The integrated indices appended to Flu A cDNA amplicons are compatible with long-read sequencing on ONT platforms and ONT Native Barcoding demultiplexing protocols. The protocol and reagents necessary for sequencing NEBNext Flu A amplicons on Oxford Nanopore Technologies platforms, can also be found here.
If the cDNA amplicons (900 bp to 2,500 bp in length) are fragmented and treated as unindexed DNA inputs, they can be used in short-read sequencing approaches. For short read sequencing on Illumina platforms, NEB recommends pairing the NEBNext Flu A Integrated Indexing Primer Module with the LunaScript® Multiplex One-Step RT-PCR Kit (NEB #E1555), NEBNext UltraExpress® FS DNA Library Prep Kit (NEB #E3340), and NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 1-5) (see neb.com/oligos). Follow the instructions in Section 1 of the NEBNext Flu A Integrated Indexing Primer Module manual to generate cDNA, and then follow the UltraExpress® FS DNA Library Prep Kit manual to prepare Illumina Flu A libraries.
