Rapid sequencing V14 - Amplicon sequencing (SQK-RBK114.24 or SQK-RBK114.96)

Oxford Nanopore Technologies
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Protocol

Rapid sequencing V14 - Amplicon sequencing (SQK-RBK114.24 or SQK-RBK114.96) VRAA_9198_v114_revH_29Nov2023

  • The fastest and simplest protocol to sequence amplicon DNA
  • For multiplexing up to 96 single species amplicon samples
  • Optimised for 500 bp – 5 kb amplicons.
  • Library preparation time ~60 minutes
  • High yield
  • Fragmentation
  • 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

  • The fastest and simplest protocol to sequence amplicon DNA
  • For multiplexing up to 96 single species amplicon samples
  • Optimised for 500 bp – 5 kb amplicons.
  • Library preparation time ~60 minutes
  • High yield
  • Fragmentation
  • 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: RAA_9198_v114_revH_29Nov2023

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 amplicon sequencing using the Rapid Barcoding Kit 24 or 96 V14

This protocol describes how to carry out rapid barcoding of amplicon DNA using the Rapid Barcoding Kit 24 or 96 V14 (SQK-RBK114.24 or SQK-RBK114.96) to sequence up to 96 single amplicon samples. This method allows you to perform your own amplicon sequencing and validation with a quick turn-around time, without the need for primers and a reference, and with competitive price per sample. Using this method, PCR amplicons ranging from 500 bp to 5 kb can be sequenced, which allows users to check that each amplicon is the correct size and no mutations have been introduced during PCR amplification.

The analysis workflow is not intended for marker gene sequencing of mixtures/communities of different organisms (e.g. 16S sequencing). In de-novo consensus mode it expects a single amplicon per barcode. When running in variant calling mode, multiple amplicons per barcode can be processed, but their sequences need to be sufficiently different from each other so that most reads only align to one of the provided references.

We recommend new users to sequence for 12 hours, although a shorter run-time (e.g. 4 hours) may be sufficient to generate enough reads per target. We suggest generating 150X or ~1500 reads per target. In most cases, this should be sufficient data to perform analysis.

After sequencing, we recommend performing downstream analysis using the EPI2ME amplicon workflow (wf-amplicon).

The results of the workflow include an interactive HTML report, FASTQ files with the consensus sequences of the amplicons, and BAM files with alignments of the input reads re-aligned against the consensus. Optionally, a reference with the expected amplicon sequences can be supplied to the workflow, in which case VCF files with variants called against that reference are additionally emitted.

Detailed instructions for setting up MinKNOW and the EPI2ME workflow are included in this protocol.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

  • Extract your DNA, 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 your primer design is correct prior to amplicon generation.
    Note: To make sure that the whole region of interest is captured when using Rapid Barcoding Kits, the primers should be designed such that they include an extra of 15-20 bp at the start and end of the actual target sequence.
  • Generate your amplicon sample(s) by PCR amplification.
  • 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
PCR clean-up AMPure XP Bead purification (or equivalent) of amplicon samples to remove PCR artifacts 25 minutes 4°C overnight
Amplicon DNA barcoding Tagmentation of the amplicon DNA using the Rapid Barcoding Kit V14 15 minutes 4°C overnight
Sample pooling and clean-up Pooling of barcoded libraries and AMPure XP Bead clean-up 25 minutes 4°C overnight
Adapter ligation 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

Amplicon V14 workflow SVG April2024

Sequencing and analysis

You will need to:

  • Start a sequencing run using the MinKNOW software, which will collect raw data from the device into basecalled reads and will perform barcode demultiplexing.
  • Start the EPI2ME software and use the EPI2ME amplicon workflow (wf-amplicon) for analysis.
重要

Compatibility of this protocol

This protocol should only be used in combination with:

