Complete genome sequence of Prevotella histicola T05-04

TL;DR

This paper presents the complete genome sequence of Prevotella histicola T05-04, a bacterium linked to human oral cancer.

Contribution

The novelty is the closed genome assembly of P. histicola T05-04 using Nanopore sequencing.

Findings

The genome was previously fragmented into 109 contigs.

Nanopore sequencing enabled the assembly of a complete, closed genome.

This provides a more accurate reference for future studies on this bacterium.

Abstract

Prevotella histicola is a non-spore forming, obligatory anaerobic, gram-negative coccobacillus bacterium originally isolated from human oral squamous cell carcinoma tissue. Previously, the available genome for P. histicola T05-04 consisted of 109 contigs. Here, we report the complete (closed) genome sequence for Prevotella histicola T05-04 assembled using Nanopore sequencing.

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ANNOUNCEMENT

The bacterial Prevotella genus is comprised of many human-associated species that are highly abundant at different sites of the body. The ecological role of Prevotella histicola as a prominent member of the oral microbiome and its relationship with other members of the microbiome has yet to be fully understood. Genome sequencing of type-strain P. histicola T05-04 will aid in future investigations of the genomic features underlying the biological role(s) of this member of the human microbiome.

The Prevotella genus (phylum, Bacteroidota; class, Bacteroidia; order, Bacteroidales; family, Prevotellaceae) consists of gram-negative, obligately anaerobic, non-spore forming bacteria. We purchased lyophilized P. histicola strain T05-04 (DSM 19854), which was reconstituted using pre-reduced medium (Brain Heart Infusion Broth from Anaerobe Systems, cat# AS-872) under anaerobic conditions at 37°C.

Before sequencing, P. histicola T05-04 was cultured in Tryptone Yeast Glucose (TYG) medium (1, 2) and incubated at 37°C for 24 hours, streak plated onto Schaedler Kanamycin Vancomycin agar with 5% sheep’s blood for 24 hours all under anoxic conditions. A single colony was cultured in TYG media, incubated for 24 hours, and then verified by Gram stain, colony PCR, and 16S rRNA Sanger sequencing. DNA was isolated using the DNeasy Blood and Tissue Kit yielding 208 ng/µL using Qubit dsDNA HS Assay and a Qubit Fluorometer. 260/280 (1.89) and 260/230 (1.87) ratios were quantified using a Nanodrop One UV-visible spectrophotometer.

DNA library preparation was performed using Oxford Nanopore Technologies Ligation Sequencing gDNA–Native Barcoding Kit 24 V14 (SQK NBD114.24, ONT, UK) according to the manufacturer’s protocol and sequenced on a FLO-MIN114 Flow Cell (ONT, UK) for 1.5 hours. DNA fragments of all sizes were enriched with Short Fragment Buffer (SKQ 1481067) without shearing. Read lengths of N_50_ 1,670 and 720.55 k raw reads were generated using Dorado 7.4.14 with a minimum Q score of 12 after raw reads were generated.

The genome sequence was assembled using FLYE 2.9.5 (3) with default parameters. Genome polishing was performed using BWA (4) with default parameters and Racon (5) using mismatching and matching bases set to 3 and −3, respectively. A final round of polishing was performed using Medaka (6) with default parameters. Final assembly of P. histicola resulted in three circular contigs 2,176,075 bp, 812,616 bp, and 6,399 bp in length. P. histicola strain F0411 has been previously described to have two chromosomes of different sizes and divergent sequences (NCBI RefSeq assembly GCF_018128125.1), which we have identified is also true for P. histicola T05-04. In addition, the third circular contig (coverage >1,000×) likely encodes for a plasmid. Reorientation of each assembled contig was performed using dnaapler (7). Final visualization of the three contigs was performed via Bandage (8). fastANI (9) using default parameters demonstrated 97% identity to Prevotella histicola. Currently, Prevotella histicola genomes are assembled to the scaffold and contig level, with only one fully assembled genome available for a different strain (NCBI RefSeq assembly GCF_018128125.1). This underscores the significance of our work to fully assemble a closed genome for P. histicola T05-04.

The 3 Mbp genome for P. histicola T05-04 has a 41.5% GC content. Final assembly coverage averaged to 232× (contig 1 = 213×, contig 2 = 266×, contig 3 = 2,345×). Sequence annotation for the genome was performed using the NCBI Prokaryotic Genome Annotation Pipeline (10) predicting 2,110 protein-coding genes and 2,430 genes in total.