Minknow software

After cells were cultivated for 48 h, purification of RNA was achieved using the RNeasy Mini Kit (Qiagen). The reverse transcription reaction was performed using the Maxima H Minus Reverse Transcriptase. For preparation of RNA sequencing, the PCR Barcoding Kit and cDNA-PCR Sequencing Kit (Oxford Nanopore Technologies) were utilized as recommended by the manufacturer. RNA sequencing was performed using the MinION Sequencing Device, the SpotON Flow Cell and MinKNOW software (Oxford Nanopore Technologies) according to the manufacturer’s instructions. Base calling was performed by Albacore implemented in the nanopore software. Only D2-reads with a quality score above 8 were used for further alignment. Reads were re-arranged in accordance to their barcode and trimmed by Porechop (https://github.com/rrwick/Porechop (accessed on 15 December 2020)). Alignment was performed by minimap2 (https://github.com/lh3/minimap2 (accessed on 15 December 2020)) and processed by sam-tools. Mapped reads were normalized by DESeq2. The expression matrix was analyzed with AutoPipe (https://github.com/heilandd/AutoPipe (accessed on 15 December 2020)) by a supervised machine-learning algorithm. Visualization was performed using VisLab Expression Data Viewer (https://github.com/heilandd/Vis_Lab1.5 (accessed on 15 December 2020)).

The purification of mRNA from total RNA samples was achieved using the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, Carlsbad, USA). The subsequent reverse transcription reaction was performed using SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Carlsbad, USA). For preparation of RNA sequencing, the Low Input by PCR Barcoding Kit and the cDNA-PCR Sequencing Kit (Oxford Nanopore Technologies, Oxford, United Kingdom) were used as recommended by the manufacturer. RNA sequencing was performed using the MinION Sequencing Device, the SpotON Flow Cell and MinKNOW software (Oxford Nanopore Technologies, Oxford, United Kingdom) according to the manufacturer’s instructions. Samples were sequenced for 48 h on two flow-cells acquire a total sequence length of 16.38 Gbp (mean read length 689 bp, ~4 million reads). Basecalling was performed by Albacore implemented in the nanopore software. Only D²-Reads with a quality Score above 8 were used for further alignment.

For library construction (Figure 5C), DNA contamination was removed from total RNA extracted using the RapidOut DNA Removal Kit (Thermo Fisher Scientific, Waltham, MA, USA). QIAseq FastSelect rRNA Plant Kit (Qiagen, Hilden, Germany) was used to remove wheat rRNA (ribodepletion) and purified with AMPure XP beads (Beckman Coulter, Brea, CA, USA). The purified RNA concentration was measured using a Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were prepared using the Direct cDNA Sequencing Kit (SQK-DCS109; Oxford Nanopore Technologies, Oxford, UK) following the manufacturer’s instructions and previous studies [31 (link),53 (link)]. The prepared libraries were loaded on one Flongle Flow Cell (FLO-FLG001, R9.4.1; Oxford Nanopore Technologies, Oxford, UK) per sample, and the sequencing run was started on a MinION device with a Flongle adapter (Oxford Nanopore Technologies, Oxford, UK). Sequencing was performed for 24 h using MinKNOW software (version 21.11.7; Oxford Nanopore Technologies, Oxford, UK) and base-calling in High-accuracy mode with Guppy (version 5.1.12; Oxford Nanopore Technologies, Oxford, UK). Sequencing adapters were removed with Geneious Prime (version 21.1.1). Viruses were identified in ‘What’s in my Pot?’ (WIMP) workflow of EPI2ME Agent (version 3.4.2) and NCBI BLASTn using FASTQ files generated by the ONT platform.

Amplicon sequencing targeting the full length of the 16S rRNA gene was performed using MinION equipped with R9.4.1 flow cell (Oxford Nanopore Technologies, Oxford, UK). A 16S rRNA sequencing library was constructed from 10 µL of extracted DNA using the 16S barcoding kit (Oxford Nanopore Technologies). The library construction was performed according to the manufacturer's instructions except that DNA amplification was carried out using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, MA, USA) with the following thermal cycling conditions: 2 min at 95 °C, 25 cycles of 20 s at 98 °C, 30 s at 60 °C, and 2 min at 72 °C, and 5 min at 72 °C. Sequencing was carried out with Oxford Nanopore's MinKNOW software and basecalls were performed using Guppy (v. 4.3.4) in fast mode using the config file dna_r9.4.1_450bps_fast.cfg. Generated FASTQ files were further analyzed for taxonomic classification using the cloud-based EPI2ME FASTQ 16S workflow with a quality score ≥ 7 for quality filtering.

Sequencing was done using the Oxford Nanopore Technologies MinION Mk1B (MIN-101B) sequencer and Spot-ON Flow Cell, R9 version (FLO-MIN106D). Oxford Nanopore’s MinKNOW software (v21.11.9) was used to read from the flow cell and base-call the reads (Additional file 2: Working Protocol 8).
During sequencing, ARTIC’s RAMPART software (v1.0.6) was used to monitor the reads on each barcode separately and the respective coverage of the Mpox genome (Additional file 2: Working Protocol 9).

For Nanopore sequencing a 1D sequencing library (SQK-LSK108) was loaded onto a FLO-MIN106 (R9.4) flowcell, connected to the MinION Mk1B (Oxford Nanopore Technology, Oxford, United Kingdom). MinKNOW software (version 1.11.5; Oxford Nanopore) was used for quality control of active pores and for sequencing. Raw files generated by MinKNOW were base called, on a local compute server (HP ProLiant DL360 G9, 2x XEON E5-2695v3 14 Cores and 256 RAM), using Albacore (version 1.2.5; Oxford Nanopore). Reads, in fastq format, with a minimum length of 1000 bps were extracted, yielding 5.45 Gigabase sequence with an average read length of 9.09 kb.