Simultaneous detection of DNA and RNA virus species involved in bovine respiratory disease by PCR-free rapid tagmentation-based library preparation and MinION nanopore sequencing

Abstract

AbstactThe Oxford Nanopore MinION Mk1B is a portable 90 g device that sequences DNA directly at 450 bases/second generating sequence reads in excess of 400 kb. Recent improvements in error rate and speed of library preparation mean that this device has considerable potential for rapid molecular bovine pathogen diagnostics. We tested the MinION for rapid untargeted detection of viral pathogens associated with bovine respiratory disease (BRD), an economically important disease often involving primary infection of the lung by one or more of a number of DNA and/or RNA viruses. We combined three foetal lung cell cultures which were infected with either Bovine Respiratory Syncytial Virus (BRSV), Bovine Herpes Virus 1 (BoHV1) or Bovine Parainfluenza Virus 3 (BPI-3). BoHV1 is a DNA virus and BPI-3 and BRSV are RNA viruses. The cell cultures were treated with DNase and RNase to deplete bovine nucleic acid prior to viral nucleic acid extraction and double-stranded cDNA synthesis. Sequencing libraries were generated by PCR-free tagmentation in under 10 minutes and loaded onto a MinION sequencer. Approximately 7,000 sequencing reads were generated and analysed using high-throughput local BLAST against the NCBI nr/nt database. The top BLAST hit for 2,937 of these reads was identified as a virus. Of these, 2,926 (99.6%) were correctly identified either as BoHV1, BRSV or BPI-3.