Metagenomic Next-Generation Sequencing

Abscess samples (a 600-μl volume of each) from all patients were each mixed with 1 g of 0.5-mm diameter glass beads and then placed on a vortex mixer for 30 min at 3,000 rpm. Next, 300 μl of each pretreated sample was subjected to DNA extraction using the TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. Purified DNA was fragmented into 200–300 bp segments using ultrasound followed by end-repair, ligation with multiplex barcode adapters, and PCR amplification to complete construction of DNA libraries. After molarities of DNA libraries were estimated using indexing PCR, DNA concentrations were determined via the DNA Qubit Assay (Thermo Fisher); meanwhile, DNA quality was evaluated electrophoretically using an Agilent 2,100 system (Agilent Technologies, Santa Clara, CA). Up to 20 qualified DNA libraries were pooled, and then pooled libraries were subjected to DNA sequencing analysis using the MGISEQ-2000 platform.