EzBioCloud: a genome-driven database and platform for microbiome identification and discovery.

With the continued evolution of DNA sequencing technologies, the role of genome sequence data has become more integral in the classification and identification of Bacteria and Archaea. Six years after introducing EzBioCloud, an integrated platform representing the taxonomic hierarchy of Bacteria and Archaea through quality-controlled 16S rRNA gene and genome sequences, we present an updated version, that further refines and expands its capabilities. The current update recognizes the growing need for accurate taxonomic information as defining a species increasingly relies on genome sequence comparisons. We also incorporated an advanced strategy for addressing underrepresented or less studied lineages, bolstering the comprehensiveness and accuracy of our database. Our rigorous quality control protocols remain, where whole-genome assemblies from the NCBI Assembly Database undergo stringent screening to remove low-quality sequence data. These are then passed through our enhanced identification bioinformatics pipeline which initiates a 16S rRNA gene similarity search and then calculates the average nucleotide identity (ANI). For genome sequences lacking a 16S rRNA sequence and without a closely related genomic representative for ANI calculation, we apply a different ANI approach using bacterial core genes for improved taxonomic placement (core gene ANI, cgANI). Because of the increase in genome sequences available in NCBI and our newly introduced cgANI method, EzBioCloud now encompasses a total of 109 835 species, of which 21 964 have validly published names. 47 896 are candidate species identified either through 16S rRNA sequence similarity (phylotypes) or through whole genome ANI (genomospecies), and the remaining 39 975 were positioned in the taxonomic tree by cgANI (species clusters). Our EzBioCloud database is accessible at www.ezbiocloud.net/db.

Assessing the utility of mitochondrial gene markers in the family Suessiaceae (Dinophyta) with phylogenomic validation.

The dinoflagellate family Suessiaceae comprises cosmopolitan species distributed across polar and tropical waters in both marine and freshwater ecosystems, encompassing free-living forms, symbionts, and parasites. Recently, species diversity within the family has rapidly expanded, now including a few species reported to cause red tides. Despite their ecological and evolutionary importance, classifying them within Suessiaceae is difficult due to the limitations of the existing molecular markers-the highly conserved small subunit ribosomal gene (SSU rDNA) and the presence of two indel regions of sequence fragments of the large subunit ribosomal gene (LSU rDNA)-resulting in poorly resolved phylogenetic relationships. We assessed mitochondrial cytochrome b (cob) and cytochrome c oxidase 1 (cox1) genes to develop robust molecular markers that can reveal the genetic diversity of the family Suessiaceae. The divergences of cob and cox1 sequences among the species in the family were greater than the SSU rDNA but less than the LSU rDNA and the ITS region. Moreover, the distinctive topology inferred from the mitochondrial genes provided high resolution among the suessiacean species. We examined the validity of the genetic markers using phylogenomics based on 2,023 core proteins. The divergence of the cob phylogeny was most consistent with that of the phylogenomic results. Taken together, the cob gene can be a novel marker reflecting topology at the genome-scale within the family Suessiaceae.

ContEst16S: an algorithm that identifies contaminated prokaryotic genomes using 16S RNA gene sequences.

Thanks to the recent advancement of DNA sequencing technology, the cost and time of prokaryotic genome sequencing have been dramatically decreased. It has repeatedly been reported that genome sequencing using high-throughput next-generation sequencing is prone to contaminations due to its high depth of sequencing coverage. Although a few bioinformatics tools are available to detect potential contaminations, these have inherited limitations as they only use protein-coding genes. Here we introduce a new algorithm, called ContEst16S, to detect potential contaminations using 16S rRNA genes from genome assemblies. We screened 69 745 prokaryotic genomes from the NCBI Assembly Database using ContEst16S and found that 594 were contaminated by bacteria, human and plants. Of the predicted contaminated genomes, 8 % were not predicted by the existing protein-coding gene-based tool, implying that both methods can be complementary in the detection of contaminations. A web-based service of the algorithm is available at www.ezbiocloud.net/tools/contest16s.

