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Introduction to the influenza whole genome sequencing protocol
This protocol has been upgraded to our Kit 14 chemistry and describes how to carry out PCR amplification and native barcoding of influenza amplicons using the Native Barcoding Kit 24 or 96 V14 (SQK-NBD114.24 or SQK-NBD114.96). There are 96 unique barcodes available, allowing the user to pool up to 96 different Influenza A and/or Influenza B samples in one sequencing experiment.
While this protocol is available in the Nanopore Community, we kindly ask users to ensure they are citing the following references, that this protocol is based on.
Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza A viruses by Bin Zhou et al., 2009. and Universal influenza B virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics by Bin Zhou et al., 2014.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Ensure you have the 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:
- Perform RT-PCR amplification on influenza samples
- 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 basecall the reads
- Demultiplex barcoded reads in MinKNOW choosing the SQK-NBD114.24 or SQK-NBD114.96 kit option
- Analyse your data using the Influenza typing workflow (wf-flu) in EPI2ME Labs.