Design and validation of a universal influenza virus enrichment probe set and its utility in deep sequence analysis of primary cloacal swab surveillance samples of wild birds

Abstract

Influenza virus infections in humans and animals are major public health concerns. In the current study, a set of universal influenza enrichment probes was developed to increase the sensitivity of sequence-based virus detection and characterization for all influenza viruses. This universal influenza enrichment probe set contains 46,953 120nt RNA biotin-labeled probes designed based on all available influenza viral sequences and it can be used to enrich for influenza sequences without prior knowledge of type or subtype. Marked enrichment was demonstrated in influenza A/H1N1, influenza B, and H1-to-H16 hemagglutinin plasmids spiked into human DNA and in cultured influenza A/H2N1 virus. Furthermore, enrichment effects and mixed influenza A virus infections were revealed in wild bird cloacal swab samples. Therefore, this universal influenza virus enrichment probe system can capture and enrich influenza viral sequences selectively and effectively in different samples, especially ones with degraded RNA or containing low amount of influenza RNA.

ProbeTools: designing hybridization probes for targeted genomic sequencing of diverse and hypervariable viral taxa

“…Consequently, practical viral genome sequencing requires Open Access *Correspondence: kevin.kuchinski@bccdc.ca targeted enrichment for confident detection and accurate genotyping, especially in high-throughput surveillance and clinical applications [1][2][3]. Hybridization probe capture methods have been used for viral target enrichment [4][5][6][7], but designing probe oligo sequences for many viruses can be a major obstacle due to extensive genomic diversity and hypervariability within and between viral taxa [8][9][10][11][12][13].…”

“…Another strategy is to align candidate probe sequences against select reference genomes to identify and retain only those probes targeting divergent genotypes [8]. Redundancy has also been addressed by constraining the design space to a limited number of representative reference genomes, selected either by manual curation or clustering [9][10][11][12]. Some of these strategies have been supplemented with multiple sequence alignments over hypervariable loci or entire genomes so that probes are designed from consensus and degenerate sequences [9,10].…”

“…H1N1, H3N2, H5N1, H7N9). Furthermore, each of these subtypes has diverged into numerous clades, many of which have been only partially characterized [12,18,19].…”

ProbeTools: Designing hybridization probes for targeted genomic sequencing of diverse and hypervariable viral taxa

“…This threatens economic disruption, future pandemic crises, and new types of seasonal influenza, which remains an important global health burden and among the ten leading causes of death worldwide [12,[21][22][23][24][25][26][27][28][29][30][31]. Consequently, surveillance of AIVs in wild birds is a cornerstone of outbreak prevention and pandemic preparedness [12,20,[32][33]. An effective panel of AIV-specific probes would be instrumental for these genomicsbased surveillance efforts.…”

“…Consequently, practical viral genome sequencing requires targeted enrichment for confident detection and accurate genotyping, especially in high-throughput surveillance and clinical applications [1][2][3]. Hybridization probe capture methods have been used for viral target enrichment [4][5][6][7], but designing probe oligo sequences for many viruses can be a major obstacle due to extensive genomic diversity and hypervariability within and between viral taxa [8][9][10][11][12][13].…”