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

Viruses

TrevorW. Alexander¹  Champika Fernando²  JanetE. Hill²  Yanyun Huang³  Maodong Zhang³  DaleL. Godson⁴ 

[1] Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Lethbridge, AB T1J 4B1, Canada;Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N5B4, Canada;Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada;Prairie Diagnostic Services Inc., Saskatoon, SK S7N5B4, Canada;

关键词: Illumina MiSeq sequencing;    influenza D virus;    nanopore GridION sequencing;    qPCR;    diagnostics;    bovine respiratory disease;   

DOI  :  10.3390/v12080814

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown