We have two PCR barcoding expansions available depending on the number of barcodes required:

• PCR Barcoding Expansion 1-12 (EXP-PBC001): up to 12 unique barcodes are available
• PCR Barcoding Expansion 1-96 (EXP-PBC096): up to 96 unique barcodes are available

These expansions are used with the Ligation Sequencing Kit V14 and recommended for users who:

• Want to multiplex up to 12 or 96 barcodes, depending on the expansion they are using.
• Would like to achieve raw read sequencing modal accuracy of Q20+ (99%) or above.
• Want to optimise their sequencing experiment for output.
• Require control over read length.
• Would like to utilise upstream processes such as size selection or whole genome amplification.

This protocol describes how to carry out PCR barcoding of gDNA using the Ligation Sequencing Kit V14 (SQK-LSK114) with the PCR Barcoding Expansion Pack 1-12 (EXP-PBC001) or 1-96 (EXP-PBC096). This protocol also outlines recommendations to PCR barcode amplicons. Using the PCR Barcoding expansions allows for up to either 12 or 96 samples to be combined and loaded onto a single flow cell. It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.

Note: For amplicon inputs, first-round PCR product with the following tailed primers are required. 5’ TTTCTGTTGGTGCTGATATTGC-[ project-specific forward primer sequence ] 3’ 5’ ACTTGCCTGTCGCTCTATCTTC-[ project-specific reverse primer sequence ] 3’

Steps in the sequencing workflow:

Prepare for your experiment You will need to:

• Extract your DNA, and check its length, quantity and purity. 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
  

Experiment workflow

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

Library preparation	Process	Time	Stop option
For gDNA: DNA end-prep or For amplicons: Incorporate tailed primers	Prepare the DNA ends for PCR adapter attachment or Perform a round of PCR to incoporate tailed primers	35 minutes	4°C overnight
For gDNA: attach barcoding adapters or For amplicons: second round of PCR to incorporate barcode sequences	Attach barcoding adapters to the DNA ends. or Complete a second round of PCR to incorporate the Oxford Nanopore barcode sequences.	40 minutes	4°C overnight
Amplify and pool samples	Amplify each barcoded sample by PCR, then pool the samples together.	15 minutes + Adjustable PCR time	4°C overnight
End-prep	Repair the DNA and prepare the DNA ends for adapter attachment	35 minutes	4°C overnight
Adapter ligation and clean-up	Attach the sequencing adapters to the DNA ends	20 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. 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:

• In the current MinKNOW software version, the PCR Barcoding Expansions are not available in "Kit selection" when setting up a sequencing run. These will be included in the next software update. For the meantime, we recommend the following:
  • Start a sequencing run in the MinKNOW software using SQK-LSK114, which will collect raw data from the device and convert it into basecalled reads.
  • Demultiplex your data post-run on MinKNOW using the barcoding option, choosing either EXP-PBC001 or EXP-PBC096 as your barcoding kit. Further information is available in the "Post-run barcoding" of the MinKNOW protocol.
• Start the EPI2ME software and select the barcoding workflow for further analysis (this step is optional).

If using amplicons, 100 ng of first-round PCR product (with tailed primers) is required per sample.

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。

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.

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。

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。

单次管装试剂：

部分试剂改为瓶装：

声明： 本产品包含由贝克曼库尔特公司（Beckman Coulter, Inc）生产的 AMPure XP 试剂，并可与试剂盒一起于-20°C 下储存（试剂稳定性将不受损害）。

请注意： DNA参照（DCS）是一段可比对到Lambda基因组的3'端、长度为3.6 kb 的标准扩增子。

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
PCR Barcode 1-12	BC1-12	Clear	12	20
Barcode Adapter	BCA	Blue stripe	1	260

Name	Acronym	Cap colour	No. of vials/plates	Fill volume per well (µl)
PCR Barcode Primer Mix plate	BC01-96	White	1 plate	24
Barcode Adapter plate	BCA	Blue	1 plate	240

The wells of the 96 tube plate correspond to the barcodes in the following way. All barcodes are supplied at 10 µM concentration and to be used at a final concentration of 0.2 µM.

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.

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。

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.

In the current MinKNOW software version, the PCR Barcoding Expansions are not available in "Kit selection" when setting up a sequencing run. These will be included in the next software update.

For the meantime, we recommend starting a sequencing run in the MinKNOW software using Ligation Sequencing Kit V14 (SQK-LSK114) to perform real-time basecalling. Once basecalling is complete, the data can be demultiplexed using post-run barcoding on MinKNOW, as outlined below.

Real-time basecalling

Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section for your device until the end of the "Completing a MinKNOW run" section.

Select Ligation Sequencing Kit (SQK-LSK114) in "Kit selection". The barcoding option will be unavailable as default. Other parameters can be kept at their default settings.

Post-run barcoding

Follow the instructions in "Post-run barcoding" of the MinKNOW protocol.

Click Analysis to open post-run options. Choose Barcoding and input your fastq data files. Select the PCR Barcoding Expansion used during library preparation in "Barcode settings" and use the default settings for other parameters.

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。

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。

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。