation sequencing V14 - Single-cell transcriptomics with 5' cDNA prepared using 10X Genomics on PromethION (SQK-LSK114) protocol.

起始DNA

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。

cDNA-PCR Sequencing Kit (SQK-PCS111) contents

SQK-PCS111 1

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Strand Switching Primer II SSPII Violet 1 20 µl
RT Primer RTP Yellow 1 10 µl
cDNA RT Adapter CRTA Amber 1 10 µl
Rapid Adapter T RAP T Green 1 10 µl
Annealing Buffer AB Orange 1 10 µl
cDNA Primer cPRM White cap, grey label 1 40 µl
Elution Buffer EB Black 1 500 µl
Short Fragment Buffer SFB Clear 1 1,800 µl
Sequencing Buffer II SBII Red 1 500 µl
Loading Beads II LBII Pink 1 360 µl
Loading Solution LS White cap, pink label 1 400 µl
Flush Buffer FB Blue 6 1,170 µl
Flush Tether FLT White cap, purple label 1 200 µl

Custom-ordered oligo sequences

Order the following HPLC-purified oligos at 100 μM, and dilute to 10 μM in TE buffer for use in the Pre-pull-down step of the library prep.

Name Sequence
[Btn]Fwd_3580_partial_read1_defined 5'-/5Biosg/CAGCACTTGCCTGTCGCTCTATCTTC
CTACACGACGCTCTTCCGATCT-3'
Rev_PR2_partial_TSO_defined 5'-CAGCTTTCTGTTGGTGCTGATATTGCAAGCAGTGGTA
TCAACGCAGAG-3'

3. 计算机要求及软件 (1)

MinION Mk1B的IT配置要求

请为MinION Mk1B配备一台高规格的计算机或笔记本电脑，以适配数据采集的速度。您可以在MinION Mk1B的IT配置要求文件中了解更多。

Software for nanopore sequencing

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data in real time and processes it into basecalls. You will be using MinKNOW for every sequencing experiment. MinKNOW can also demultiplex reads into folders for each barcode found in Oxford Nanopore library preparation kits, and basecall/demultiplex data after a sequencing run has completed. MinKNOW use For instructions on how to run the MinKNOW software, please refer to the relevant section in the MinKNOW protocol.

EPI2ME (optional)

The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You can the EPI2ME platform if you would like further analysis of your data post-basecalling. Please note that EPI2ME does not currently offer a workflow for single-cell transcriptomics analysis. EPI2ME installation and use For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to the EPI2ME Platform protocol.

Guppy (optional)

The Guppy command-line software can be used instead of MinKNOW for basecalling and demultiplexing reads into folders for each barcode found in Oxford Nanopore library preparation kits. You can use it if you would like to re-analyse old data, or integrate basecalling into your analysis pipeline. Guppy installation and use If you would like to use the Guppy software, please refer to the Guppy protocol.

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。

4. Pre-pull-down PCR

材料
  • 10 ng of cDNA amplicons prepared using 10X Genomics Next GEM Single Cell 3' Kits (V3.1)
  • Custom ordered-oligo at 10 μM: [Btn]Fwd_3580_partial_read1_defined (sequence provided in Equipment and Consumables)
  • Custom-ordered oligo at 10 μM: Rev_PR2_partial_TSO_defined (sequence provided in Equipment and Consumables)
耗材
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
  • Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml thin-walled PCR tubes
仪器
  • 热循环仪
  • 迷你离心机
  • Hula混匀仪（低速旋转式混匀仪）
  • 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 pipette and tips
  • P2 移液枪和枪头

Set up the following biotin tagging reaction in a 0.2 ml thin-walled PCR tube:

Reagent Volume
cDNA template 10 ng, x μl
[Btn]Fwd_3580_partial_read1_defined, 10 μM 2 μl
Rev_PR2_partial_TSO_defined, 10 μM 2 μl
Nuclease-free water 21-x μl
LongAmp Hot Start Taq 2X Master Mix 25 μl
Total 50 μl

Amplify using the following cycling conditions:

Cycle step Temperature Ramp rate Time No. of cycles
Initial denaturation 94°C max 3 min 1
Denaturation

Annealing ramp-down

Annealing

Extension		 94°C

66°C down to 58°C

58°C

65°C		 max

0.2°C/s

max

max		 30 sec

40 sec

50 sec

6 mins

4
Final extension 65°C max 10 min 1
Hold 4°C - ∞ -

Resuspend the AMPure XP beads by vortexing.

将样品转至干净的1.5 ml Eppendorf DNA LoBind 离心管中。

Add 40 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.

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。

Prepare 500 μl of fresh 70% ethanol in nuclease-free water.

