Improved influenza A whole-genome sequencing protocol

Abstract

Influenza A virus poses significant public health challenges due to its high mutation rate and zoonotic potential. Whole-genome sequencing (WGS) is crucial for monitoring and characterizing these viruses. Oxford Nanopore Technologies (ONT) and Illumina next-generation sequencing platforms are commonly used, with ONT being advantageous for its long-read capabilities, portability, and unique ability to access raw data in real-time during sequencing, making it suitable for rapid outbreak responses. This study optimizes the ONT Ligation Sequencing Influenza A Whole Genome protocol by refining RT-PCR kits, primers, and purification methods, and evaluating automation for high-throughput processing. The alternative RT-PCR kits, combined with alternative primers, significantly improved read depth coverage and reduced short, untargeted reads compared to the original ONT protocol. The improvement was particularly evident in the minimum read depth coverage of polymerase segments, which often face challenges with achieving uniform coverage, displaying higher coverage at the 5' and 3' termini, and lower coverage in the central regions. This optimized protocol for targeted influenza A WGS not only enhances sequencing quality and efficiency, but is applicable to all NGS platforms, making it highly valuable for studying influenza adaptation and improving surveillance. Additionally, this protocol can be further refined and adapted for the sequencing of other pathogens, broadening its utility in various pathogen monitoring and response efforts.

Keywords: Illumina; MinION; NGS; RT-PCR; WGS; influenza; nanopore; next-generation sequencing.

Figure 1

Sequencing summary for comparison of RT-PCR kits' performance on complete genome and polymerase segments. Average mapped avian influenza A reads for the complete genome (A) and polymerase segments (D) of eight different influenza viruses. Average avian influenza A genome mean read depth for the complete genome (B) and polymerase segments (E). Average avian influenza A minimum read depth for the complete genome (C) and polymerase segments (F). P-value is defined as follows: *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

Figure 2

Sequencing summary for comparison of Tuni and Opti primer sets' performance on complete genome and polymerase segments. Average mapped avian influenza A reads for the complete genome (A) and polymerase segments (D) of six different avian influenza viruses. Average avian influenza A genome mean read depth for the complete genome (B) and polymerase segments (E). Average avian influenza A minimum read depth for the complete genome (C) and polymerase segments (F). P-value is defined as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 3

Read length vs average read quality kernel density estimation distribution plot of sequenced reads prepared with four different purification kits.

Figure 4

Sequencing summary for comparison of four amplicon purification kits' performance on complete genome and polymerase segments. Average mapped avian influenza A reads in the complete genome (A) and in polymerase segments (D) of six influenza viruses. Average avian influenza A genome mean read depth in complete genome (B) and in polymerase segments (E). Average avian influenza A minimum read depth in complete genome (C) and in polymerase segments (F). P-value is defined as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 5

Sequencing summary for comparison of manual and automated amplicon purification performance on complete genome and polymerase segments. Average mapped avian influenza A reads in the complete genome (A) and in polymerase segments (D). Average avian influenza A genome mean read depth in complete genome (B) and in polymerase segments (E). Average avian influenza A minimum read depth in complete genome (C) and in polymerase segments (F). P-value is defined as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.