  • Rapid Barcoding Kit 24 V14 (SQK-RBK114.24)
  • Rapid Barcoding Kit 96 V14 (SQK-RBK114.96)
  • R10.4.1 flow cells (FLO-MIN114)
  • Flow Cell Wash Kit (EXP-WSH004)
  • Flow Cell Priming Kit V14 (EXP-FLP004)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)
  • Rapid Adapter Auxiliary V14 (EXP-RAA114)
  • MinION Mk1C - MinION Mk1C IT requirements document
  • MinION Mk1B - MinION IT Requirements document

2. Equipment and consumables

材料
  • 50 ng amplicon DNA per sample (500 bp – 5 kb amplicon size)
  • 快速条形码测序试剂盒-24 V14 （SQK-RBK114.24）或 快速条形码测序试剂盒-96 V14 （SQK-RBK114.96）
耗材
  • MinION及GridION测序芯片
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881) (or equivalent for DNA purification)
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
  • （非必需）牛血清白蛋白（BSA）（50 mg/mL）（例如 Invitrogen™ UltraPure™ BSA （50 mg/mL）， AM2616）
  • 新制备的80%乙醇（用无核酸酶水配制）
  • 无核酸酶水（如ThermoFisher，AM9937）
  • Qubit™ 分析管（Invitrogen, Q32856）
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
  • 2 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
  • Hula混匀仪（低速旋转式混匀仪）
  • 微孔板离心机，如Fisherbrand™ 微孔板迷你离心机（Fisher Scientific, 11766427）
  • Microfuge
  • 涡旋混匀仪
  • 热循环仪或恒温加热仪
  • Qubit荧光计（或用于质控检测的等效仪器）
  • 磁力架
  • 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
  • P2移液枪和枪头
  • 多通道移液枪和枪头
  • 盛有冰的冰桶
  • 计时器
重要

Ensure you have correctly designed your primer sequence prior to amplicon generation and library preparation.

Due to the nature of how Rapid Barcoding Kits reads are sequenced and limitations of correctly assembling terminal ends of linear sequences, small truncations (~10-20 bp) at both ends of the generated consensus can occur.

This means that part of the primer sequence (or potentially the whole primer and a few more bases depending on primer length) might be missing from the consensus sequence produced by the analysis workflow.

To make sure that the whole region of interest is captured when using Rapid Barcoding Kits, the primers should be designed such that they include an extra of 15-20 bp at the start and end of the actual target sequence.

For this protocol, you will need 50 ng of single species amplicon DNA per sample.

We recommend performing a purification step following the PCR reaction used to generate your amplicons. Please use standard methods appropriate for the amplicon size, such as AMPure XP beads. This step ensures the removal of proteins, salts, dNTPs, and primers, which could potentially impact the library preparation and subsequent analysis workflow.

Please note that while alternative methods exist, they have not been validated by our internal teams.

This method is intended for barcoding single species amplicon samples and is not suitable for mixed species amplicons.

重要

The wf-amplicon workflow is optimised for 500 bp – 5 kb amplicons.

Sequencing amplicons <500 bp or >5 kb may result in sub-optimal performance.

Further work is ongoing to broaden the range of amplicons compatible with this end-to-end workflow.

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测序芯片三个月之内进行，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。

Rapid Barcoding Kit 24 V14 (SQK-RBK114.24) contents

Single-use tubes format:

RBK114.24 tubes

Bottle format:

SQK-RBK114.24 tubes v1

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 Barcoding Kit 96 V14 (SQK-RBK114.96) contents

RBK114.96 tubes (1)

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.

3. Library preparation

材料
  • 50 ng amplicon DNA per sample (500 bp – 5 kb amplicon size)
  • 快速连接条形码（RB01-24）或 快速连接条形码盘（RB01-96）
  • 快速测序文库接头（RA）
  • 接头缓冲液（ADB）
  • AMPure XP 磁珠（AXP）
  • Elution Buffer (EB)
耗材
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881) (or equivalent for DNA purification)
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
  • 无核酸酶水（如ThermoFisher，AM9937）
  • 新制备的80%乙醇（用无核酸酶水配制）
  • Qubit™ 分析管（Invitrogen, Q32856）
  • 1.5 ml Eppendorf DNA LoBind离心管
  • 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
  • 2 ml Eppendorf DNA LoBind 离心管
仪器
  • 热循环仪
  • 微孔板离心机，如Fisherbrand™ 微孔板迷你离心机（Fisher Scientific, 11766427）
  • Microfuge
  • 涡旋混匀仪
  • Hula混匀仪（低速旋转式混匀仪）
  • 磁力架
  • 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
  • 多通道移液枪和枪头
  • 盛有冰的冰桶
  • 计时器
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。

