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

It was observed that the single read cluster composed of less than 5 reads (Run1, sample #4, GA98) yielded a poor-quality consensus sequence, consistent with the known individual read error rate of MinION sequencing. High-quality consensus sequences were obtained from the other read clusters (>5 reads per cluster). Each of the obtained consensus sequences were compared to GenBank databases using BLAST, which revealed >99% sequence identity to respective IBV isolates Table 4 ). As described in the Sanger Sequencing section, two samples (#8 and #9) contained only one IBV isolate per the non-MinION assays, and the AmpSeq results were consistent with these findings. Non-MinION assays showed that seven samples contained two or more isolates per sample. Six samples (#1, #3, #4, #6, #7, and #10) contained two isolates per sample (Table 4 ). Of those six samples, AmpSeq detected both isolates in four samples (#4, #6, #7, and #10), one isolate in one sample (#3), and no IBV in one sample (#1; as mentioned above, sample #1 was the sample with the highest Cq value). Of note, sample #10 contained two isolates from the same lineage and AmpSeq was able to identify both isolates within this sample.