将样品瞬时离心，并静置于磁力架上待磁珠和液相分离。保持离心管在磁力架上不动，用移液枪吸去清液。

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

Briefly 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 pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and incubate for 2 minutes at room temperature.

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

Remove and retain 10 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

5. Pull-down

耗材
  • 1 M Tris-HCl, pH 7.5
  • 5 M NaCl (Sigma, 71386)
  • 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
  • M280 streptavidin, 10 μg/μl (Invitrogen, 11205D)
  • 15 ml Falcon tubes
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml thin-walled PCR tubes
仪器
  • Vortex mixer
  • 迷你离心机
  • Hula混匀仪（低速旋转式混匀仪）
  • 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 pipette and tips
  • P2 移液枪和枪头

Prepare 4 ml of 2X wash/bind buffer (10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA).

Reagent Stock concentration Final concentration Volume
Tris-HCl pH 7.5 1 M 10 mM 40 μl
NaCl 5 M 2 M 1600 μl
EDTA 0.5 M 1 mM 8 μl
Nuclease-free water - - 2352 μl
Total - - 4000 μl

Transfer 3.5 ml of the 2X wash/bind buffer to a fresh 15 ml Falcon tube and add 3.5 ml of nuclease-free water to make 7 ml of 1X wash/bind buffer (5 mM Tris-HCl pH 7.5, 1 M NaCl, 0.5 mM EDTA).

Resuspend the M280 streptavidin beads (10 μg/μl) by vortexing.

Transfer 5 μl of the streptavidin beads to a clean 1.5 ml Eppendorf DNA LoBind tube.

Add 1 ml of 1X wash/bind buffer and vortex the beads with buffer for 5 seconds. Pellet the beads on a magnet for two minutes, then pipette off the supernatant.

Repeat the previous step two more times for a total of three washes.

Resuspend the beads in 10 μl of 2X wash/bind buffer to achieve a final bead concentration of 5 μg/μl.

重要

It is critical that 2X buffer is used for this step. Using 1X buffer will result in inefficient binding.

Add 10 μl of 5 μg/μl prepared beads (50 μg beads total) to the tube with 10 μl of biotinylated cDNA.

Incubate on a Hula mixer (rotator mixer) for 20 minutes at room temperature.

Add 1 ml of 1X wash/bind buffer and vortex the DNA and beads with buffer for 5 seconds. Pellet the beads on a magnet for two minutes, then pipette off the supernatant. Take care to not aspirate any of the beads.

Repeat the previous step two more times for a total of three washes.

Add 200 μl of 10 mM Tris-HCl pH 7.5 and vortex the beads for 5 seconds.

Spin down and place the tube back on the magnet for three minutes. Pipette off the supernatant.

Remove the tube from the magnetic rack and resuspend the pellet in 20 μl of nuclease-free water. Vortex for 5 seconds and briefly spin down to collect the amplicon-bead conjugate.

6. Post-pull-down PCR

材料
  • cDNA Primer (cPRM)
  • Elution Buffer from the Oxford Nanopore kit (EB)
耗材
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
  • Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 0.2 ml thin-walled PCR tubes
  • 1.5 ml Eppendorf DNA LoBind 离心管
仪器
  • 热循环仪
  • Vortex mixer
  • Hula混匀仪（低速旋转式混匀仪）
  • 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）

In a 0.2 ml thin-walled PCR tube, prepare the following PCR reaction:

Reagent Volume
cPRM 1 μl
Nuclease-free water 4 μl
LongAmp Hot Start Taq 2X Master Mix 25 μl
Total 30 μl

Resuspend the amplicon-bead conjugate by pipetting and then transfer 20 μl of the conjugate into the 0.2 ml thin-walled PCR tube containing the PCR reaction. Mix by pipetting.

Do not spin down the tube; transfer immediately to the thermal cycler and amplify using the following cycling conditions:

| Cycle step | Temperature | Time | No. of cycles | | ---------- | ---------- | ---------- | ---------- | ---------- | | Initial denaturation | 94°C | 3 min | 1 | | Denaturation

Annealing

Extension | 94°C

62°C

65°C | 15 s

15 s

6 min |

4 | | Final extension | 65°C | 10 min | 1 | | Hold | 4°C | ∞ | - |

Resuspend the AMPure XP beads by vortexing.

将样品转至干净的1.5 ml Eppendorf DNA LoBind 离心管中。

Add 40 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.

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。

Prepare 500 μl of fresh 70% ethanol in nuclease-free water.

