Pathogen DNA/RNA Extraction and NGS Library Preparation

To perform NGS, the first step was extraction of nucleic acid. The corresponding kit to extract pathogen DNA or RNA, and then Benzonase (Qiagen) and Tween 20 were used to remove the human-derived sequences [8 (link)] using the Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA), which removed ribosomal RNA following RNA extraction. Complementary DNA (cDNA) was then produced using reverse transcriptase and deoxyribonucleotide triphosphate (dNTPs). DNA and cDNA were used to construct the library, using the appropriate library preparation kit [9 (link)]. The Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for library quality assessment. The samples were then sequenced for 75 cycles of single-end data using a sequencer, and each library eventually produced 20 million reads. In this process, blood samples from healthy people at a concentration of 10⁵ cells/mL were used as negative controls, and sterile deionized water was used as non-template controls [10 (link)].