Complete Genome Sequences of Three Phocaeicola vulgatus Strains Isolated from a Healthy Japanese Individual

Phocaeicola vulgatus, one of the most numerically predominant Bacteroides species, has been extensively studied for its critical roles in infectious diseases as well as in other aspects of human health (1–4). Despite their clinical importance, Bacteroides species are most likely underrepresented in public genome databases, especially in terms of complete genomes of strains isolated from humans. Thus, there exists an urgent need for high-quality, well-established genome data to support studies on microbiome diversity and function. Hence, we sequenced and assembled the complete genomes of three P. vulgatus strains isolated from a healthy Japanese volunteer.

The fecal sample was collected from a volunteer who had normal bowel activity and no history of antibiotic use during the 3 months prior to the study. Samples were collected with Puritan fecal Opti-Swab CB-206 and cultured in an anaerobic atmosphere on Bacteroides bile esculin (BBE) agar (Kyokuto) and BBE with ceftazidime (30 mg/L) for 48 h at 37°C. Colonies of more than 1-mm diameter were subcultured on Gifu anaerobic medium agar (Nissui) under the same conditions. The isolates were identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (MALDI Biotyper [MBT]). P. vulgatus strains were incubated in ABCM broth (Eiken Chemical) in an anaerobic chamber (Hirasawa) at 35°C for 12 h. Bacterial pellets were obtained by centrifugation. Total DNA was extracted using a NucleoBond high-molecular-weight (HMW) DNA kit (Macherey-Nagel). The DNA was quantified using Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kits (Thermo Fisher) and qualified with a NanoDrop instrument (Thermo Fisher) at an optical density of 260/280 nm (OD_260/280), and fragments were checked by electrophoresis. The same qualified DNA templates were used for both long-read and short-read sequencing.

Genome assembly was conducted using a hybrid approach combining Nanopore long-read sequencing and DNBseq short-read sequencing, similar to a previously described method (5). For long-read sequencing, a library was constructed using a ligation sequencing kit (SQK-LSK-109; Oxford Nanopore Technologies [ONT]), and sequencing was performed using a GridION X5 system (ONT) on a FLO-MIN106 flow cell. Long-read sequence data were base called using Guppy v.4.2.3. The raw reads were subjected to trimming and quality filtering using Porechop v.0.2.4 (https://github.com/rrwick/Porechop) and Filtlong v.0.2.0 (minimum length 100 bp) (https://github.com/rrwick/Filtlong). For short-read sequencing, the MGIEasy FS DNA library prep set (MGI Tech) was used for library construction. Subsequently, 2 × 150-bp paired-end sequencing was performed using the DNBSEQ-G400 platform (MGI Tech). The raw sequencing reads were processed using fastp v.0.20.1 (6) for trimming adapters and low-quality data, and approximately 3.5 million read pairs (1.0 Gbp) were sampled using SeqKit v.0.16.1 (7). High-quality short-read and long-read sequences were assembled using Unicycler v.0.4.8 (8) with default settings. The assembled contig graph was confirmed using Bandage v.0.8.1 (9), and the integrity of the assembled genomic data was confirmed using CheckM v.1.1.3 (10). Annotation of the assembled genomes was performed using the DDBJ Fast Annotation and Submission Tool (DFAST) (https://dfast.nig.ac.jp/). Default parameters were used for all software unless otherwise specified.

The genome information is summarized in Table 1. According to CheckM, all three of the obtained genomes were 99.25% complete with no contamination.

This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant 20H00561) and the Collaboration Program at ROIS Joint Support-Center for Data Science Research (grant 012RP2021).

The study was approved by the Ethics Committee of Gifu University (Gifu, Japan; approval number 2019-164), and the study participant provided written informed consent.