重要

PCR clean-up of amplicon DNA samples

We recommend performing a purification step following the PCR reaction used to generate your amplicons. Please use standard methods appropriate for the amplicon size, such as AMPure XP beads. This step ensures the removal of proteins, salts, dNTPs, and primers, which could potentially impact the library preparation and subsequent analysis workflow.

Please note that while alternative methods exist, they have not been validated by our internal teams.

设定热循环仪的程序：30℃两分钟，后接80℃两分钟。

请根据下表，使用相应方法对各试剂进行解冻、离心和混匀：

试剂 1. 室温下解冻 2. 迷你离心机瞬时离心 3. 吹打混匀
快速连接条形码（RB01-24）或 快速连接条形码盘（RB01-96） 未冻结 ✓ ✓
快速测序文库接头（RA） 未冻结 ✓ ✓
AMPure XP磁珠（AXP） ✓ ✓ 临使用前，吹打或涡旋混匀
洗脱缓冲液（EB） ✓ ✓ ✓
接头缓冲液（ADB） ✓ ✓ 涡旋混匀

Prepare the DNA in nuclease-free water, as follows. Approximately 50 ng of amplicon DNA is required in 9 µl of volume for each sample for barcoding.

  • Dilute your amplicon DNA samples with nuclease-free water to approximately 50 ng. See the table below for dilutions:
Starting Conc. Volume of DNA Volume of nuclease-free water Total volume
100 ng/µl 2 µl 34 µl 36 µl
90 ng/µl 2 µl 31 µl 33 µl
80 ng/µl 2 µl 27 µl 29 µl
70 ng/µl 3 µl 35 µl 38 µl
60 ng/µl 2 µl 20 µl 22 µl
50 ng/µl 2 µl 16 µl 18 µl
40 ng/µl 5 µl 31 µl 36 µl
30 ng/µl 5 µl 22 µl 27 µl
20 ng/µl 5 µl 13 µl 18 µl
10 ng/µl 10 µl 8 µl 18 µl
<5.56 ng/µl 9 µl 0 µl 9 µl
  • Pipette mix the dilutions, and spin down briefly.
  • Add 9 µl of volume for each sample into a 0.2 ml PCR tube or plate.

为计划上样于同一测序芯片的各样本选择不同的条形码。一个实验最多可为96个样本添加条码并混样测序。

请注意： 每个样本使用一种条形码。

In 0.2 ml thin-walled PCR tubes or plate, mix the following reagents. The Rapid Barcodes can be transferred using a multichannel pipette:

Reagent Volume
50 ng template DNA 9 μl
Rapid Barcodes (RB01-96, one for each sample) 1 μl
Total 10 μl

充分吹打混匀管中试剂，再瞬时离心。

将PCR管或96孔板在30℃下孵育两分钟，然后在80℃下孵育两分钟，随后短暂置于冰上冷却。

将PCR管或96孔板瞬时离心，收集管/板底的液体。

Pool all the barcoded samples into a clean 1.5 ml Eppendorf DNA LoBind tube, noting the total volume.

. Volume per sample For 12 samples For 24 samples For 48 samples For 96 samples
Total volume 10 μl 120 μl 240 μl 480 μl 960 μl

Resuspend the AMPure XP beads (AXP) by vortexing.

To the entire pooled barcoded sample, add an equal volume of resuspended AMPure XP Beads (AXP) and mix by flicking the tube.

