Nextera xt library preparation kit

Libraries for NGS were prepared from 1 ng cfDNA using the Nextera XT library preparation Kit (Illumina) according to the manufacturer’s protocol, with the exception that the final elution after bead clean-up was carried out in 34 μl of resuspension buffer (Illumina). A further contamination control was added by using 5 μl of molecular biology grade water (5 Prime, Germany) as template for the Nextera XT library preparation Kit (Illumina). Sequencing of the libraries was performed on a HiSeq2500 (Illumina), resulting in 25–30 million 100-bp single end reads, on average, per sample. Since samples V6, V22, and P6 were initially sequenced with considerably higher numbers of reads, those samples were randomly reduced in silico to 30 million representative subsampled reads.

DNA was extracted using the QIAmp DNA stool minikit (Qiagen, Crawley, West Sussex, United Kingdom)^{40 (link)} following the manufacturer’s instructions with minor modifications and stored at – 80 °C until its use. Prior to shotgun sequencing, a Qubit High Sensitivity DNA assay (BioSciences, Dublin, Ireland) was used to determine the total DNA concentration, and purity was assessed by the 260/280 and 260/230 absorbance ratios using a spectrophotometer NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, Delaware). Paired-end sequencing libraries were prepared from the extracted DNA using the Illumina Nextera XTLibrary Preparation Kit (Illumina Inc., San Diego, California) followed by sequencing 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.

Immediately before (day 20) and after enrofloxacin treatment (day 24), and 2 weeks after antibiotic withdrawal (day 37), one bird per replicate was sacrificed and digesta samples from the cecum were collected in Eppendorf cups, immediately snap frozen (liquid N2), and stored at −20°C. The animals were euthanized with pentobarbital IV (sodium pentobarbital 20%, Kela, Hoogstraten, Belgium) dosed at approximately 100 mg/kg.
DNA was extracted using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Antwerp, Belgium), which as shown in our recent studies, has minimum effects on the microbial community structure and promising results in terms of the DNA integrity for chicken cecum samples (Wegl et al., 2021 (link)). The purity of the DNA was assessed by evaluating the 260/280 and 260/230 absorbances using NanoDrop^® (ND-1000, Thermo Fisher Scientific, Merelbeke, Belgium), while Quantus^® (Promega, Leiden, The Netherlands) was used to estimate the DNA concentration. Finally, DNA integrity was visually validated by gel electrophoresis. The DNA was sent to LGC Genomics GmbH, Berlin, Germany, where paired-end sequencing libraries were constructed using the Illumina Nextera XT Library Preparation Kit (Illumina Inc., San Diego, CA) followed by sequencing on the Illumina NextSeq 500 platform using high-output chemistry (2 × 150 bp) according to the manufacturer’s instructions.