Influenza viruses are divided into four distinct types, A, B, C, and D. While influenza A and influenza B are primarily responsible for seasonal epidemics of disease in humans, influenza A virus (IAV) is the only species known to cause pandemics (https://www.who.int/news-room/fact-sheets/detail/influenza-(seasonal)). IAV is further differentiated into subtypes based on the combination of hemagglutinin (HA) and neuraminidase (NA) surface proteins present. There are currently 18 HA and 11 NA subtypes described (https://www.cdc.gov/flu/about/viruses-types.html). Influenza is an enveloped virus with a genome composed of eight negative-sense, single-stranded RNA segments encoding 10 major proteins. These include the subtype related HA and NA proteins, the nucleoprotein (NP), the three subunits of an RNA-dependent RNA polymerase complex (PB1, a transcriptase; PB2, which recognizes 5’ caps; PA, an endonuclease), the matrix (M1) and membrane (M2) proteins, which share one segment, as do the non-structural protein (NS) and the nuclear export protein.Whole genome sequencing (WGS) strategies have emerged in recent years and have been used to inform the global influenza surveillance program [1], [2], [3]. The use of WGS to obtain all eight complete influenza segments is advantageous for monitoring and analyzing the complete genetic makeup of the virus to understand its evolution, tracking the spread of different strains, and informing vaccine development (https://www.cdc.gov/flu/php/viruses/genetic-characterization.html). Conserved RNA termini found in all eight negative-sense, single-stranded RNA segments of the influenza genome have enabled the design of 5’-tailed universal primer sets, allowing for efficient WGS PCR amplification [4], [5]. During viral replication, however, defective interfering particles (DIPs) containing defective viral genomes (DVGs) that share the same universal termini as the full-length genomic segments are generated [6]. The presence of these DVGs potentially leads to imbalanced amplification of the eight influenza genome segments during WGS PCR [7]. In this study, to achieve our national influenza surveillance goal, we evaluated several optimization strategies to promote more balanced amplification of all influenza segments prior to Oxford Nanopore Technologies (ONT) library preparation and sequencing (Table 1). Specifically, we tested primers, PCR cycling conditions, and enzymes to mitigate amplification bias, as well as different size-selection bead ratios to enrich for PCR amplicons corresponding to full-length gene segments prior to library preparation. ONT sequencing was chosen as the WGS strategy as it enables real-time data analysis, rendering it particularly beneficial for surveillance and diagnostic efforts.