Sau3a

Inverse PCR was carried out as described by Williams et al [30 (link)], using Sau3A partial digestion of PPCD1 DNA followed by circularization and amplification with primers flanking the breakpoints identified by the prior Roche-Nimblegen CGH analysis. Briefly, 0.6 μg PPCD1 genomic DNA was partially digested with Sau3A (New England Biolabs, Ipswich MA) and ligated with T4 DNA ligase (Promega, Madison WI) at a concentration of 3 ng/μl. PCR primers were designed using Primer3 software. Oligonucleotides were obtained from Integrated DNA Technologies (Coralville IA). PCR was carried out with High Fidelity Polymerase (Roche, www.lifescience.roche.com) according to the manufacturer’s recommendations. Cycling conditions were 94°, 2 min; [94°, 30 sec; 62°, 40 sec; 72°, 1 min] for 40 cycles; 72°, 5 min. PCR products were cloned into pGEMTEasy (Promega, Madison WI). DNA from individual clones was isolated using the FastPlasmid Miniprep kit (5 Prime, www.5prime.com) and sequenced by Sanger sequencing. Breakpoints were identified by divergence from the wildtype genomic sequence at a position other than a Sau3A restriction site.

The metagenomic library was prepared by Apharmas (American Pharma Source LLC, Rockville, MD 20850, USA). For this, the genomic DNA of 1,110 clinical E. coli isolates from GEAR-base was pooled and digested with Sau3A (New England Biolabs, Massachusetts, USA). The digested DNA was size-fragmented twice by agarose gel electrophoresis, and the DNA fragments were selected, purified, and ligated into the pACYC184 vector, pre-cut with BamHI and Shrimp Alkaline Phosphatase (New England Biolabs, Massachusetts, USA). Ligated plasmids were transformed into E. coli DH5-alpha (New England Biolabs, Massachusetts, USA) and E. cloni 10G supreme (Lucigen, Wisconsin, USA). About 50% of the cells of the primary library were used to extract plasmid DNA, which was subsequently column purified forming the metagenomic plasmid library. For QC, PCR was conducted by using pACYC184 specific primers (see Supplementary Table S2). Additionally, an aliquot of the plasmid library was subjected to NGS to further assess and verify homogenous coverage of the pooled 1,110 E. coli genomes.

The genomic DNA of M. abscessus was isolated from a log phase culture using the CTAB method [23 (link)]. The DNA was digested with Sau3A (NEB, Ipswich, MA, USA) and run on 1.5% agarose gel. DNA fragments ranging from 200 to 1500 bp in size were eluted using a gel elution kit (Qiagen, Valencia, CA, USA). The eluted DNA was ligated into the pJEM11 vector, predigested with BamH1, and dephosphorylated using calf intestinal phosphatase (NEB, Ipswich, MA, USA) with an insert-to-vector ratio of 2:1. A ligation mixture was used to transform the chemically competent E. coli DH5α cells and the selected transformants on a Luria Bertani (LB) agar plate containing kanamycin (50 μg/mL).