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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/634600 doi: bioRxiv preprint First, the basecalled reads were aligned to the IBV-lineage-typing database using BLASTn and reads were clustered based on the read sequence alignment to the respective prototype sequence of IBV lineage. These read clusters were used to interpret presence of IBV genotypes and lineages in the samples. For isolate level identification of IBV, the lineage-based read clusters were individually aligned to the All-IBV database, which produced sub-clusters of reads. Each of these read sub-clusters potentially represents a different isolate and was further used for interpretation. Knowing that MinION sequencing has a high sequencing error rate in individual reads, further steps were added in the data analysis algorithm to obtain a more accurate consensus sequence. Therefore, these read sub-clusters were mapped, using Geneious mapper in Geneious software v11.1.3. (Biomatters, Ltd, Auckland, New Zealand), to the IBV-lineagetyping database (a reference FASTA file) to obtain consensus sequences from each sub-cluster.