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 schematic diagram of the workflow of MinION data analysis is presented in Fig. 1 . For Nanopore sequencing data, pre-processing steps were performed to prepare data for downstream analysis. Briefly, Nanopore reads (FAST5) were basecalled using Albacore v 2.02 (https://github.com/Albacore/albacore) with following parameters (read_fast5_basecaller.py -i /Input_reads.fast5 --recursive -t 4 -s /Output_files --flowcell FLO-MIN107 --kit SQK-LSK108o fastq). Porechop (https://github.com/rrwick/Porechop) was used for adapter trimming, (default setting), barcode-based demultiplexing (default settings), and to trim an additional 21 nucleotides, representing the S1 primer sequences (porechop -i Input_file.fastq --extra_end_trim 21 -b ./output_demultiplexed files.fastq). After barcode and adapter removal, reads were analyzed with a script-based, 2-step data analysis protocol, which includes Centrifuge-kreport as taxonomic read classifier 24 using the sequences in the above mentioned BLAST databases.