Shotgun metagenome sequencing

DNA was extracted from approximately 0.1 gram of stool using the PowerFecal DNA isolation kit by MO BIO (MO Bio Laboratories) per manufacturer recommendations. DNA samples were diluted to approximately 200 ng/mL and Salinibacter ruber genomic DNA was added at a final concentration of 1.4 ng/mL as an internal standard to all samples. Sequencing libraries were generated from microbial DNA using the Nextera XT protocol (Illumina). Sequencing was performed on an Illumina NextSeq500 machine using 150-bp DNA paired end reads to a depth of approximately 4 G base pairs per sample. Data can be accessed at SRA accession PRJNA644399. Raw sequence data was de-multiplexed and converted to fasta format and subjected to downstream analysis. Paired-end sequencing reads from each sample were aligned with Kraken v2⁴¹ against a database consisting of the human genome and approximately 40,054 bacterial, fungal, viral and parasitic genomes. The database was derived from the RefSeq genome database (ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/), human genome (GR38Ch; ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/latest_assembly_versions/), and draft genomes from NCBI Assemblies and PATRIC. Comparison of the overall microbiome composition between groups was performed by multi-response permutation procedure (MRPP) using the Vegan package in R⁴². Data from the human microbiome project 2²⁷ was analyzed with respect to phytase gene abundance in which patient stool was subjected to metagenomic shotgun sequencing and analyzed using the Humann2 pipeline⁴³. Normalized counts were aligned (https://ibdmdb.org/tunnel/public/HMP2/WGS/1818/products; Merged Tables; ecs.tsv.gz) and associated metadata was downloaded from The Inflammatory Bowel Disease Multi’omics Database. Normalized reads aligning to phytase (EC 3.1.3.26) were compared between UC and non-IBD patients (all samples or the last collected sample per subject) by unpaired t-test.