Metagenomics Analysis of Swabs and Sponges

Swab samples were extracted using bead-beating with Lysing Matrix E beads (MP Biomedicals, Burlingame, CA) in RLT-Plus lysis buffer from the AllPrep DNA/RNA isolation kit (Qiagen). DNA extraction was completed using the AllPrep DNA/RNA isolation kit per manufacturer's protocol. Sponge samples were first eluted as previously described,²⁶ (link) then extraction was performed as described above for swabs. Barcoded libraries were prepared using the Qiagen FX DNA Library Kit following manufacturer's protocols and sequenced using Illumina NextSeq500 2×150bp v2 chemistry to a target depth of 10 million read pairs per sample. Positive controls comprising our bacterial mock community and negative controls were also sequenced to a lower target depth of 500,000 reads per sample. Reads from all samples including controls were preprocessed and quality filtered using Trim Galore (available at https://github.com/FelixKrueger/TrimGalore). Host-derived reads were removed using KneadData (available at https://bitbucket.org/biobakery/kneaddata). Taxonomic classification and quantification at the species level was performed using kraken 2 and Bracken.²⁷ (link)^,28 (link) Gene family abundance, pathway abundance, and pathway coverage was calculated using HUMAnN2.²⁹