New England Biolabs, E3436S, NEBNext Flu A Integrated Indexing Primer Module

NEBNext Flu A Integrated Indexing Primer Module is intended for use in cDNA synthesis, amplification, and library preparation upstream of Influenza A (Flu A) sequencing. Libraries generated with the Primer Module are meant for sequencing with the Oxford Nanopore Technologies Related Categories Infectious Diseases & ARTIC Sequencing,, Influenza Sequencing,, Next Generation Sequencing Library Preparation FAQ Q: Which additional reagents are needed for cDNA synthesis and amplification with the NEBNext Flu A Integrated Indexing Primer Module (NEB #E3436)? A: NEB recommends pairing the NEBNext Flu A Integrated Indexing Primer Module with the LunaScript® Multiplex One-Step RT-PCR Kit (NEB #E1555). A protocol for the use of these reagents for Flu A amplicon generation can be found here. Q: Where can I find NEBNext Flu A Integrated Indexing Primer (NEB #E3436) sequences? A: The NEBNext Flu A Integrated Indexing primer sequences can be found here: https://github.com/nebiolabs/NEBNextFluA. Q: Which Flu A strains can be targeted with the NEBNext Flu A Integrated Indexing Primers (NEB #E3436)? A: The universal Influenza A targeting aspect of the primers included in this kit can be used for identification and sequencing of a range of Influenza A strains, such as H3N2, H1N1, and H5N1, from various host sources (e.g., Human, Avian, Bovine, etc.). Q: Which library prep reagents and protocols can be used for sequencing cDNA amplicons made with the NEBNext Flu A Integrated Indexing Primer Module (NEB #E3436)? A: 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. Q: Is there a recommended Ct value-range for Flu A inputs when using the NEBNext Flu A Integrated Primer Module (NEB #E3436)? A: NEB recommends inputs of at least 1,000 genome copies. As Ct values vary by assay type and the genome target of the assay, we do not have a specific Ct value range. Q: What is the minimum read depth needed for Flu A sequencing when using the NEBNext Flu A Integrated Primer Module (NEB #E3436)? A: For ONT sequencing on a MinION flow cell, NEB recommends a minimum read depth of 100,000 reads per Flu A library. For Illumina sequencing, NEB recommends a minimum read depth of 0.5 M PE reads per Flu A library. Q: What sample types can the NEBNext Flu A Integrated Indexing Primer Module (NEB #E3436) be used with? A: Protocols for use with the NEBNext Flu A Integrated Indexing Primer Module have been validated with high quality, commercially available gRNA templates. Additional sample types that have been tested include cell culture RNA isolates and RNA extracted from clinical samples (e.g. nasopharyngeal swabs). Q: What extraction methods are recommended for use in the NEBNext Flu A sequencing workflow when using the NEBNext Flu A Integrated Primer Module (NEB #E3436)? A: RNA isolated using NEB’s Monarch® Mag Viral DNA/RNA Extraction Kit (NEB #T4010S) and Monarch® Spin RNA Isolation Kit (Mini) (NEB #T2110) are recommended. Other bead-based and column-based RNA extraction approaches can also be used. Q: The ONT SQK-LSK114 kits include AMPure® XP Beads, but are additional beads needed to run the NEBNext Flu A Integrated Indexing Primer Module (NEB #E3436) ONT® library prep workflow? A: As of late 2025, ONT® SQK-LSK114 kits come with 1,200 µl of AMPure XP beads, a sufficient volume for running the NEBNext Flu A Integrated Indexing ONT® library prep workflow. SQK-LSK114-XL kits do not come with sample purification beads.