This protocol has been upgraded to our Kit 14 chemistry and describes how to carry out PCR amplification and native barcoding of influenza amplicons using the Native Barcoding Kit 24 or 96 V14 (SQK-NBD114.24 or SQK-NBD114.96). There are 96 unique barcodes available, allowing the user to pool up to 96 different Influenza A and/or Influenza B samples in one sequencing experiment.

While this protocol is available in the Nanopore Community, we kindly ask users to ensure they are citing the following references, that this protocol is based on.

Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza A viruses by Bin Zhou et al., 2009. and Universal influenza B virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics by Bin Zhou et al., 2014.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

• Ensure you have the 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

Prepare your library

You will need to:

• Perform RT-PCR amplification on influenza samples
• Prepare the DNA ends for adapter attachment
• Ligate native barcodes supplied in the kit to the DNA ends
• Ligate sequencing adapters supplied in the kit to the DNA ends
• Prime the flow cell, and load your DNA library into the flow cell

Sequencing

You will need to:

• Start a sequencing run using the MinKNOW software, which will collect raw data from the device and basecall the reads
• Demultiplex barcoded reads in MinKNOW choosing the SQK-NBD114.24 or SQK-NBD114.96 kit option
• Analyse your data using the Influenza typing workflow (wf-flu) in EPI2ME Labs.

A minimum volume of 1 µl is required per sample input of Influenza A or B.

If performing typing of an unknown sample, 2 µl of input will be required:

• 1 µl input for the Influenza A primer mix
• 1 µl for the Influenza B primer mix

This protocol outlines how to carry out PCR amplification and native barcoding of influenza amplicons from multiple samples on a 96-well plate using the Native Barcoding Kit 24 or 96 V14 (SQK-NBD114.24 or SQK-NBD114.96).

When processing multiple samples simultaneously, we recommend making master mixes with an additional 10% of the volume. We also recommend using a template-free pre-PCR hood for making up the master mixes, and a separate template pre-PCR hood for handling the samples. It is important to clean and/or UV irradiate these hoods between sample batches. Furthermore, to track and monitor cross-contamination events, it is important to run a negative control reaction at the reverse transcription stage using nuclease-free water instead of sample, and carrying this control through the rest of the prep.

All post-PCR procedures must be carried out in a separate area to the pre-PCR preparation, with dedicated equipment for liquid handling in each area.

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.

Influenza A primer sequences described in the protocol originated from: Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza A viruses by Bin Zhou et al., 2009.

Influenza B primer sequences described in the protocol originated from: Universal influenza B virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics by Bin Zhou et al., 2014.

请注意： 我们正在将试剂盒中的条形码包装更改为96孔板形式。这一改动将减少塑料浪费，并支持自动化应用。

孔板形式

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°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。

管装形式

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°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。

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）。

条形码孔板中的条形码是按照列进行排序的。

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：

Below is the full list of our native barcode (NB01-96) sequences. The first 24 unique barcodes are available in the Native Barcoding Kit 24 V14 (SQK-NBD114.24). The Native Barcoding Kit 96 V14 (SQK-NBD114.96) include the first 24 native barcodes, with the additional 72 unique barcodes. The native barcodes are shipped at 640 nM.

In addition to the barcodes, there are also flanking sequences which add an extra level of context during analysis.

Barcode flanking sequences:

Forward sequence: 5' - AAGGTTAA - barcode - CAGCACCT - 3' Reverse sequence: 5' - GGTGCTG - barcode - TTAACCTTAGCAAT - 3'

Native barcode sequences

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。

MinION Mk1C是一款集计算功能和触控屏幕于一体的便携式测序分析仪，它无需依赖任何额外设备，即可生成并分析纳米孔测序数据。您可以在 MinION Mk1C的IT配置要求文件中了解更多。

Sequencing on a MinION Mk1D requires a high-spec computer or laptop to keep up with the rate of data acquisition. For more information, refer to the MinION Mk1D IT requirements document.

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.

For instructions on how to run the MinKNOW software, please refer to 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 will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.

