Abstract

Adding/removing text [Throat samples immersed inactivating preservation solution were mixed with lysis buffer (Thermo Scientific, Massachusetts, United States), protease K (Yeasen, Shanghai, China), and binding buffer (Thermo Scientific, Massachusetts, United States). This mixture was subjected to mechanical disruption via a vortex mixer and beads for a duration of 30 s. Meanwhile, a positive control and a negative control were set up to monitor the whole experiment process (from the nucleic acid extraction step) of thecapturebased NGS. The positive control consisted of a mixture of Staphylococcus aureus (103 CFU/mL) and Peripheral Blood Mononuclear Cells (PBMCs) at a concentration of 105 cells/mL, derived from healthy donors. The negative control comprised PBMCs (105 cells/mL) obtained from healthy donors. Following this, a simultaneous extraction of Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) was performed utilizing the VAMNE Magnetic Pathogen DNA/RNA Extraction Kit (Vazyme,Nanjing,China). Nucleic acid quantification was conducted employing a Qubit 3.0 fluorometer, utilizing the double-stranded DNA (dsDNA) and RNA high sensitivity assay kits. For the synthesis of cDNA and the preparation of the sequencing library, the HieffNGS®C37P4 OnePot cDNA & gDNA Library Prep Kit (Yeasen, Shanghai, China) was utilized, adhering to the manufacturer's instructions. Targetsequence enrichment was achieved by incubating the samples with a set of 1,872 microorganism-specific probes (GenePlus, Beijing, China) for an approximate duration of 4 h, followed by an 18-cycle PCR amplification of the captured products. The probes target 1,124 bacteria, 218 fungus, 157 DNA viruses, 317 RNA viruses, and 56 parasites. The amplified products were then processed to form DNA nanoballs (DNBs). Sequencing was executed on the DNBSEQ-G99 platform with 100-bp single-end reads, targeting a sequencing depth of 5 million reads.]. A correction has been made to the section [2 Methods, 2.2 Sampl