. Volume per sample For 12 samples For 24 samples For 48 samples For 96 samples
Volume of AXP 10 μl 120 μl 240 μl 480 μl 960 μl

将离心管置于Hula混匀仪（低速旋转式混匀仪）上室温孵育10分钟。

Prepare at least 3 ml of fresh 80% 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 1.5 ml of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

重复上述步骤。

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约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). Incubate for 10 minutes at room temperature.

将离心管静置于磁力架上至少一分钟，直到磁珠和液相分离，且洗脱液澄清无色。

Remove and retain 15 µl of eluate 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
CHECKPOINT

取1µl洗脱样品，用Qubit荧光计定量。

Transfer 11 µl of the sample into a clean 1.5 ml Eppendorf DNA LoBind tube.

Note: We recommend transfering a maximum of 800 ng of the DNA library. If necessary, take forward only the necessary volume for 800 ng of DNA library and make up the rest of the volume to 11 µl using Elution Buffer (EB).

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）。

轻弹离心管以充分混合，并瞬时离心。

Incubate the reaction for 5 minutes at room temperature.

步骤结束

制备好的文库即可用于芯片上样。请在上样前，始终将文库置于冰上。

4. Priming and loading the MinION and GridION Flow Cell

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
  • 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）
  • Nuclease-free water
  • 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 (1)

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。

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）。

按下表制备测序芯片的预处理液，室温下吹打混匀。

请注意： 我们正在将部分试剂的包装形式由单次管装改为瓶装。请按照与您所用试剂盒包装相对应的说明操作。

单次使用管装： 向一整管测序芯片冲洗液（FCF）中加入5µl 50mg/ml的牛血清白蛋白（BSA）及 30µl 测序芯片系绳（FCT）。

瓶装： 请另拿一支适当体积的离心管制备测序芯片预处理液：

试剂 体积（每张芯片）
测序芯片冲洗液 (FCF) 1,170 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 1,205 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

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，我们建议使用文库颗粒（LIB）。然而，对于粘度较高的文库，可以考虑使用文库溶液（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：

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。

5. Data acquisition and basecalling

How to start sequencing

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

MinKNOW can be used and set up to sequence in multiple ways:

  • On a computer either direcly or remotely connected to a sequencing device.
  • Directly on a GridION, MinION Mk1C or PromethION 24/48 sequencing device.

For more information on using MinKNOW on a sequencing device, please see the device user manuals:

To start a sequencing run on MinKNOW:

1. Navigate to the start page and click Start sequencing. start

2. Fill in your experiment details, such as name and flow cell position and sample ID. Grid start seq

3. Select the Rapid Barcoding Kit 24 V14 (SQK-RBK114.24) or Rapid Barcoding Kit 96 V14 (SQK-RBK114.96). Edit 3

4. Change the run limit to 12 hours by clicking "Options" and changing the run limit value to 12. The other run settings can be left at the defaults.
Amplicon run setup 1

plasmid seq rbk114 v1

Note: We recommend a run-time of 12 hours for new users to ensure sufficient data is generated for downstream analysis. However, shorter run-times may be sufficient depending on your sample input and analysis requirements.
If using custom sequencing time, for optimal results, we recommend allowing the sequencing run to generate 1000-1500 reads per sample before proceeding to the analysis workflow.

5. Set up basecalling and barcoding using the following parameters:

  • Toggle basecalling to ON.
  • Next to "Models", click Edit options and choose High accuracy basecaller (HAC) from the drop-down menu.
  • Toggle barcoding to ON.
  • Keep all other options at their default settings.
  • Click Continue to output and continue.

Amplicon run setup 3

6. Set up the output format and filtering as follows:

  • Select either .POD5 or .FAST5 (legacy) as the output format.
  • Ensure .FASTQ is selected for basecalled reads.
  • Ensure filtering is ON and read splitting is enabled. Other parameters can be kept to their default settings.
  • Click Continue to final review to continue.

Amplicon run setup 4

7. Click Start on the Review page to start the sequencing run. Amplicon run setup 5

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。

6. 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。

7. Downstream analysis using EPI2ME

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.

Further information about the available EPI2ME workflows can be found here, along with the Quick Start Guide to start your first bioinformatic workflow.

