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

Salman L. Butt; Eric C. Erwood; Jian Zhang; Holly S. Sellers; Kelsey Young; Kevin K. Lahmers; James B. Stanton

For example, cost and time efficiency of a sequencing protocol can be improved through multiplexing of more samples in a single sequencing run. 3, 47 In this study, samples (Run 1 = 10, Run 2 = 5) were simultaneously multiplexed (i.e., pooled and then sequenced in one run) while maintaining IBV genotyping from data collected. Since the MinION flow cells were not exhausted, and can be washed and re-used, the AmpSeq method also has the potential for sequential multiplexing. This would decrease the need to hold samples for weeks while waiting for the cost-optimal number of samples for simultaneous multiplexing. Alternatively, if detection of all isolates within a given sample is preferred, the sequencing can be run longer to increase sensitivity. The single protocol nature of AmpSeq, the ability to obtain S1 gene sequence results, real-time data analysis, and flexibility of testing design makes MinION-based AmpSeq a viable sequencing protocol for typing of IBV isolates.