Long-Read Sequencing and Genome Assembly

High molecular weight DNA was sheared in a Covaris g-TUBE (Covaris, Woburn, MA, USA) to obtain an average fragment size of 10 kb. After shearing the DNA, size distribution was checked on a Fragment Analyzer (Advanced Analytical Technologies, Ames, IA, USA). Five hundred nanograms of the DNA was used to prepare a SMRTbell library with the PacBio SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer’s recommendations. No size selection was applied. The pooled bar-coded libraries were sequenced with v3.0/v3.0 chemistry and diffusion loading on a PacBio Sequel instrument (Pacific Biosciences, Menlo Park, CA, USA) at 600 min movie length, pre-extension time of 120 min, using one SMRT cell 1 M v3.
Genome assembly was performed using CANU 2.0 with the option ‘pacbio-raw’ and a defined expected genome size of 5 Mbp^{72 (link)}. The circularization of the genomes was achieved using Circlator v.1.5.5 with default parameter settings^{73 (link)}. Genes were predicted using Prokka v.1.14.6^{74 (link)} but, during the submission of the sequenced genomes, reassigned through the NCBI Prokaryotic Genome Annotation Pipeline (PGAP, version 4.11). Sequencing details and NCBI accession numbers are summarized in Supplementary Data file 4.