Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples.

Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
Salman L ButtJames B Stanton

Abstract

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accu...Continue Reading

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Citations

Mar 10, 2021·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·Laura Goodman, Kevin Lahmers

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Datasets Mentioned

BETA
FJ904716.1
AF274439.1

Methods Mentioned

BETA
PCR
genotyping
MinION sequencing
electrophoresis
AmpSeq
amplicon sequencing

Software Mentioned

ClustalW
BLAST
MEGA
QIIME
AmpSeq
Biomatters
Mothur
Kraken
Porechop
MinION

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