Fecal Metagenomics: Microbiome Analysis

Fecal microbiome was analyzed as previously described^{53 (link)}. DNA was isolated using the MagAttract PowerMicrobiome DNA/RNA EP Kit (Qiagen) in an Eppendorf epMotion 5073 automated liquid handling system. Metagenomic libraries were prepared using 30 μl of DNA with the Nextera DNA Flex Library Prep Kit (Illumina). Library preparation was initiated with tagmentation and post-tagmentation cleanup followed by amplification of tagmented DNA and cleanup using Sample Purification Beads for both cleanup steps. The individual libraries were diluted and pooled at a concentration of 7 nM to make the final library. This pool was normalized to 1.8 pM, spiked-in with 1% PhiX sequencing control and sequenced on an Illumina NextSeq 500 instrument. For sequence analysis, MetaPhlAn2 was used to obtain relative abundances. Principal coordinates analysis plots were generated using the Canberra distance metric based on resulting species-level classifications^{54 (link)}. For estimating percentages of Human Microbiome Project (HMP) body site origin, the SourceTracker^{55 (link)} approach was applied by training on MetaPhlAn data from the HMP. Alpha values were tuned via cross-validation, and SourceTracker analyses were performed on genus-level taxonomic classifications.