UACG: Up-to-Date Archaeal Core Genes and Software for Phylogenomic Tree Reconstruction.

In the post-genomic era, phylogenomics is a powerful and routinely-used tool to discover evolutionary relationships between microorganisms. Inferring phylogenomic trees by concatenating core gene sequences into a supermatrix is the standard method. The previously released up-to-date bacterial core gene (UBCG) tool provides a pipeline to infer phylogenomic trees using single-copy core genes for the Bacteria domain. In this study, we established up-to-date archaeal core gene (UACG), comprising 128 genes suitable for inferring archaeal phylogenomic trees. To test the gene set, we selected the Haloarcula genus and scrutinized its phylogeny. The phylogeny inferred using the UACG tool was consistent with the orthoANIu dendrogram, whereas the 16S rRNA gene phylogeny showed high intragenomic heterogeneity resulting in phylogenetic discrepancies. The software tool using the UACG set is available at https://www.ezbiocloud.net/tools/uacg .

UBCG2: Up-to-date bacterial core genes and pipeline for phylogenomic analysis.

Phylogenomic tree reconstruction has recently become a routine and critical task to elucidate the evolutionary relationships among bacterial species. The most widely used method utilizes the concatenated core genes, universally present in a single-copy throughout the bacterial domain. In our previous study, a bioinformatics pipeline termed Up-to-date Bacterial Core Genes (UBCG) was developed with a set of bacterial core genes selected from 1,429 species covering 28 phyla. In this study, we revised a new bacterial core gene set, named UBCG2, that was selected from the more extensive genome sequence set belonging to 3,508 species spanning 43 phyla. UBCG2 comprises 81 genes with nine Clusters of Orthologous Groups of proteins (COGs) functional categories. The new gene set and complete pipeline are available at http://leb.snu.ac.kr/ubcg2 .

Phylogeny Trumps Chemotaxonomy: A Case Study Involving Turicella otitidis.

The genus Turicella was proposed to harbor clinical strains isolated from middle-ear fluids of patients with otitis media. 16S rRNA phylogeny showed that it belonged to the mycolic acid-containing actinobacteria, currently classified in the order Corynebacteriales, and was closely related to the genus Corynebacterium. A new genus was proposed for the organisms as unlike corynebacteria they lacked mycolic acids and had different menaquinones. Here, we carried out large-scale comparative genomics on representative strains of the genera Corynebacterium and Turicella to check if this chemotaxonomic classification is justified. Three genes that are known to play an essential role in mycolic acid biosynthesis were absent in Turicella and two other mycolate-less Corynebacterium spp., explaining the lack of mycolic acids resulted from the deletion of genes and does not confer any phylogenetic context. Polyphasic phylogenetic analyses using 16S rRNA, bacterial core genes and genes responsible for synthesizing menaquinones unequivocally indicate that Turicella is a true member of the genus Corynebacterium. Here, we demonstrate that menaquinone and mycolic acid that have been used as critical taxonomic markers should be interpreted carefully, particularly when genome-based taxonomy is readily available. Based on the phylogenetic analysis, we propose to reclassify Turicella otitidis as Corynebacterium otitidis comb. nov.

UBCG: Up-to-date bacterial core gene set and pipeline for phylogenomic tree reconstruction.

Genome-based phylogeny plays a central role in the future taxonomy and phylogenetics of Bacteria and Archaea by replacing 16S rRNA gene phylogeny. The concatenated core gene alignments are frequently used for such a purpose. The bacterial core genes are defined as single-copy, homologous genes that are present in most of the known bacterial species. There have been several studies describing such a gene set, but the number of species considered was rather small. Here we present the up-to-date bacterial core gene set, named UBCG, and software suites to accommodate necessary steps to generate and evaluate phylogenetic trees. The method was successfully used to infer phylogenomic relationship of Escherichia and related taxa and can be used for the set of genomes at any taxonomic ranks of Bacteria. The UBCG pipeline and file viewer are freely available at https://www.ezbiocloud.net/tools/ubcg and https://www.ezbiocloud.net/tools/ubcg_viewer , respectively.