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

A total of 10 clinical swab samples (Run 1) were processed for Sanger sequencing to confirm the presence of IBV lineages. Based on the MinION results, primers targeting GIV-L1 and GI-L1 (GA98 and Conn and primer sets, respectively) were created. The PCR products were cloned and multiple (6-24) clones from each sample were submitted for Sanger sequencing and results are summarized in Table 3 . Using GIV-L1 serotype primers showed that 9 out of 10 samples were positive with 1 sample (#5) negative for IBV (which is consistent with the pan-IBV RT-qPCR results for this sample). Consensus sequences from multiple clones obtained by using GIV-L1 primers showed the presence of GA98 (GA/A9dvaccinated) in 7 of 9 IBV-positive samples. DE072 was detected in samples #1 and #9 as the lone GIV-1 isolate but was also detected in three samples (#2, and #10) that also contained GA98 (Table 4 ). By using the GI-L1 primer set, which amplified Conn and Mass type IBVs, 3 of 9 IBV-positive samples were All rights reserved. No reuse allowed without permission.