Spoton flow cell

Bacterial genomic DNA was extracted from 10 ml overnight cultures of P. distasonis APCS2/PD using phenol/chloroform with precipitation in 3 M sodium acetate and cold absolute ethanol for the generation of long- and short-read sequences for hybrid assembly. DNA was quantified using Qubit BR DNA Assay Kit. Short-read shotgun sequencing of the extracted DNA was carried out using the Accel-NGS 1S Plus DNA Library Kit (Swift Biosciences, MI, USA) and Illumina HiSeq 4000 technology following manufacturer’s instructions. Long-read Oxford Nanopore library preparation was performed according to the manufacturer protocol for Rapid Barcoding Sequencing Kit (SQK-RPK004; Oxford Nanopore Technologies, UK) with the adaptions described by Guerin et al., (2021).^{23 (link)} Pooled samples were loaded into SpotON Flow Cell (Oxford Nanopore Technologies, UK) and MinION sequenced for 48 hours (Oxford Nanopore Technologies,UK). Hybrid assembly of quality-filtered and trimmed Illumina and raw Nanopore reads was conducted by hybridSPAdes (v1.13.1).^{91 (link)} Nine scaffolds greater than 1 kb were generated and were manually curated, joined, and circularized using CLC Sequence Viewer. The assembled and circularized genome and associated plasmid were then submitted to NCBI Prokaryotic Genome Annotation Pipeline. The GenBank file for the genome and associated plasmid was visualized using Proksee.^{72 (link)}

The purification of mRNA from total RNA samples was achieved using the Dynabeads mRNA Purification kit (Thermo Fisher Scientific). The subsequent reverse transcription reaction was performed using SuperScript IV reverse transcriptase (Thermo Fisher Scientific). Preparation for RNA sequencing was carried out using the low-input PCR barcoding kit and the cDNA-PCR Sequencing kit (Oxford Nanopore Technologies), which 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). Only D²-Reads with a quality score above eight were used for further alignment. The quality score is measured based on the basecalling algorithm albacore (Nanopore), defined as −10 × log10 (probability of incorrect base call). Reads were rearranged in accordance to their barcode and trimmed using Porechop (https://github.com/rrwick/Porechop), with alignment performed using minimap2 (https://github.com/lh3/minimap2) and postprocessed by sam-tools. Mapped reads were normalized by DESeq (Love et al, 2014 (link)). The expression matrix was analyzed with AutoPIPE (https://github.com/heilandd/AutoPipe), a supervised machine-learning algorithm and visualized in a heat map as described previously (Henrik Heiland et al, 2019) (link).

The purification of mRNA from total RNA samples was achieved using the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The subsequent reverse transcription reaction was performed using SuperScript IV reverse transcriptase (Thermo Fisher Scientific). For preparation of RNA sequencing, the Low Input by PCR Barcoding Kit and the cDNA-PCR Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK) 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) according to the manufacturer’s instructions. Base calling was performed by Albacore implemented in the nanopore software (Oxford Nanopore Technologies). 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). Alignment was performed by minimap2 (https://github.com-/lh3/minimap2) and processed by sam-tools. Mapped reads were normalized by DESeq2 [32 (link)]. The expression matrix was analyzed with AutoPipe (https://github.com/heilandd/AutoPipe) by a supervised machine-learning algorithm and visualized in a heatmap. (Data available in GEO: in preparation).