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.