VLP2
Approximately 5 ml saliva was sampled from each of five periodontally healthy people. Five salivary specimens were pooled into one sample and precipitated VLPs using PEG 8000. Briefly, approximately 25 ml pooled saliva was homogenized in equal volumes of PBS solution and then centrifuged at 4,500 g for 30 min to remove cellular debris. The supernatant was serially filtered through 0.45 and 0.22 μm filters. The filtrate was stored overnight at 4 °C after adding PEG 8000 at a final concentration of 10%, and subsequently centrifuged at 13,000 g for 30 min to gather pellets. DNA was isolated from the resultant pellets and amplified with REPLI-g Midi polymerase (Qiagen). Isolated DNA was quantified using a Qubit Fluorometer (Invitrogen) before sheared into about 180 bp fragments with a Covaris Focused-ultrasonicator (Covaris). Libraries were prepared on the sheared fragments using a TruSeq Sample Preparation Kit v2 (Illumina) according to the manufacturer's instructions. Fragment size and integrity of the libraries were assessed by a Fragment Analyzer (Advanced Analytical Technologies). Libraries were sequenced with 2×101 bp paired-end (PE) on an Illumina HiSeq 2000 platform.