Megaruptor 2

Genomic DNA was sheared to 20 kb fragments using the Megaruptor 2 (Diagenode) and size selected with a 10 kb cut-off using the BluePippin system (Sage Science). 4 µg of sheared and size-selected DNA was then used for Nano-OTS library preparation as described by Höijer et al.^{33 (link)}. A detailed description of all the steps of the Nano-OTS protocol is available from protocols.io (https://www.protocols.io/view/nano-ots-bp5smq6e). The sequences for all 23 gRNAs investigated by Nano-OTS are available in Supplementary Table S1. To increase coverage, two separate libraries were prepared and sequenced on one R9.4.1 flow cell each. Guppy v4.0 was used for base calling.

SMRTcell libraries were constructed for samples passing quality control (Supplementary Data 2) according to the manufacturer’s instructions of the SMRTcell Express Prep kit v2.0 following the Low DNA Input Protocol (Pacific Biosciences, Menlo Park, CA) as described in^{74 (link)}. Genomic DNA was sheared to 20-kb fragments using Megaruptor 2 (Diagenode, Belgium) and then bead-size selected with AMPure PB beads (Pacific Biosciences) to remove <3-kb SMRTbell templates. SMRT sequencing was performed on the Sequel System II with Sequel II Sequencing kit 2.0 (Sequel Sequencing kit 2.1 for Sequel I system, see below) in ‘circular consensus sequencing’ (i.e., CCS) mode, 30 h movie time with pre-extension and Software SMRTLINK 8.0. Samples were barcoded using the Barcoded Overhang Adapters Kit-8A, multiplexed, and sequenced (3 samples/SMRT Cell) at the Genome Technology Center (RGTC) of the Radboud university medical center (Nijmegen, the Netherlands). Four samples were instead sequenced on the Sequel I system at BGI Genomics Co. Ltd. (Shenzhen, China) (Supplementary Data 2). In this case, one SMRT Cell was run for each sample.

Based on the results of short-read sequencing (see below), twenty-five KPC producers were selected to be sequenced using long-read sequencing technology, to help close the whole plasmid sequences. These isolates were selected as representatives of all different hospitals, bacterial species, STs, replicon profiles and KPC alleles.
Genomic DNA was extracted from the clinical isolates using NucleoSpin Microbial DNA kit (Macherey–Nagel, Germany). Whole genome sequencing (WGS) was performed on the Sequel I platform (Pacific biosciences, Menlo Park, CA, United States). Microbial multiplexing protocol was used for the library preparation according to the manufacturer instructions for Sheared DNA. DNA shearing was performed using the Megaruptor 2 (Diagenode, Liege, Belgium) using long hydropores producing 10 kb long inserts. No size selection was performed during the library preparation. The Microbial Assembly pipeline offered by the SMRT Link v9.0 software was used to perform the assembly and circularization with minimum seed coverage of 30X. Assembled sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).

Genomic DNA was extracted from the four clinical isolates using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany). Whole genome sequencing (WGS) was performed on the Sequel I platform (Pacific biosciences, Menlo Park, CA, United States). Microbial multiplexing protocol was used for the library preparation according to the manufacturer instructions for Sheared DNA. DNA shearing was performed using the Megaruptor 2 (Diagenode, Liege, Belgium) using long hydropores producing 15kb long inserts. No size selection was performed during the library preparation. Microbial Assembly pipeline offered by the SMRT Link v8.0 software was used to perform the assembly and circularization with minimum seed coverage of 30×. Assembled sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Antibiotic resistant genes, plasmid replicons, mobile elements and multilocus sequence types (MLST) were determined through uploading the assembled sequences to ResFinder 4.1 and CARD (Zankari et al., 2012 ; Alcock et al., 2020 (link)), PlasmidFinder (Carattoli et al., 2014 ), ISfinder (Siguier et al., 2006 (link)), and MLST 2.0 (Larsen et al., 2012 ), respectively. Comparative genome alignment was done using Mauve v.2.3.1.² and BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011 (link)). Diagrams and gene organization were sketched using Inkscape 0.92.4³.

Genomic DNA (5 µg) was sheared to ~5–25-kilobase fragments using Megaruptor^® 2 (Diagenode, B06010002) and was then size-selected (10–30 kilobases) with a BluePippin device (Sage Science, MA) to remove the small DNA fragments. Subsequently, genomic libraries were prepared using the Ligation Sequencing 1D Kit (SQK-LSK108, Oxford Nanopore, UK). End-repair and dA-tailing of DNA fragments were performed using the Ultra II End Prep module (New England Biolabs, E7546L), according to manufacturer’s recommended protocols. Finally, the purified dA-tailed sample was incubated with blunt/TA ligase master mix (#M0367, NEB), tethered with 1D adapter mix from the SQK-LSK108 Kit (Oxford Nanopore Technologies), and purified. The resulting library was sequenced on R9.4 flow cells using GridION X5.

Leaves of Abrus precatorius were flash-frozen in liquid nitrogen after propagation and harvesting by Matthew Johnson at the School of Plant Sciences at University of Arizona. Samples were shipped to Los Alamos National Lab on dry ice. The origin of this cultivar is from Abrus precatorius 010017 collected by S. Bentz in 2001 at Mount Carbine, S 16°31′, E 145°08′, Queensland, Australia. High Molecular weight genomic DNA was extracted from the leaf material using a modified CTAB (cetyl trimethylammonium bromide) extraction protocol. Genomic DNA was analyzed for DNA quality and subsequently sheared using the Megaruptor2 instrument (Diagenode, Ougrée, Belgium) with a target size of 60 kb. Multiple SMRT bell templates were prepared according to PacBio (Pacific Biosciences, Menlo Park, CA, USA) SMRT bell template prep protocol for templates longer than 15 kb. Libraries were size-selected on Blue Pippin (Sage Science, Beverly, MA, USA) instrument using marker S1, with 7 and 10 kb lower cutoff. Size selected DNA was collected and purified using AMPure PB beads (Beckman Coulter, Indianapolis, IN, USA). Sequencing primer was annealed and DNA polymerase bound to the SMRT bell templates. The libraries were sequenced on PacBio RSII DNA sequencer using C4/P6 chemistry.

Based on the results of short-read sequencing (see below), 19 VIM producers were selected to for long-read sequencing, to help close the whole plasmid sequences. These isolates were selected as representatives of all different hospitals, bacterial species, STs, replicon profiles and bla_VIM alleles.
Genomic DNA was extracted from the clinical isolates using NucleoSpin Microbial DNA kit (Macherey–Nagel, Germany). Whole genome sequencing (WGS) was performed on the Sequel I platform (Pacific biosciences, Menlo Park, CA, United States). Microbial multiplexing protocol was used for the library preparation according to the manufacturer instructions for Sheared DNA. DNA shearing was performed using the Megaruptor 2 (Diagenode, Liege, Belgium) using long hydropores producing 10 kb long inserts. No size selection was performed during the library preparation. The Microbial Assembly pipeline offered by the SMRT Link v9.0 software was used to perform the assembly and circularization with minimum seed coverage of 30X. Assembled sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).