For the analysis of amplicon sequences, we recommend using the wf-amplicon workflow which requires Nextflow and Docker or Singularity to be installed before running the workflow.

For installation instructions please click here.

重要

The wf-amplicon workflow is optimised for 500 bp – 5 kb amplicons.

Sequencing amplicons <500 bp or >5 kb may result in sub-optimal performance.

We recommend generating 150X or 1500 reads per target, which should be enough to perform the analysis in most cases.

Further work is ongoing to broaden the range of amplicons compatible with this end-to-end workflow.

Open the EPI2ME app using the desktop shortcut.

Navigate to the workflow downloads page. Click on the wf-amplicon workflow to download and confirm to install.

wf-amplicon-install-confirm_2

Navigate to the Workflows tab and click on wf-amplicon.

Select-wf-amplicon_3

Click on "Run this workflow" to open the launch wizard.

wf-amplicon-run_4

Set up your run by selecting your sequencing data in the "Input Options".

wf-amplicon-select-input-data_5

To speed up the analysis, you can set "Downsampling size" to 500.

Unless your amplicons are very long (>5 kb), this should provide sufficient coverage.

Amplicon wf downsampling new dec2023

可选操作

The amplicon workflow can be run with a sequence reference file if required.

Set up the reference FASTA by uploading the file in the following location:

wf-amplcion-reference_5.5

For the remaining parameter options we recommend keeping the default settings.

Click "Launch workflow".

Ensure all parameter options have green ticks.

wf-amplicon-launch_6

Once the workflow finishes, a report will be produced.

Amplicon workflow report

The primary outputs of the workflow include:

· an interactive HTML report with tables and plots detailing the results. · FASTQ files (one per barcode) with the de-novo consensus sequence and per-base consensus qualities (as calculated by Medaka). · BAM files (one per barcode) of input reads re-aligned against the consensus.

Example reports:

· When a reference file has been uploaded, reads are aligned to the reference (containing the expected sequence for each amplicon) for variant calling. An example of a sample variant calling report can be viewed here. · When no reference file has been uploaded, the amplicon’s consensus sequence is generated de novo. An example of sample de novo consensus report can be viewed here.


Report contents

The report consists of several sections. The introduction section gives a brief overview of key results for the individual samples analysed, while the preprocessing section illustrates the number of reads removed during downsampling / filtering. It also contains read length and quality histograms as well as a plot showing base yield vs. read length.

The remaining sections of the report depend on the mode in which the workflow was run. In variant calling mode, they summarise the mapping and variant calling stages of the workflow, showing the depth of coverage of aligned reads and providing further details on the variants called. In de novo consensus mode, results of the draft consensus QC stage and re-alignment of input reads against the consensus are described. Additionally, a depth of coverage plot of the re-aligned reads along the consensus is shown.

8. Issues during DNA/RNA extraction and library preparation

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。

The VolTRAX run terminated in the middle of the library prep

Observation Possible cause Comments and actions
The green light was switched off

or

An adapter was used to connect the VolTRAX USB-C cable to the computer		 Insufficient power supply to the VolTRAX The green LED signals that 3 A are being supplied to the device. This is the requirement for the full capabilities of the VolTRAX V2 device. Please use computers that meet the requirements listed on the VolTRAX V2 protocol.

The VolTRAX software shows an inaccurate amount of reagents loaded

Observation Possible cause Comments and actions
The VolTRAX software shows an inaccurate amount of reagents loaded Pipette tips do not fit the VolTRAX cartridge ports Rainin 20 μl or 30 μl and Gilson 10 μl, 20 μl or 30 μl pipette tips are compatible with loading reagents into the VolTRAX cartridge. Rainin 20 μl is the most suitable.
The VolTRAX software shows an inaccurate amount of reagents loaded The angle at which reagents are pipetted into the cartridge is incorrect The pipetting angle should be slightly greater than the cartridge inlet angle. Please watch the demo video included in the VolTRAX software before loading.

9. 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。

Last updated: 4/29/2024

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