Epi2me desktop agent

500 ng of poly(A)-selected mRNA was used as input for the Direct RNA Sequencing Kit (SQK-RNA002, Oxford Nanopore Technologies), used as directed with a modified reverse transcription (RT) step. Marathon reverse transcriptase (kindly gifted from Dr. Kathleen Collins) was used for the RT instead of Superscript III. The RT reaction was performed in 1X first strand buffer (20 mM Tris-HCl pH 7.5, 75 mM KCl, and 5 mM MgCl₂), with 0.8 mM dNTPs, 8 mM DTT, and 20 μM Marathon reverse transcriptase. The RT reaction was incubated at 37°C for 50 min then 70°C for 10 min. Downstream steps were followed according to kit instructions. The library was loaded onto an R9.4.1 flow cell (FLO-MIN106, Oxford Nanopore Technologies) and sequenced on a minION (MIN-101B, Oxford Nanopore Technologies). MinKNOW (v22.05.5, Oxford Nanopore Technologies) was run without live base calling for 72 hours. Bases were called from fast5 files using Guppy (v6.0.1, Oxford Nanopore Technologies). Reads were aligned to the S288C reference genome (SacCer3) using the EPI2ME Desktop Agent (v3.5.6, Oxford Nanopore Technologies). Bam files were visualized directly in Integrated Genomics Viewer (IGV, Broad Institute).

Oxford Nanopore sequencing libraries of FPV-COMB and FPV-CAMs were prepared using SQK-LSK108 and SQK-LSK308 kits respectively as per the manufacturer’s instructions. Sequencing was allowed to run for 24 hours on a MinION MK-1b device fitted with a FLO-MIN107 (R9.5 chemistry) flow cell. Raw 1D data in fast5 format was base called using guppy_basecaller with basecall model of high accuracy (version 4.0.15) (Oxford Nanopore Technologies) using the University of Melbourne High-Performance Cluster, Spartan [22 ]. Porechop (version 0.2.3) (https://github.com/rrwick/Porechop) was used to trim the adapter sequences. FASTQ WIMP (version 2021.03.05) analysis was performed using the EPI2ME Desktop agent (version 3.3.0) (Oxford Nanopore Technologies) to investigate the taxonomic diversity of both isolates (FPV-COMB and FPV-CAMs). Nanopore reads with a minimum length of 1000 bp were extracted from a set of FASTQ files using Filtlong (version 0.2.0) (https://github.com/rrwick/Filtlong). Trimmed sequence reads were mapped against the Gallus gallus reference genome (GenBank NC_006088) using Minimap2 (2.17-r941) [23 (link)] to remove chicken genome-associated reads from the data. Unmapped reads from this step were mapped against a Staphylococcus aureus reference genome (GenBank AP017922.1) to remove the major bacterial taxa sequenced.