title: Y25_saliva
design: For each saliva sample, a volume of 1 mL was extracted using Qiagen Blood Mini Kit according to the manufacturers instructions. Libraries from saliva DNA were prepared with the Accel-NGS 2S Plus DNA Kit (Swift Biosciences), following the manufacturers instructions. Libraries were either prepared manually or automated on a Bravo NGS Workstation (Agilent). The starting amount of DNA for library preparation was normalized to 5ng. DNA shearing was performed on either a Covaris S220 or E220 focused-ultrasonicator with the following settings: Peak Power: 175, Duty Factor: 5.0, Cycles/Burst: 200, Run Time: 90s. All libraries were dual-barcoded, using the 2S Dual Indexing Kit (Swift Biosciences). For PCR amplification, which selectively enriches for library fragments that have adapters ligated on both ends, the PCR cycles were normalized to 8 for all libraries with a starting amount of 5ng of DNA. Finished libraries were quantitated using Promega's QuantiFluor dsDNA assay and the average library size was determined on an Agilent Tapestation 4200. Library concentrations were then normalized to 4nM and validated by qPCR on a QuantStudio-3 real-time PCR system (Applied Biosystems), using the Kapa library quantification kit for Illumina platforms (Kapa Biosystems). Libraries were then pooled at equimolar concentrations and sequenced on an Illumina HiSeq2500 sequencer in rapid mode and a read-length of 251bp paired-end (Illumina V2 Rapid sequencing reagents).