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

One area that is problematic for a test such as AmpSeq is to detect all the isolates that may be present. While AmpSeq did detect all isolates in 4 samples, in 3 cases not all of the isolates were detected. A potential explanation to partial detection of multiple IBV genotypes could be the relative abundance of IBV genotypes in these clinical samples. It could also be that amplification of these IBV genotypes by serotype-specific RT-qPCR assays is more efficient due to small targeted fragment size (e.g., 120 bp for PCR and ~1600 bp for AmpSeq) and better primer alignment to the target (e.g., degenerate bases are used in the S1 primers used for AmpSeq). One complicating factor of the current AmpSeq protocol is that the IBV target sequence 27 was not originally designed for high specificity, especially from clinical samples. As such, a high proportion of total reads were non-IBV reads (e.g., often mapping to the chicken genome, data not shown), consistent with the extra bands visible in the original report for these All rights reserved. No reuse allowed without permission.