Easysampler collection kit

Stool samples were collected by participants at home prior to the clinic visit using an EasySampler collection kit (ALPCO) and put into faecal collection tubes containing DNA/RNA Shield buffer (Zymo Research). A total of N = 1001 stool samples were collected and processed for shotgun metagenomic sequencing of which n = 854 with snacking data were included in this secondary analysis. In brief, DNA was isolated by QIAGEN Genomic Services using the DNeasy 96 PowerSoil Pro kit and libraries were prepared for 300-bp paired-end reads and sequenced using the Illumina NovaSeq 6000 platform with the S4 flowcell according to the manufacturer’s protocols. The microbiome methodology including DNA extraction and sequencing, metagenome quality and pre-processing, microbiome taxonomic and functional potential profiling and metagenomic assembly have been previously described [21 ] and have been updated to MetaPhlAn 4.0 [38 (link)] for taxonomic profiling of species-level genome bins (SGBs) [39 (link)], and to HUMAnN 3.5 for functional profiling [40 (link)].

Participants collected a stool sample at home prior to their clinical visit using the EasySampler collection kit (ALPCO). Upon receipt at the laboratory, samples were homogenised, aliquoted and stored at − 80 °C in Qiagen PowerBeads 1.5-ml tubes (Qiagen) as previously described [27 ].