Ampure magnetic beads

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).

A total of 255 saliva samples were used for DNA extraction and sequencing, from participants whose parents had consented to oral microbiota analysis. Genomic DNA was extracted from saliva samples in a clean facility at the University of Adelaide using the Roche High Pure PCR Template Preparation Kit (Roche Life Sciences). Two extraction blank controls (EBCs, i.e., empty tubes) were included for every 22 saliva samples. The V4 region of the bacterial 16S rRNA gene was amplified using uniquely barcoded reverse primers for each sample, as previously described [34 (link)]. No-template controls (NTCs) were processed alongside each amplification. Amplified, barcoded DNA was quantified using Qubit (ThermoFisher Scientific), pooled at equal relative concentrations, cleaned using Ampure magnetic beads (New England Biolabs), and quantified using TapeStation (Agilent). Paired-end 150 bp sequencing was performed using an Illumina MiSeq.