WGS of Bacteroides fragilis from clinical specimens
All B. fragilis isolates were subjected to Oxford Nanopore sequencing. Bacterial DNA was extracted using the QIAamp BiOstic Bacteremia DNA Kit (Qiagen, Hilden, Germany) following the manufacturers protocol. Sequencing libraries were prepared using the Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS) protocol for cell cultures with the Rapid Barcoding Kit 96 V14 (SQK-RBK 114.96; Oxford Nanopore Technologies, Oxford, UK). The libraries were sequenced using the R10.4.1 flow cells on the GridION MK1 platform for 72 h. Basecalling was performed using Dorado Basecall Server v7.8.3 in super-accurate (SUP) mode and reads with a Phred quality score (Q score) less than 10 were filtered out. Sequencing yield and quality were assessed with NanoPlot v1.46.2 [19]. Quality-filtered reads were mapped against the B. fragilis NCTC 9343 reference genome (RefSeq accession number GCF_000025985.1) using minimap2 v2.30 [20]. Per-base coverage was calculated with modsdepth v0.3.12 [21], yielding a mean coverage depth of 139.1  48.75. Raw sequencing reads were assembled using Hybracter v0.11.2 [22], and the sequencing assemblies were analyzed using Bactopia v3.2.0 [23]. Species identification was performed using the Genome Taxonomy Database Toolkit (GTDB-Tk) v2.4.0 [24], and the assembly quality was assessed using CheckM v1.2.3 [25]. Multi-locus sequence typing (MLST), which was based on seven core gene fragments (groL, rpoB, recA, dnaJ, rprX, prfA and fusA), was conducted using mlst v2.23.0 [26]. Antibiotic resistance genes were identified using AMRFinderPlus v4.0.19 [27]. For cfiA+ BF, the genomic location of cfiA was also determined by AMRFinderPlus, and the upstream region was searched for potential insertion sequences with reference to the ISfinder database (https://isfinder.biotoul.fr, last update: 21 November 2025, access date: 2 December 2025) [28]. The sequencing data of all isolates have been deposited in the National Center for Biological Information (NCBI) BioProject database under the accession number PRJNA1372458.