Nextera xt chemistry

DNA was isolated from bacterial cultures using the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany) using the protocol for Gram-positive bacteria according to the instructions of the manufacturer. The amount of input DNA was quantified on a Lunatic instrument (Unchained Labs, Pleasanton, CA, USA). Sequencing libraries were prepared using Nextera XT chemistry (Illumina Inc., San Diego, CA, USA) for a 300-bp paired-end sequencing run on an Illumina MiSeq sequencer. Samples were sequenced to aim for minimum coverage of 80-fold using Illumina’s recommended standard protocols. The resulting FASTQ files were first quality trimmed and then de novo assembled using SPAdes version 3.9.0. Contigs were filtered for a minimum coverage of 5× and a minimum length of 200 bp using SeqSphere+ software v6.0.0 (Ridom, Münster, Germany).

Frozen bacterial isolates were cultured overnight at 37°C in brain heart infusion broth and genomic DNA was extracted using the Metagenomic DNA isolation Kit for Water (Epicentre, Madison, WI). Samples were prepared using Nextera XT chemistry (Illumina, Inc., San Diego, CA) and were sequenced using Illumina Miseq paired-end read technology using 300 base read lengths. Five strains were selected from the outbreak isolates to serve as references and their reads were de novo assembled using SPAdes v. 3.9 [8 (link)]. The complete genome counterparts for these strains have been reported in a previous work [9 (link)]. Two draft and three complete, unrelated reference genomes were also downloaded from NCBI and included in this analysis making a total of 15 assessed reference genomes (Table 1).

Lower bowel microbiota analysis was performed from fecal pellets collected from each animal both at the beginning (after the acclimatization period, a) and shortly before the end of the treatment experiment, b. 16S rRNA amplicon sequencing was performed for the identification of microbiota composition. The preparation of total DNA, 16S amplicon library preparations, microbiota sequencing (Illumina MiSeq Sequencer) and final bioinformatics analysis were done using Illumina Nextera XT chemistry and the bacterial 16S rDNA v3-v4 region-specific primers for 16S amplicon generation as previously described (80 (link)) (see Supplemental Materials and Methods [Text S1] for details). Briefly, paired sequencing reads obtained from 16S amplicons on a MiSeq instrument (Illumina) were collected, analyzed and searched for various bacterial taxonomic groups, down to genus or species level, using a standardized software pipeline comparing against several appropriate bacterial 16S databases. Principal coordinates analysis (PCoA) on OTU classifications was used for the visualization of beta-diversity. Beta-diversity analysis was performed on subsampled data using Bray-Curtis coefficient. PCoA (beta diversity) and alpha diversity (Shannon-Wiener index) were calculated using mothur version 1.39.5. Significance of overall diversity was calculated using AMOVA.

Prior to WGS, a primary species identification from single colonies was performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker, Billerica, MA, United States) and MBT Compass IVD software 4.1.60 (Bruker) as described by Halbedel et al. (2020) (link).
As for WGS, DNA was isolated from bacterial cultures with the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions for Gram-positive bacteria. The amount of input DNA was quantified on a Lunatic instrument (Unchained Labs, Pleasanton, CA, United States). Nextera XT chemistry (Illumina Inc., San Diego, CA, United States) was used to prepare sequencing libraries for a 300 bp paired-end sequencing run on an Illumina MiSeq sequencer. Samples were sequenced to achieve a minimum 80-fold coverage using recommended standard protocols by Illumina. The resulting FASTQ files were quality trimmed and de novo assembled with the SPAdes version 3.9.0. Contigs were filtered for a minimum of fivefold coverage and 200 bp minimum length with SeqSphere+ software v. 7.8.0 (Ridom, Münster, Germany) (Jünemann et al., 2013 (link)).