Minion r9.4.1 flow cell

Reconstruction of the bostrycoidin pathway was performed and described previously [9 ]. An identical workflow was performed for reconstruction of the bikaverin pathway, where all three genes were codon optimized and synthesized. The PPTase genes (Table 1) were codon optimized and synthesized (GenScript Biotech, NJ, USA), before introduction into the pESC-LEU plasmids containing either fsr1 or bik1 as visualized in Fig. 2 along with all codon optimized gene sequencing (Additional file 1: Figure S1). To validate the plasmid constructs, all final plasmids were sequenced, using a R.9.4.1 MinION Flow Cell (Oxford Nanopore Technologies, Oxford, United Kingdom) in accordance to previous work [9 ].Table 1

Overview of the PPTases used in this study

PPTase	Origin	Size (aa)	Accession number
Gsp	B. brevis	242	CAA53988.1
Sfp	B. subtilis	224	WP_101501862.1
SpPPT1	S. pombe	258	SPAC17C9.02c
NpgA	A. nidulans	344	AN6140.2
FgPPT1	F. graminearum	309	FGSG_08779
FsPPT1	F. solani	315	NECHADRAFT_35672
FvPPT1	F. verticillioides	292	FVEG_01894

Fig. 2

Schematic overview of the biomolecular workflow from target gene clusters, through codon optimization and final cluster reconstruction in plasmids and subsequent transformation into S. cerevisiae BY4743

Fifty weevils from the rearing colony, aged between 2.5 and 3.5 months, were used to extract gDNA using the method described above. The concentration was assessed on the Qubit fluorometer (Invitrogen) system using the Qubit dsDNA HS Kit. Prior to library preparation, extraction quality was confirmed on the Agilent 2100 Bioanalyzer using the Agilent DNA 7500 Kit. Libraries were then prepared for both long‐read and short‐read sequencing. The sequencing libraries for the Oxford Nanopore MinION were prepared using the Ligation Sequencing Kit (SQK‐LSK110) following the manufacturer’s recommendations. The resulting library was sequenced on R9.4.1 MinION flow cells (Oxford Nanopore Technologies) for 72 h or to pore exhaustion. A total of five libraries, from the same DNA extraction, were generated and sequenced. Short‐read sequencing library preparation was carried out at McGill and Genome Québec Innovation Center (Montréal, Québec, Canada) and sequenced (paired‐end 2 × 150 bp) on a single lane of NovaSeq 6000 (Illumina).

Sequencing libraries for Oxford Nanopore MinION were prepared, using the direct cDNA Sequencing kit (SQK-DSC109) with the Native Barcoding Expansion 1–12 (EXP-NBD104), following the manufacturer recommendations post cDNA synthesis (Oxford Nanopore Technologies, Oxford, UK). A sequencing library consisted of nine individually amplified female transcriptomes (six on Essex and three on PI88788) from the same population. Before multiplexing the final library, barcoded sample quality and average size were assessed with the Agilent 2100 Bioanalyzer using the Agilent DNA 7500 Kit (Agilent Technologies). A total of 20 fmol of each barcoded sample were pooled before adding 5.0 µL of sequencing adapters. The final library was split and sequenced on two R9.4.1 MinION flowcells (Oxford Nanopore Technologies) for a duration of 72 h or to pore exhaustion. A total of three independent libraries, one for each population sampled, were prepared and sequenced in the same fashion.

We extracted genomic DNA from all the presumptively identified Acinetobacter baumannii complex isolates, prepared double-stranded genomic DNA libraries, and sequenced the libraries on an Illumina platform as previously described (7 (link)). After preliminary analyses of the short-read whole-genome sequencing (WGS) data, we selected representatives of the different A. baumannii lineages identified in our data set and carried out long-read whole-genome sequencing of these isolates using the Oxford Nanopore technology to obtain completely assembled genomes for comprehensive analyses. Genomic DNA was reextracted from the selected isolates using the A&A Genomic Mini AX Bacteria+ kit (A&A Biotechnology, Gdańsk, Poland) to obtain less fragmented DNA. Long-read sequencing libraries were then generated using the Rapid Barcoding Sequencing kit (SQK-RBK004) and sequenced on a MinION Flow Cell (R9.4.1) with MinKNOW version 22.08.9 (Oxford Nanopore Technologies, Inc., Oxford, United Kingdom). We then carried out superaccuracy base calling and demultiplexing on the generated reads using Guppy version 6.3.8.

Using ATCC33701 gDNA, the complete genome sequence was obtained by combining sequencing data from both Illumina MiSeq (Illumina, San Diego, CA, USA) and MinION (Oxford Nanopore Technologies, Oxford, UK) sequencers. Illumina sequencing was performed according to the manufacturer’s instructions. Briefly, an index-tagged library was prepared using the Nextera XT DNA Library Preparation Kit (Illumina), and 300-bp paired-end reads were sequenced on an Illumina MiSeq instrument. Nanopore sequencing was also performed according to the manufacturer’s instructions. Briefly, a DNA library was prepared using a Ligation Sequencing Kit and a Native Barcoding Expansion 1–12 (Oxford Nanopore Technologies). The prepared library was subsequently loaded into a MinION flow cell (R9.4.1; Oxford Nanopore Technologies). The MinION sequencing run was performed over 48 h. Base-calling and barcoding were performed using Guppy v2.3.7 (Oxford Nanopore Technologies). Hybrid assembly of the MiSeq and MinION reads was performed using Unicycler v0.4.2 (28 (link)). Complete genomes were annotated using DFAST (https://dfast.nig.ac.jp/).