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Native Barcoding Expansion 1-12 and 13-24 features
These kits are recommended for users who:
- wish to multiplex samples to reduce price per sample
- want to optimise their sequencing experiment for throughput
- require control over read length
- are interested in utilising upstream processes such as size selection or whole genome amplification
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Introduction to the ASFV Native Barcoding protocol
This protocol describes an efficient, low-cost method to sequence ASFV at scale. The method uses tiled PCR amplification of the virus to obtain good coverage, while enabling sample multiplexing to reduce run costs.
The kits used are the Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114), in conjunction with the Ligation Sequencing Kit (SQK-LSK109). There are 24 unique barcodes if using both expansion kits, allowing the user to pool up to 24 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:
- 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
Prepare your library
You will need to:
- Amplify the viral genome using tiled primers, purify and pool the amplicons
- Repair the DNA, and prepare the DNA ends for barcode 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
- Assemble and analyse the ASF genome using bioinformatics tools of your choice (recommendations included in the "Downstream analysis" sections of the protocol)