将样品瞬时离心，并静置于磁力架上待磁珠和液相分离。保持离心管在磁力架上不动，用移液枪吸去清液。

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

将离心管瞬时离心后置于磁力架上。用移液枪吸走残留的乙醇。让磁珠在空气中干燥30秒，但不要干至表面开裂。

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

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

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

Dispose of the pelleted beads

Quantify 1 µl of eluted sample using a Qubit fluorometer - recovery aim >50 ng total.

7. Adapter addition

材料
  • Elution Buffer from the Oxford Nanopore kit (EB)
  • Rapid Adapter T (RAP T)
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

Analyse 1 µl of sample using the Agilent Bioanalyzer. Determine the average amplicon size from this data, and use this to calculate the input sample volume for the next step.

Calculate the required sample volume for 35 fmol and dilute this into 12 μl of EB.

Add 0.5 μl of Rapid Adapter T (RAP T) to the amplified cDNA library.

Mix well by pipetting and spin down.

Incubate the reaction for 5 minutes at room temperature.

步骤结束

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

8. Priming and loading the SpotON flow cell

材料
  • Sequencing Buffer II (SBII)
  • Loading Beads II (LBII)
  • Flush Buffer (FB)
  • Flush Tether (FLT)
耗材
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 无核酸酶水（如ThermoFisher，AM9937）
仪器
  • MinION device
  • SpotON Flow Cell
  • 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
  • MinION 及GridION 测序芯片遮光片
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。

Using the Loading Solution

We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water. Note: some customers have noticed that viscous libraries can be loaded more easily when not using Loading Beads II.

Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), Flush Tether (FLT) and Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down the SBII and FLT at room temperature.

Mix the Sequencing Buffer II (SBII), Flush Buffer (FB), Flush Tether (FLT) and Loading Solution (LS, if using) tubes by vortexing. Spin down the SBII and FLT at room temperature.

Prepare the flow cell priming mix: Add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing.

Open the MinION Mk1B lid and slide the flow cell under the clip.

Press down firmly on the flow cell to ensure correct thermal and electrical contact.

Flow Cell Loading Diagrams Step 1a

Flow Cell Loading Diagrams Step 1b

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。

Slide the priming port cover clockwise to open the priming port.

Flow Cell Loading Diagrams Step 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

Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting.

重要

The Loading Beads II (LBII) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

In a new tube, prepare the library for loading as follows:

Reagent Volume per flow cell
Sequencing Buffer II (SBII) 37.5 µl
Loading Beads II (LBII), mixed immediately before use, or Loading Solution (LS), if using 25.5 µl
DNA library 12 µl
Total 75 µl

Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer II (SBII) and Loading Beads II (LBII) because the fuel in the buffer will start to be consumed by the adapter.

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。

9. Data acquisition and basecalling

纳米孔数据分析概览

有关纳米孔数据分析的完整概述，包括碱基识别和次级分析，请参阅 数据分析 文档。

如何开始测序

MinKNOW软件负责仪器控制，数据采集和实时碱基识别。如您已在计算机上安装MinKNOW，则可选择以下几种途径开展测序：

1. 使用计算机上的MinKNOW进行实时数据采集和碱基识别

请按照 MinKNOW 实验指南 的说明：从“开始测序”部分起，到“MinKNOW运行结束”部分止。

2. 使用GridION进行实时数据采集和碱基识别

请参照 GridION 用户手册 中的说明。

3. 使用MinION Mk1C测序仪进行实时数据采集和碱基识别

请参照 MinION Mk1C 使用指南中的说明。

4. 使用PromethION测序仪进行实时数据采集和碱基识别

请参照 PromethION 使用指南 或 PromethION 2 Solo 使用指南中的说明。

5. 使用计算机上的MinKNOW进行数据采集，过后再用NinKNOW进行线下碱基识别

请按照 MinKNOW 实验指南 中的说明：从“开始测序”部分起，到“MinKNOW运行结束”部分止。 当您设置实验参数时，请将 碱基识别 选项设为“关”。 测序实验结束后，请按照 MinKNOW 实验指南的本地分析 部分操作。

10. Downstream analysis

EPI2ME provides a Nextflow-based workflow for the analysis of single-cell sequencing data.

The workflow, wf-single-cell, processes the FASTQ format sequence data prepared by the MinKNOW software. The workflow screens each sequence read for 10X cell barcode information and assigns reads to a cell of origin. A subset of sequences from “true” cells are dynamically filtered on the basis of the number of assigned sequence reads. These sequences are mapped to the reference genome, and tables of both gene and transcript abundance are prepared for each cell. These "cell barcode x gene" or transcript abundance information are used to prepare the familiar UMAP plots that may show the stratification of the cell types present within the sample.

For more information on this workflow, follow the link to the GitHub documentation.

11. 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。

12. 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。

13. 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: 12/6/2023

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