The taxonomic composition of the human microbiome of CRC patients and healthy donors (human reads removed)
In this study, the beneficial effect of ketogenic diet in the context of colorectal cancer was analyzed using a CRC mouse model with a humanized gut microbiome. For this purpose, single homogenized human stool samples from five healthy donors and one CRC patient were analyzed by shotgun sequencing to generate a baseline for the establishment of humanized microbiome to be transferred into mice. Briefly, fecal DNA was isolated from these samples using the DNeasy PowerSoil DNA Isolation Kit (Qiagen), according to the kit manual, except that samples were homogenized using the TissueLyzerII for five minutes at 40Hz instead of intense vortexing. The extracted DNA was further purified and concentrated using the DNA Clean and Concentrator Kit (Zymo Research). This DNA was thawed and refrozen once at -80°C for 16s sequencing before being thawed again for WGS. 200 ng of DNA was used for metagenomic library preparation using the Next Generation Sequencing DNA Library Prep Kit (Westburg), according to the user manual using an average insert size of libraries of 450?base pairs. Following quantification and quality control (Qubit® dsDNA BR Assay Kit (Thermofisher Scientific); Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent Technologies)), sequencing was performed on a NextSeq500 instrument using 2×150 base pair read lengths. The achieved 5HD mix recapitulated the compositions of the individual donor samples, as evidenced by family, genus and species level analysis of whole genome shotgun sequencing. The mix was further used to colonize germ-free and antibiotic-treated SPF mice. Human reads were filtered out using KneaDdata using hg37 and GHM13.