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.

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 three months 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

Note: We are in the process of reformatting our kits with single-use tubes into a bottle format.

Single-use tubes format:

Bottle format:

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.

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.

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. 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.

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 Bioinformatics section of the Resource centre. 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.