Rs 2

Genomic DNA from BiotGm was prepared using the procedure reported^{66 (link)}. The BiotGm genome was sequenced using the Pacific Biosciences RS II (Pacific Biosciences, Menlo Park, CA, USA) sequencing platform. The sequencing depth was ~200-fold coverages. De novo assembly of PacBio reads was carried out using RS_HGAP_Assembly.3 protocol included in SMRT portal. Genome annotation was performed by the NCBI Prokaryotic Genome Automatic Annotation Pipeline. Composition vector tree (CVTree) phylogenetic analysis was performed using a free, web-based tool-the Bacillus Typing Bioinformatics Database (https://btbidb.com)^{67 (link)}. The genome data of other B. thuringiensis strains were selected in the database to generate the phylogenetic tree.

Genomic DNA extraction, library preparation, and whole-genome sequencing on a NextSeq 500 platform (Illumina, Inc.), using 2 × 150-bp chemistry, was performed as previously described (24 (link)). One isolate (designated NZ-SC16875) underwent genomic DNA extraction with the GenElute bacterial genomic DNA kit (Sigma-Aldrich), followed by sequencing on the RSII (Pacific Biosciences) with P6-C4 chemistry according to the 20-kb template preparation using the BluePippin size selection system protocol (Pacific Biosciences). Sequences were initially analyzed using the Nullarbor pipeline (T. Seeman; https://github.com/tseemann/nullarbor). Phage regions were detected using Phaster (25 (link)). Maximum likelihood trees were inferred using IQ-TREE (26 (link)). Recombination was detected using Gubbins (version 2.2.0-1) (27 (link)), and population structure was investigated using hierarchical Bayesian analysis with hierBAPS (28 (link)). Additional information on bioinformatic analyses is provided in the Supplemental Methods.

Isolates were grown on Eugon agar from frozen culture. After 48–72 hours of incubation at 37°C in 5% CO₂, colonies were selected and plated on to fresh Eugon agar to ensure pure culture for extraction. DNA was extracted (Epicentre Masterpure kit, Epicentre, Madison, WI) at 48–72 hours postinoculation and sequenced using NexteraXT library prep (Illumina, San Diego, CA) on a Miseq (Illumina). For long read sequencing DNA was extracted (DNeasy Blood and Tissue kit, Qiagen, Hilden, Germany) libraries prepared with 15kb-20kb insert size (BluePippin kit) and sequenced on an RS II (Pacific Biosciences, Menlo Park, CA) with 2 SMRT cells per sample.

WGS was performed on the NextSeq 500 (Illumina) using 2 × 150 bp chemistry. One isolate (S80062MN28) was subjected to long-read sequencing on the RS-II (Pacific Biosciences). Bioinformatic approaches and analyses included assembly, annotation and comparative genomics. The global S. aureus phylogeny was inferred using publicly available genomes. Antibiotic resistance and virulence gene detection was performed using the NCBI antimicrobial resistance database (ncbi: updated 20 September 2018) and the virulence factor database (vfdb: updated 14 August 2018). All sequence data generated for this study have been made publicly available through the European Nucleotide Archive, project accession PRJEB31151.

S. rimosus ATCC 10970 was sequenced using a hybrid approach combining three next-generation sequencing platforms to balance their shortcomings and strengths; the Illumina GAIIx sequencer was used for obtaining short-read sequences of high quality, and the Pacific Biosciences RS II (PacBio) sequencer was used for obtaining the long reads needed for tackling repetitive regions scattered throughout the genome. These were used together for genome sequencing, while Oxford Nanopore technology was solely used for the sequencing of the plasmids.
Short-read sequencing was performed at Macrogen, Inc. (Daejeon, Republic of Korea). At least 1 μg gDNA was used to construct the short-read genome-sequencing library with the TruSeq DNA PCR-fREE LT kit (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s instruction.
Long-read genome sequencing for strain ATCC 10970 was performed with PacBio technology at Macrogen, Inc. (Daejeon, Republic of Korea). At least 2 μg gDNA was used as input for PacBio genome sequencing library preparation. The sequencing library was prepared according to a guide for preparing 20-kb SMRTbell template prep kit (Pacific Biosciences, Menlo Park, CA, USA). The templates were sequenced using SMRT sequencing. Library preparation and its quality control were performed at Macrogen, Inc.

The complete genome sequence was determined for E. hirae R17, and published previously (Peng et al., 2016a). The genome of E. hirae R17 was sequenced using the Pacific Biosciences RS II sequencing platform (Pacific Biosciences, Menlo Park, CA, USA). Single‐molecule real‐time (SMRT^®) sequencing was conducted using the C4 sequencing chemistry and P6 polymerase with one SMRT^® cell. De novo assembly of the PacBio reads were carried out using continuous long reads (CLR) following the hierarchical genome assembly process (HGAP) workflow (PacBioDevNet; Pacific Biosciences) as available in SMRT^® Analysis v2.3 (Chin et al., 2013). The functions of predicted proteins were annotated based on homologs (using SwissProt, http://www.uniprot.org/uniprot/), and clusters of ortholog groups were determined (using COG, http://www.ncbi.nlm.nih.gov/COG/). The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was employed to identify coding sequences (CDS) based on the best‐placed reference protein set and GeneMarkS+. The complete genome sequence of E. hirae R17 was deposited in GenBank under the accession number CP015516 (chromosome) and CP015517 (plasmid pRZ1).