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® (ONT) Native Barcoding workflow.
Compatible with various Influenza A strains (e.g., H3N2, H1N1, H5N1) from various hosts (e.g., Human, Avian, Bovine)
Integrated indexing enables pooling of up to 48 samples following RT-PCR
For laboratories interested in sequencing the Influenza A genome, streamlined and specific cDNA synthesis is an essential first step. The NEBNext Flu A Integrated Indexing Primer Module is a curated set of 48 plated primer pairs for use in RT-PCR upstream of NGS library preparation. Downstream, the libraries prepared are compatible ONT Native Barcoding*. The positioning of the integrated indices in the primer sequences decreases the risk for variants to impact amplification efficiency. Refer to the product manual for the full list of required materials not supplied.
The NEBNext Flu A Integrated Indexing Primer Module has undergone extensive QCs to ensure that the primer pairs are balanced, well-aligned with clinically relevant Flu A sequences, and free of cross contamination. In-house testing includes the full recommended workflow, combining RT-PCR, NGS library prep, and sequencing followed by analysis of sequencing metrics.
Due to its modular nature, the NEBNext Flu A Integrated Indexing Primer Module provides flexibility for maximal ease of use. Full information on the primer sequences included can be found at https://github.com/nebiolabs/NEBNextFluA.
*See FAQs for information about sequencing libraries prepared with the NEBNext Flu A Integrated Indexing Primer Module on Illumina® sequencers.
Figure 1: NEBNext Flu A Integrated Indexing Primer Module library prep workflow for Oxford Nanopore Technologies sequencing
Figure 2: Technical overview of NEBNext Flu A Integrated Indexing Primer Module library prep for Oxford Nanopore Technologies sequencing
The NEBNext Flu A Integrated Indexing Primer Module is designed to be paired with LunaScript® Multiplex One-Step RT-PCR Kit (NEB #E1555) for streamlined cDNA synthesis and targeted amplification, followed by library preparation with Oxford Nanopore Technologies Ligation Sequencing Kit (SQK-LSK114).
Note: The optional use of these Flu A cDNA amplicons as unindexed inputs for Illumina library prep is also supported using the NEBNext UltraExpress® FS DNA Library Prep Kit (NEB #E3340). Figure 3: NEBNext Flu A Integrated Indexing Primer Module paired with LunaScript Multiplex One-Step RT-PCR Kit for cDNA synthesis and amplification provides robust amplicon yields for Influenza A genome sequencing
Indexed full-length cDNA genome segments were generated from 10,000 copies of Influenza H3N2 gRNA (ATCC VR-1680) templates, with 1 ng Universal Human Reference RNA (UHR) background. The one-step cDNA synthesis and amplification reaction was applied using the NEBNext Flu A Integrated Indexing Primers paired with the LunaScript Multiplex One-Step RT-PCR Kit (NEB #E1555). Indexed amplicons were subsequently pooled and cleaned up prior to ONT® library preparation (SQK-LSK114). TapeStation® HSD5000 gel image (A) and trace of pooled influenza H3N2 template with 10,000 copies of Influenza A RNA (B). The table in Panel B identifies the different segments of the Influenza A genome and their approximate sizes. Figure 4: NEBNext Flu A Integrated Indexing Primer Module generates high overall genome coverage of Influenza A with Oxford Nanopore Technologies sequencing
Indexed full-length cDNA genome segments were generated from 10,000 copies of Influenza H3N2 gRNA (ATCC VR-1680) templates, with 1 ng Universal Human Reference RNA (UHR) background. The one-step cDNA synthesis and amplification reaction was applied using the NEBNext Flu A Integrated Indexing primers paired with the LunaScript Multiplex One-Step RT-PCR Kit (NEB #E1555). Indexed amplicons were subsequently pooled and cleaned up prior to ONT library preparation (SQK-LSK114). Libraries were loaded onto a FLO-PRO114M Flow Cell (R.10.4.1) and sequenced on a PromethION®. Sequencing reads were downsampled to 200,000 reads with seqtk and aligned with MiniMap2 to a composite Influenza consensus reference genome. A) Boxplot showing the mean mapped read counts per genome segment for each of 48 barcoded samples (n = 2). B) IGV visualization of read coverage across Influenza A genome segments from a single barcode (0–10,000 log scale). Figure 5: NEBNext Flu A Integrated Indexing Primers can detect multiple Influenza A strains from a broad range of genome copies
Indexed full-length cDNA genome segments were generated from non-template controls (NTCs), H3N2 gRNA (ATCC VR-1882) or Influenza H1N1 gRNA (ATCC VR-1894) using NEBNext Flu A Integrated Indexing Primers paired with the LunaScript Multiplex One-Step RT-PCR Kit (NEB #E1555). Genome copy number inputs of 100, 1,000 and 10,000 were used for both gRNA control templates, with 1 ng Universal Human Reference RNA (UHR) background. A one-step cDNA synthesis and amplification reaction was applied and indexed amplicons were subsequently pooled and cleaned up prior to ONT library preparation (SQK-LSK114). Libraries were loaded onto a MinION® Flow Cell (R.10.4.1) and sequenced on a GridION®. Sequencing reads were downsampled to 200,000 reads with seqtk and aligned with MiniMap2 to a composite Influenza consensus reference genome. A) TapeStation HSD5000 gel image of the post-cleanup pooled cDNA samples and individual samples (NTCs and Influenza A samples). B) Heatmap of the average number of reads mapping to Influenza A genome segments across the Influenza A gRNA templates utilized.
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