Minion mk1b sequencing

According to the manufacturer’s instructions, library preparation was performed using the Rapid Barcoding Sequencing Kit (SQK-RBK004) (Oxford Nanopore Technologies). The constructed libraries were loaded into R9.4.1 (FLO-MIN106D) flow cells and sequenced on a MinION Mk1B sequencing device for approximately 24 h using the MinKNOW software 22.03.5 (Oxford Nanopore Technologies). We sequenced a random selection of three plasmid DNA samples twice, obtained from the same bacterial cultures, to determine the intrinsic variations of sequencing and bioinformatic analyses. Basecalling was carried out with Guppy 6.0.6 (https://community.nanoporetech.com) in a fast basecalling model. Raw data were demultiplexed, and adapters and barcodes were trimmed using Porechop 0.2.4 with default parameters (26 ). Then, raw reads were filtered with Filtlong 0.2.1 using a minimum read length threshold of 1 kbp and keeping 95% of the best reads (measured by bases). Filtered reads metrics were assessed using NanoPlot 1.40.0 (27 (link)).

Whole genome sequencing was carried out using the Illumina platform with a paired-end sequencing strategy (HiSeq-PE150) by a commercial service provider (Novogene Co., Ltd., Singapore). Quality inspection was performed using FastQC v0.11.8 and MultiQC v1.7 [87 (link)]. Sequencing using the Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK) platform was performed at the Monash University, Malaysia’s Genomics Facility. Briefly, high quality genomic DNA was subjected to Ligation Sequencing Kit SQK-LSK109 library preparation based on the manufacturer’s protocol. The DNA library was then sequenced using a MinION R10.3 flow cell on a MinION Mk1B sequencing device (Oxford Nanopore Technologies). The quality of the reads was determined using CheckM 1.2.0 (https://github.com/Ecogenomics/CheckM accessed on 1 September 2022). Base calling was conducted using Guppy v3.2.10 using a high accuracy configuration and configured to include trimming adapter sequences.
Hybrid assembly of the Illumina and Nanopore sequence data was performed using Unicycler v0.5.0 [88 (link)]. The assembled genome sequence was annotated using PROKKA [89 (link)] and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [90 (link)] upon submission to GenBank.