Amplification of bacterial DNA
16S rRNA genes were amplified with primers targeting the V4 region using the standardized Earth Microbiome 16S Illumina Amplicon protocol15. Samples were processed in batches with appropriate negative controls to ensure there were no contaminants arising from the DNA extraction kits as described earlier16. Following clean up, the amplicon fragment lengths were assessed for quality using Tapestation (Agilent, Santa Clara, CA, USA). DNA was quantified for each amplicon using the Qubit dsDNA HS Assay kit with a Qubit 4.0 fluorometer (Invitrogen). Each DNA library was normalized to a DNA concentration of 4 nM and then pooled to contain 5 μL DNA from each sample. The quality of the pooled DNA sample was assessed on Tapestation and demonstrated an average base pair length of 401 bp. Using the Qubit method described above, the pooled DNA was quantified to an average 1.61 ng/μL.