Flowcell r9

In order to recover large-size DNA, genomic DNA of CREC strains were purified from liquid culture using the Genomic DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, US), according to the standard protocol (https://files.zymoresearch.com/protocols/). In the next step, rapid barcoding sequencing kit (SQK-RBK004 version 6, Oxford Nanopore) was used according to the manufacturer’s protocol for genomic DNA. The sequencing was carried out on a portable MinION device using a flow cell R9.4.1(Oxford Nanopore). Local basecalling was performed using the Guppy (version 3.1.5) (Oxford Nanopore) with the option enabled to trim the sequencing adapters. NanoFilt program (version 2.0.0) was used to filter out the reads with Phred-score < 7 and a length < 1000 and the statistics of the reads and quality scores were extracted with NanoStat (version 0.8.0). Finally, NanoLyse software (version 0.5.0) was used to remove the control DNA [38 (link)].

The sequencing of PCR-cDNA libraries was carried out using a MinION device (Oxford Nanopore Technologies, MIN-101B) equipped with a compatible Flow Cell R9.4.1 (Oxford Nanopore Technologies, FLO-MIN106D). Each loading library consisted of one cellular library and one EV library in a 2:1 ratio, respectively, and to a final loading concentration of ~ 100 fmol. This ratio was chosen as cellular sequencing libraries were expected to be comparatively more complex to EV sequencing libraries. Briefly, 66.66 fmol of cell library was combined with 33.33 fmol of EV library, and the volume was adjusted to 11 µL of Elution Buffer (EB). Next, 1 µL of Rapid Adapter (RAP) was added to the combined libraries, followed by a room temperature incubation of 5 min. The flow cell was then primed and loaded following the manufacturer’s recommendations. Primary data acquisition was performed using the MinION Software (Oxford Nanopore Technologies, MinION Release 22.05.5) with default parameters, but no local basecalling. The sequencing of the loaded libraries was allowed to proceed for 72 h, after which the resulting FAST5 files were further processed bioinformatically.

DNA isolation was performed by the CTAB method [27 (link)]. WGS was carried out using the Nextera DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and MiSeq Reagent Kits v3 (Illumina, San Diego, CA, USA) for the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Long reads were obtained using the Rapid Barcoding Kit RBK004 and flowcell R9.4.1 on the MinION platform (Oxford Nanopore, Oxford Science Park, GB). Basecalling was performed with Guppy ver. 5.0.16 (Oxford Nanopore, Oxford Science Park, UK) with default parameters [28 ]. Short and long raw reads were used to obtain the hybrid assembly of the strain using Unicycler ver. 0.4.7 software (The University of Melbourne, Melbourne, Australia) with default settings that included primary filtering and quality control [29 (link)]. Annotation was carried out by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) ver. 5.3 (National Center for Biotechnology Information, Bethesda, MD, USA) [30 (link)].