Dna prep kit

For genome-wide association studies and population genetics, whole genome sequencing was conducted on 365 C. difficile clinical isolates. Of these isolates, 99 were sequenced at University of Texas Dallas Genome Center from multiplexed DNA libraries prepared by the Illumina DNA Prep Kit and sequenced by paired-end 2 × 150 bp on an Illumina NextSeq™500 platform. The remaining 266 isolates were sequenced at SeqCenter, LLC Pittsburgh, using an Illumina DNA Prep Kit and IDT 10 bp UDI indices and sequenced on an Illumina NextSeq 2000 producing 2x151bp reads. Post-sequencing, the metrics from the sequencers were used to guarantee that the number of bases with a quality score of Q30 or higher meets or exceeds the total ordered. The data was demultiplexed and adapters removed using bcl2fastq (v2.20.0.422). Other isolates sequenced in this study were done as above at SeqCenter, LLC.

Total DNA of erythromycin-resistant colonies was purified using the DNeasy Blood & Tissue Kits (Qiagen). Genomic DNA library preparation and sequencing were performed at the SeqCenter (Pittsburgh, PA) by using Illumina NextSeq 2000 and NovaSeq 6000 technology. Sample libraries were prepared using the Illumina DNA Prep kit and IDT 10 bp unique dual indices (UDI). Demultiplexing, quality control and adapter trimming were performed with BCL Convert (Version 4.1.5). To analyze genetic alterations by HGT, paired-end sequencing reads (2 × 151 bp) were assembled by SPAdes (Version 3.15.2)^{62 (link)}. The sequencing reads (2 × 151 bp) were also mapped to the plasmid bearing the thyA gene (shown in Fig. 4a) and the plasmid with transgenes (shown in Fig. 5a), using Bowtie 2 (Version 2.4.5)^{63 (link)}. Raw sequencing data and assembled genomes have been deposited at DDBJ/ENA/GenBank under the accession number: PRJNA754595.

Library preparation, short- and long-read sequencing (Illumina and Oxford Nanopore technologies, respectively), and de novo assembly were performed by the Microbial Genome Sequencing Center (MiGS; Pittsburgh, PA, USA). For DMS3 and JG024 phage reboot clone and PA14 and PAO1 host defense system deletion verification, Illumina NextSeq 2000 sequencing was performed, presented in Table S5. Illumina paired-end reads (2 × 151 bp) were obtained using the Illumina DNA Prep Kit, IDT 10 bp UDI indices, and the Illumina NextSeq 2000 platform (80 (link)). Demultiplexing, quality control, and adapter trimming were performed by MiGS with bcl-convert (v4.0.3). Quality control was checked using FastQC (v0.11.5) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC (v1.11).
ONT sequencing libraries for JG024 WT were prepared using Oxford Nanopore’s “Genomic DNA by Ligation” kit (Oxford Nanopore Technologies, Oxford, UK) and sequenced on a MinION R9 flow cell. Base calling for ONT long reads was performed using Guppy HAC basecalling mode (v4.2.2) (81 (link)). bcl2fastq v2.20.0.445 (82 ) and Porechop v0.2.3_seqan2.1.1 (83 (link)) were used for quality control and adapter trimming for Illumina and ONT sequencing, respectively.