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.

How to QC your input DNA

It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.

For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.

Chemical contaminants

Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.

We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.

For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

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

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.

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

Plate format

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
DNA Control Sample	DCS	Yellow	2	35
Native Adapter	NA	Green	1	40
Sequencing Buffer	SB	Red	1	700
Library Beads	LIB	Pink	1	600
Library Solution	LIS	White cap, pink label	1	600
Elution Buffer	EB	Black	2	500
AMPure XP Beads	AXP	Clear cap, light teal label	1	6,000
Long Fragment Buffer	LFB	Orange	1	1,800
Short Fragment Buffer	SFB	Clear	1	1,800
EDTA	EDTA	Blue	1	700
Flow Cell Flush	FCF	Clear cap, light blue label	1	8,000
Flow Cell Tether	FCT	Purple	1	200
Native Barcode plate	NB01-24	-	2 plates, 3 sets of barcodes per plate	15 µl per well

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.

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

Vial format

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Native Barcodes	NB01-24	Clear	24 (one per barcode)	20
DNA Control Sample	DCS	Yellow	2	35
Native Adapter	NA	Green	1	40
Sequencing Buffer	SB	Red	1	700
Library Beads	LIB	Pink	1	600
Library Solution	LIS	White cap, pink label	1	600
Elution Buffer	EB	Black	2	500
AMPure XP Beads	AXP	Clear cap, light teal label	1	6,000
Long Fragment Buffer	LFB	Orange	1	1,800
Short Fragment Buffer	SFB	Clear	1	1,800
EDTA	EDTA	Blue	1	700
Flow Cell Flush	FCF	Clear cap, light blue label	1	8,000
Flow Cell Tether	FCT	Purple	1	200

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.

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

These expansions provide extra library preparation and flow cell priming reagents to allow users to utilise any unused barcodes for those running in smaller subsets.

Both expansion packs used together will provide enough reagents for 12 reactions. For customers requiring extra EDTA to maximise the use of barcodes, we recommend using 0.25 M EDTA and adding 4 µl for library preps using the SQK-NBD114.24 kit.

Native Barcode Auxiliary V14 (EXP-NBA114) contents:

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.

Sequencing Auxiliary Vials V14 (EXP-AUX003) contents:

For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the Library Solution (LIS).

For a full overview of nanopore data analysis, which includes options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

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

There are several options for further analysing your basecalled data:

1. EPI2ME workflows

For in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in EPI2ME, which are available in the EPI2ME section of the Community. The platform provides a vehicle where workflows deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code and example data.

2. Research analysis tools

Oxford Nanopore Technologies' Research division has created a number of analysis tools, that are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.

3. Community-developed analysis tools

If a data analysis method for your research question is not provided in any of the resources above, please refer to the resource centre and search for bioinformatics tools for your application. Numerous members of the Nanopore Community have developed their own tools and pipelines for analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.

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.