Ileal Digesta and Fecal Microbiome Profiling

All ileal digesta and fecal samples obtained at postmortem were subjected to 16S rRNA gene metabarcoding targeting the V3 hypervariable region, as described previously (30 (link)). DNA concentrations of the purified libraries were then measured using a Qubit 3.0 fluorometer (Thermo Fisher Scientific, United Kingdom) using a Qubit double-stranded DNA high-sensitivity assay kit (Thermo Fisher Scientific). Using the readings obtained by the Qubit instrument, four library pools were constructed using equimolar concentrations of DNA from each sample. A reagent-only control and mock bacterial community (HM-782D; BEI Resources, ATCC, Manassas, VA) were included as part of each sequencing run to assess background contamination, sequencing error rate, and inter-run variability. On submission to the sequencing center (Edinburgh Genomics, United Kingdom), library pools were quantified using a Quant-iT PicoGreen double-stranded DNA assay kit (Thermo Fisher Scientific) to ensure a sufficient yield for sequencing. Sequencing was carried out using an Illumina MiSeq platform (Illumina, USA), using V2 chemistry and producing 250-bp paired-end reads. The generated sequences (with primers removed) are available publicly through the European Nucleotide Archive (ENA) under accession number PRJEB33396.