Multiomic Pathogen DNA Extraction and Sequencing

DNA was extracted from all samples using a QIAamp® UCP Pathogen DNA Kit (Qiagen) following the manufacturer’s instructions. Human DNA was removed using Benzonase (Qiagen) and Tween20 (Sigma) [6 (link)]. 10 nanograms DNA samples were used for library construction through Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) [7 (link)]. Library was qualitatively assessed by Qubit dsDNA HS Assay kit, followed by High Sensitivity DNA kit (Agilent) on an Agilent 2100 Bioanalyzer. Library pools were then loaded onto an Illumina Nextseq 550Dx sequencer for 75 cycles of single-end sequencing to generate approximately 20 million reads for each library. For negative controls, we also prepared peripheral blood mononuclear cell(PBMC) samples with 10⁵ cells/mL from healthy donors in parallel with each batch, using the same protocol, and sterile deionized water was extracted alongside the specimens to serve as non-template controls (NTC) [7 (link), 8 (link)]. DNA-free water went through DNA extraction and mNGS analysis as a blank control group to assess the degree of background contamination associated with DNA extraction kit and sequencing reagents together.