Dental Calculus Metagenomic Analysis

Genomic DNA was extracted from dental calculus in clean laboratory facilities at the Australian Centre for Ancient DNA using an in-house in-solution silica-binding method [34 (link)]. Extraction blank controls (EBCs) were processed alongside samples for each extraction, with an average of two EBCs for every five samples. Barcoded amplicon libraries targeting the V4 region of the prokaryotic 16S ribosomal RNA (rRNA) encoding gene region were constructed as previously published [35 (link)], with no-template amplification controls processed alongside the biological samples. Double-stranded DNA was quantified for each sample using Qubit (ThermoFisher Scientific). Polymerase chain reaction (PCR) products were pooled at equal relative concentrations, cleaned using Ampure magnetic beads (New England Biolabs) and quantified using TapeStation (Agilent) and quantitative PCR, then combined into a single DNA sequencing library. Paired-end 150 bp sequencing was performed on an Illumina MiSeq at the Australian Genome Research Facility (AGRF).