This protocol enables preparation of Influenza A sequencing libraries for Oxford Nanopore Technologies sequencing platforms through a streamlined workflow:

1. One-Step RT-PCR with NEBNext iiMS Flu A Primers (indexed primers 1-48)
2. Sample Pooling of indexed amplicons
3. A-tailing with Klenow Fragment
4. Adapter Ligation with ONT adapters
5. Final Cleanup and library preparation for sequencing

The integrated indexing approach allows multiplexing of up to 48 samples with pooling immediately after the initial RT-PCR step.

• Integrated indexing: Barcodes incorporated during initial RT-PCR
• High multiplexing: Up to 48 samples per run
• Optimized for ONT: Compatible with Ligation Sequencing Kit V14 (SQK-LSK114/SQK-LSK114-XL)
• Comprehensive coverage: Universal primers target all Influenza A segments
• Flexible input: Recommended ≥1,000 copies of Influenza viral genome

This protocol uses NEBNext iiMS Flu A Primers for amplification and indexing. The forward primer contains an index sequence suitable for demultiplexing on the ONT platorm using standard ONT demultiplexing. Sequences are available in primer_sequences.fa. A plate map with barcodes is also available: plate_map.tsv.

To have NEBNext iiMS Influenza A libraries automatically demultiplexed on ONT platforms, you must select the Native Barcoding Sequencing Kit 96 (SQK-NBD-114-96) demultiplexing option when configuring the sequencing run, despite utilizing the Ligation Sequencing Kit V14 (SQK-LSK114) during library preparation.

This kit produces full length influenza A segment sequences that can be aligned to a reference genome using minimap2 or another long read aligner. Consensus sequences can be generated using ViralConsensus or Samtools consensus.