Journal
VACCINES
Volume 10, Issue 8, Pages -Publisher
MDPI
DOI: 10.3390/vaccines10081359
Keywords
whole genome sequencing (WGS); next-generation sequencing (NGS); clinical specimen; molecular surveillance
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Funding
- NANOMAX Bandiera project (2013-2015) by ItalianMinistry for Education, University and Research [G42I12000180005]
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The study successfully validated a rapid and reliable high-throughput sequencing protocol for obtaining the complete genome sequence of A(H3N2) influenza viruses directly from clinical specimens, providing important reference for influenza surveillance and outbreak investigation.
(1) Background: Over the last few years, there has been growing interest in the whole genome sequencing (WGS) of rapidly mutating pathogens, such as influenza viruses (IVs), which has led us to carry out in-depth studies on viral evolution in both research and diagnostic settings. We aimed at describing and determining the validity of a WGS protocol that can obtain the complete genome sequence of A(H3N2) IVs directly from clinical specimens. (2) Methods: RNA was extracted from 80 A(H3N2)-positive respiratory specimens. A one-step RT-PCR assay, based on the use of a single set of specific primers, was used to retro-transcribe and amplify the entire IV type A genome in a single reaction, thus avoiding additional enrichment approaches and host genome removal treatments. Purified DNA was quantified; genomic libraries were prepared and sequenced by using Illumina MiSeq platform. The obtained reads were evaluated for sequence quality and read-pair length. (3) Results: All of the study specimens were successfully amplified, and the purified DNA concentration proved to be suitable for NGS (at least 0.2 ng/mu L). An acceptable coverage depth for all eight genes of influenza A(H3N2) virus was obtained for 90% (72/80) of the clinical samples with viral loads >10(5) genome copies/mL. The mean depth of sequencing ranged from 105 to 200 reads per position, with the majority of the mean depth values being above 103 reads per position. The total turnaround time per set of 20 samples was four working days, including sequence analysis. (4) Conclusions: This fast and reliable high-throughput sequencing protocol should be used for influenza surveillance and outbreak investigation.
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