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Native Barcoding Expansion 96 features
This kit is recommended for users who:
- wish to multiplex up to 96 samples to reduce price per sample
- need a PCR-free method of multiplexing to preserve additional information such as base modifications
- want to optimise their sequencing experiment for throughput
- require control over read length
- would like to utilise upstream processes such as size selection or whole genome amplification
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Introduction to the Native Barcoding Expansion 96 protocol
This protocol describes how to carry out native barcoding of amplicons using the Native Barcoding Expansion 96 (EXP-NBD196) in conjunction with the Ligation Sequencing Kit (SQK-LSK109). There are 96 unique barcodes available, allowing the user to pool up to 96 different samples in one sequencing experiment. It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- 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
Prepare your library
You will need to:
- 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 convert it into basecalled reads
- Demultiplex barcoded reads in MinKNOW or Guppy basecalling, choosing the EXP-NBD196 kit option