For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to this link.

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** ：

** 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。

• When handling the primer stocks and derivatives, use a clean template-free PCR hood.
• When handling the samples and/or a positive control, use a clean template-addition PCR hood.

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。

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 的说明操作：从“开始测序”部分起，到“MinKNOW运行结束”部分止。

2. 使用MinION Mk1B/Mk1D测序仪进行实时数据采集和碱基识别

请按照 MinION Mk1B 用户手册 或 MinION Mk1D 用户手册中的说明操作。

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 用户手册中的说明操作。

4. 使用GridION进行实时数据采集和碱基识别

请参照 GridION 用户手册 中的说明操作。

5. 使用PromethION测序仪进行实时数据采集和碱基识别

请参照 PromethION 用户手册 或 PromethION 2 Solo 用户手册中的说明操作。

6. 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 中的说明操作：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。

The analysis of the FASTQ format sequence data is performed using a Nextflow workflow called Influenza Typing Workflow (wf-flu). The use of the Nextflow software has been integrated into the EPI2ME Labs software that we recommend for running our downstream analysis methods.

Alternative methods for downstream analysis are available using your device terminal or command line, however we only suggest this for experienced users.

The workflow processes the basecalled and demultiplexed DNA sequence data output generated by MinKNOW:

• The sequences are filtered for a minimum length and quality thresholds (200 nucleotides and Q9 respectively) prior to sequence alignment to the CDC multi-fasta Influenza reference.
• The alignment is performed using the Minimap2 software.
• Depth of coverage across the mapped sequences is measured using Samtools before genetic variants are called using Medaka.
• A coverage-masked consensus sequence is prepared for each sample using bcftools.
• The influenza strain typing is then performed using the abricate software with an insaflu database.

The influenza strains included in the database are listed in the project documentation pages for the Influenza Typing Workflow.

The workflow returns a per-run HTML-format summary report along with a CSV file of typing results. Additional files that include mapping BAM files and VCF files of Medaka variants are also included in the workflow output.

For more information, please refer to the Influenza workflow blog.

The EPI2ME application provides a clean interface to accessing bioinformatics workflows, and is our recommended method in performing your post-sequencing analysis.

Follow the instructions in the EPI2ME Installation guide to install the application on your device.

For more information on how to use EPI2ME, refer to the EPI2ME Quick Start guide.

Ensure you have installed the wf-flu workflow prior to the first analysis set-up.

In the EPI2ME Labs home page, scroll down to the "Install workflows" section and click on epi2me-labs/wf-flu:

If you have already installed the wf-flu workflow, ensure you are using the latest version.

Updating the workflow can be done directly through EPI2ME Labs by navigating to the wf-flu workflow page and clicking Update Workflow:

The wf-flu analysis requires FASTQ sequence data that has already been demultiplexed.

Reads will be demultiplexed during sequencing if you are following the recommended "Required settings in MinKNOW". However, demultiplexing can also be done post-sequencing using the MinKNOW software.

For more information and guides on demultiplexing using MinKNOW, refer to the "Post-run analysis" section in our MinKNOW Protocol.

The expected input for wf-flu is a folder of folders as shown below. Each of the barcode folders should contain the FASTQ sequence data and files may either be uncompressed or gzipped.

$ tree -d FluFastq/

FluFastq/

├── barcode01

├── barcode02

├── barcode03

├── barcode04

├── barcode05

├── barcode06

└── unclassified

The wf-flu analysis outputs will be written to the Working Directory folder specified in the EPI2ME Labs Settings tab. The location of this folder is specified in the wf-flu run Instance parameters preceeded by out_dir.

However, these files can also be accessed directly in the EPI2ME Labs application from the completed analysis page for your run:

The "Working Directory" can be specified in the EPI2ME Labs "Settings" tab and defines where the workflow intermediate files and outputs are stored.

This folder will accumulate a significant number of files that correspond to raw BAM files, other larger intermediates and analysis results files. We recommend this folder to be routinely cleared.

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。