This protocol requires the use of two kits to generate the dual barcoded libraries:

• PCR Barcoding Expansion 1-96 (EXP-PBC096): up to 96 unique barcodes are available. These PCR barcodes are used as the primary "inner" barcodes.
• Native Barcoding Kit 24 V14 (SQK-NBD114.24): up to 24 unique barcodes are available. These native barcodes are used as the secondary "outer" barcodes.

These kits are used in successive order and recommended for users who:

• Want to multiplex up to 2,304 samples, depending on the barcodes they are using from each expansion.
• Would like to achieve raw read sequencing modal accuracy of Q20+ (99%) or above.
• Require control over read length.
• Would like to utilise upstream processes such as size selection or whole genome amplification.

This protocol allows massively parallel sequencing of up to 2,304 samples (gDNA or amplicons) on a single flow cell. It uses both the PCR Barcoding Expansion 1-96 (EXP-PBC096) and the Native Barcoding Kit 24 V14 (SQK-NBD114.24).

Samples are initially PCR barcoded with the PCR Barcoding Expansion 1-96 (EXP-PBC096), allowing up to 96 samples to be pooled together. There can be up to 24 pools of 96 samples. Each pool of 96 samples will then undergo secondary barcoding through the ligation of one of 24 native barcodes using the Native Barcoding Kit 24 V14 (SQK-NBD114.24). After secondary barcoding, all pools are combined into a single library for sequencing.

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

Library preparation

You will need to:

• Prepare the DNA ends for adapter attachment
• Attach barcoding adapters supplied in the 96 PCR Barcoding kit to the DNA ends
• Amplify each barcoded sample by PCR, then pool the 96 samples together (up to 24 pools of 96 samples can be generated)
• Prepare the DNA ends of your pooled samples for native barcode attachment
• Ligate native barcodes to the DNA ends for each pool of 96 samples
• Pool up to 24 native barcoded libraries together
• Attach sequencing adapters supplied in the kit to the DNA ends of your combined dual barcoded libraries
• Prime the flow cell, and load your dual barcoded DNA library into the flow cell

Sequencing and analysis

You will need to:

• In the current MinKNOW software version, we recommend setting up live basecalling during the sequencing run without live barcoding. Demultiplexing of the dual barcoded reads will be carried out post-run:
  • Start a sequencing run in the MinKNOW software using SQK-LSK114, which will collect raw data from the device and basecall the reads.
  • Demultiplex your run post-sequencing unsing the MinKNOW software.
• 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。

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.

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，并减低EDTA的浓度。

管装试剂及高浓度装EDTA：

较高浓度装EDTA的管盖为透明色。

瓶装试剂并减低EDTA浓度：

较低浓度装EDTA的管盖为蓝色。

请注意： 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℃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。

上述扩展包旨在提供额外的建库及测序芯片预处理试剂，方便用户使用条形码测序试剂盒中剩余的少部分条形码运行测序实验。

当联用时，两扩展包内试剂可满足12次反应。如果您在此过程中需要额外的 EDTA，我们建议使用浓度为0.25M的EDTA。如果您使用不超过24种条码进行建库，则建议为每个样本添加4 µl的EDTA；如果使用25至96种条码进行建库，则建议添加2 µl。

免扩增条形码扩展包（EXP-NBA114）内容物：

请注意： 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℃储存（试剂稳定性将不受损害）。

测序辅助扩展包 V14（EXP-AUX003）内容物：

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。

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. Instructions for this can be found in the MinKNOW protocol.

We recommend setting up real-time basecalling as follows:

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.

We currently recommend performing demultiplexing for the dual barcoding protocol post-sequencing using the Guppy software.

For a complete guide please refer to our Guppy protocol

Barcode demultiplexing in Guppy for dual barcoding:

To demultiplex your reads by barcode, use the dual barcoding configuration file, specifying the barcode kit as "EXP-DUAL00":

guppy_barcoder --input_path <folder containing FASTQ and/or FASTA files> --save_path <output folder> --config configuration_dual.cfg --barcode_kits "EXP-DUAL00"

For more information and instructions on how to use Guppy, please refer to the relevant sections of the protocol:

Barcode demultiplexing Quick Start

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。