Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples

• Published in: Viruses Vol. 12; no. 8; p. 814
• Main Authors: Zhang, Maodong, Huang, Yanyun, Godson, Dale L., Fernando, Champika, Alexander, Trevor W., Hill, Janet E.
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 28.07.2020; MDPI
• Subjects: Animals; Antibodies, Viral - blood; Bioinformatics; bovine respiratory disease; Cattle; Cattle Diseases - diagnosis; Cattle Diseases - virology; Computational Biology; Dairy cattle; Deoxyribonucleic acid; diagnostics; DNA; Enzymes; Genome, Viral; Genomes; high-throughput nucleotide sequencing; High-Throughput Nucleotide Sequencing - methods; Illumina MiSeq sequencing; Influenza; Influenza D virus; Laboratory animals; Library collections; Metagenome; Metagenomics; nanopore GridION sequencing; Nanopores; Next-generation sequencing; nose; Orthomyxoviridae Infections - diagnosis; Orthomyxoviridae Infections - veterinary; Orthomyxoviridae Infections - virology; qPCR; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Respiratory tract; Respiratory Tract Infections - diagnosis; Respiratory Tract Infections - veterinary; Respiratory Tract Infections - virology; RNA, Viral - genetics; Sensitivity and Specificity; Thogotovirus - genetics; Thogotovirus - isolation & purification; Viruses; United States > US; bovine respiratory disease; influenza D virus; nanopore GridION sequencing; diagnostics; qPCR; Illumina MiSeq sequencing
• DOI: 10.3390/v12080814
• ISSN: 1999-4915

High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: What’s In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.