Copycontrol fosmid autoinduction solution

The pooled clones of Fosmid paired-end sequencing library were cultured and induced to a high copy number by the 500× Copy Control Fosmid Autoinduction Solution (Epicentre) at 37 °C overnight (16–20 h) with 12.5 μg/mL chloramphenicol and 50 μg/mL carbenicillin and shaking (225–250 rpm). Plasmid DNA was extracted using the plasmid large constructed kit (Qiagen) according to the manufacturer’s instructions, digested with I-SceI, and separated on agarose gel. The paired-end fragment fractions of 5–10 kb were recovered, electroeluted and purified. The final samples were concentrated by Ultra-0.5 centrifugal filter devices (Amicon) to a final amount of 30 μg and sent to Frasergen Company for sequencing on the PacBio Sequel platform.

A total of 22 fosmid clones with positive enzyme activities were selected, cultured and induced to a high copy number using the 500 × Copy Control Fosmid Autoinduction Solution (Epicentre, Madison, United States) at 37°C overnight (16–20 h) with 12.5 μg/ml chloramphenicol and vigorous shaking (225–250 rpm). Fosmid DNA was extracted using a GenElute Plasmid Miniprep Kit (Sigma-Aldrich, Missouri, United States) according to the manufacturer’s protocol. The quality of extracted DNA was checked using a Nanodrop and sent to BGI Genomics for whole-genome sequencing. The E. coli whole genome resequencing was performed using the paired-end 150 bp method using the DNBSEQ^™ platform.

As previously mentioned, for fosmid DNA isolation, clones were grown in 100-ml flasks containing LB-Cm broth medium supplemented with 2 μl/ml of CopyControl Fosmid Autoinduction solution (Epicentre) for higher DNA yields, followed by incubation at 37°C for 12 to 16 h and with continuous stirring at 150 rpm. Fosmid DNA was extracted using the FosmidMax DNA purification kit (Epicentre) according to the manufacturer’s instructions and was then subjected to restriction analysis using ApaI enzyme (Thermo Scientific) to determine if different DNA insert sequences were present. Subsequently, fosmids were sequenced on the Illumina NextSeq 500, with a NextSeq 500/550 High Output reagent kit v2 (300 cycles), in accordance with the standard Illumina sequencing protocols. The KneadData pipeline (v. 0.6.1) (http://huttenhower.sph.harvard.edu/kneaddata) was used to perform quality trimming using Trimmomatic (v. 0.38) (42 (link)), and sequences mapping to the cloning host and vector sequences were removed using bmtagger (v. 3.101) (ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/bmtagger/). The remaining reads were assembled using Unicycler (v. 0.4.7) (43 (link)), annotated with Prokka (v. 1.13) (44 (link)), and antibiotic resistance was profiled using the Resistance Gene Identifier web portal (45 (link)).