Sigma genelute bacterial genomic dna kit

Genomic DNA was extracted using the Sigma GenElute bacterial genomic DNA kit (Sigma-Aldrich; St. Louis, MO). All the genomes were sequenced using paired-end 500 bp insert libraries on the Illumina HiSeq. 4000 and the resulting 150 bp Illumina reads were assembled using SPAdes v.3.7.1^{49 (link)}. The final assemblies were filtered to contain only contigs that were ≥500 bp in length and had ≥5 × k-mer coverage. E. coli strain YDC107 was also sequenced using long-read sequencing to obtain a complete genome assembly, including any possible plasmids as previously described⁵⁰ .

DNA was extracted from bacterial isolates using the Sigma Genelute Bacterial genomic DNA kit (Arklow, Wicklow, Ireland). The primers used in this study are listed in Additional file 1. Universal primers 27 F and 1492R [23 (link)] were used to amplify the 16S rRNA gene in a 50 μl reaction mixture consisting of 45 μl Platinum High Fidelity Supermix (Invitrogen, USA), each primer at 25 μM, 20 ng of template DNA and water to make the reaction up to 50 μl. Amplification conditions for the PCR included an initial denaturation step of 94°C for 2 min, followed by 35 cycles of 94°C for 20 s, 52°C for 30 s, and 68°C for 2 min and a final extension step of 68°C for 10 min. PCR products were checked for size and purity on a 1% (w/v) agarose gel using gel electrophoresis. PCR products were purified with the QIAquick PCR purification kit (Qiagen, USA). DNA sequencing of the amplified 16S rRNA gene region was carried out by Beckmann Coulter Genomics (Takely, UK). Sequence alignments were performed using the ClustalW application in BioEdit [66 (link)]. MEGA (version 5) [67 (link)] was used to construct trees by using the neighbour-joining algorithm and the Kimura two-parameter substitution model. Branch support was measured by 1,000 replicate bootstrap tests for each analysis.

Genomic DNA of each ETEC isolate was extracted from overnight cultures using a Sigma GenElute bacterial genomic DNA kit (Sigma-Aldrich; St. Louis, MO). The genomes were sequenced using paired-end 500-bp insertion libraries and an Illumina HiSeq 4000 system. The 150-bp Illumina reads were assembled using SPAdes v.3.7.1 (49 (link)), and the final assemblies were filtered to contain only contigs that were ≥500 bp in length and had ≥5× k-mer coverage. The assembly metrics are provided in Table S1 in the supplemental material. Additional long-read genome sequencing was performed on a Pacific Biosciences RS II platform (PacBio) as previously described (50 (link), 51 (link)). The characteristics of the complete assemblies are listed in Table S3.