title: 16S rRNA-seq of human urine: PPROM25
design: Microbial DNA was extracted from urine samples using the QIAamp DNA Microbiome Kit (QIAGEN, Germany), following the manufacturers protocol designed to enrich microbial DNA while minimizing host DNA contamination. Briefly, urine samples were centrifuged to concentrate microbial cells, and host DNA was selectively degraded using Buffer RDD and Benzonase. Bacterial cell lysis was facilitated by Proteinase K and mechanical disruption with the Qiagen TissueLyser II (QIAGEN, Germany). DNA was then purified using QIAamp UCP Mini spin columns and eluted with Buffer AVE. DNA concentration and purity were measured using a Maestrogen UV-VIS Spectrophotometer (MaestroGen Inc., Taiwan), and the samples were stored at 20C or 80C. Microbial community profiling was performed by amplifying the V3V4 regions of the 16S rRNA gene using universal primers, generating ~460 bp amplicons. Library preparation involved two-step PCR with KAPA HotStart PCR Mix (Roche, Switzerland) and Nextera XT Index Kit (Illumina, San Diego, CA, USA) for sample barcoding. PCR products were purified using AMPure XP magnetic beads (Beckman Coulter, USA). Libraries were quantified by qPCR and normalized before pooling. Sequencing was conducted on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) using Sequencing by Synthesis (SBS) technology. Raw reads were processed with Illumina's Real-Time Analysis software and converted to FASTQ format using bcl2fastq v1.8.4.