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

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.

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.

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.

Flow cell	Minimum number of active pores covered by warranty
Flongle Flow Cell	50
MinION/GridION Flow Cell	800
PromethION Flow Cell	5000

We are in the process of reformatting the barcodes provided in this kit into a plate format. This will reduce plastic waste and facilitates automated applications.

Plate format

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Rapid Adapter	RA	Green	1	15
Adapter Buffer	ADB	Clear	1	100
AMPure XP Beads	AXP	Amber	2	1200
Elution Buffer	EB	Black	1	500
Sequencing Buffer	SB	Red	1	700
Library Beads	LIB	Pink	1	600
Library Solution	LIS	White cap, pink label	1	600
Flow Cell Flush	FCF	Clear cap, light blue label	1	8000
Flow Cell Tether	FCT	Purple	1	200
Rapid Barcode plate	RB01-24	-	2 plates, 3 sets of barcodes per plate	5 µl per well

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.

Vial format

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Rapid Adapter	RA	Green	1	15
Adapter Buffer	ADB	Clear	1	100
AMPure XP Beads	AXP	Amber	2	1,200
Elution Buffer	EB	Black	1	500
Sequencing Buffer	SB	Red	1	700
Library Beads	LIB	Pink	1	600
Library Solution	LIS	White cap, pink label	1	600
Flow Cell Flush	FCF	Blue	6	1,170
Flow Cell Tether	FCT	Purple	1	200
Rapid Barcodes	RB01-24	Clear	24	15

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.

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.

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:

• GridION user manual
• MinION Mk1C user manual
• PromethION user manual
• PromethION 2 Solo user manual

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To start a sequencing run on MinKNOW:

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

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

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

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.

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.

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.

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

After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.

After sequencing and basecalling, the data can be analysed. For further information about options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

In the Downstream analysis section, we outline further options for analysing your data.

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.

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.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.