Fusobacterium Genomics Using MinION and Illumina Sequencing Enables Genome Completion and Correction

Abstract

Understanding the virulence mechanisms of human pathogens from the genus Fusobacterium has been hindered by a lack of properly assembled and annotated genomes. Here we report the first complete genomes for seven Fusobacterium strains, as well as resequencing of the reference strain Fusobacterium nucleatum subsp. nucleatum ATCC 25586 (total of seven species; total of eight genomes). A highly efficient and cost-effective sequencing pipeline was achieved using sample multiplexing for short-read Illumina (150 bp) and long-read Oxford Nanopore MinION (>80 kbp) platforms, coupled with genome assembly using the open-source software Unicycler. Compared to currently available draft assemblies (previously 24 to 67 contigs), these genomes are highly accurate and consist of only one complete chromosome. We present the complete genome sequence of F. nucleatum subsp. nucleatum ATCC 23726, a genetically tractable and biomedically important strain and, in addition, reveal that the previous F. nucleatum subsp. nucleatum ATCC 25586 genome assembly contains a 452-kb genomic inversion that has been corrected using our sequencing and assembly pipeline. To enable genomic analyses by the scientific community, we concurrently used these genomes to launch FusoPortal, a repository of interactive and downloadable genomic data, genome maps, gene annotations, and protein functional analyses and classifications. In summary, this report provides detailed methods for accurately sequencing, assembling, and annotating Fusobacterium genomes, while focusing on using open-source software to foster the availability of reproducible and open data. This resource will enhance efforts to properly identify virulence proteins that may contribute to a repertoire of diseases that includes periodontitis, preterm birth, and colorectal cancer.IMPORTANCEFusobacterium spp. are Gram-negative, oral bacteria that are increasingly associated with human pathologies as diverse as periodontitis, preterm birth, and colorectal cancer. While a recent surge in F. nucleatum research has increased our understanding of this human pathogen, a lack of complete genomes has hindered the identification and characterization of associated host-pathogen virulence factors. Here we report the first eight complete Fusobacterium genomes sequenced using an Oxford Nanopore MinION and Illumina sequencing pipeline and assembled using the open-source program Unicycler. These genomes are highly accurate, and seven of the genomes represent the first complete sequences for each strain. In summary, the FusoPortal resource provides a publicly available resource that will guide future genetic, bioinformatic, and biochemical experiments to characterize this genus of emerging human pathogens.

Keywords: Fusobacterium; Fusobacterium nucleatum; Illumina; MinION; cancer; colorectal cancer.

FIG 1

Phred analysis of Illumina and MinION reads for the F. nucleatum subsp. nucleatum ATCC 25586 genome. (A) Q scores for Illumina reads. (B) Q scores (Phred quality) for MinION reads 20 to 80 kb in length. (C) Q scores for MinION reads up to 20 kb in length.

FIG 2

Statistics and mapping of F. necrophorum funduliforme 1_1_36S MinION long reads. After complete genome assembly, MinION reads were mapped to the F. necrophorum funduliforme 1_1_36S genome using Geneious version 9.1.4 software.

FIG 3

Genome assembly and annotation of eight Fusobacterium genomes from seven species. Short-read-only and complete genome assembly representations were created using Bandage (21).

FIG 4

Analysis of Fusobacterium nucleatum ATCC 23726 genomes compared to previous builds. (A) Alignment of the complete F. nucleatum subsp. nucleatum ATCC 23726 genome with the 67-contig draft assembly (GenBank accession number ADVK01000000). (B) Confirmation of a 452-kb genomic inversion in the previous F. nucleatum subsp. nucleatum ATCC 25586 genome assembly (GenBank accession number GCA_000007325.1).