Floria: fast and accurate strain haplotyping in metagenomes.

SUMMARY: Shotgun metagenomics allows for direct analysis of microbial community genetics, but scalable computational methods for the recovery of bacterial strain genomes from microbiomes remains a key challenge. We introduce Floria, a novel method designed for rapid and accurate recovery of strain haplotypes from short and long-read metagenome sequencing data, based on minimum error correction (MEC) read clustering and a strain-preserving network flow model. Floria can function as a standalone haplotyping method, outputting alleles and reads that co-occur on the same strain, as well as an end-to-end read-to-assembly pipeline (Floria-PL) for strain-level assembly. Benchmarking evaluations on synthetic metagenomes show that Floria is > 3× faster and recovers 21% more strain content than base-level assembly methods (Strainberry) while being over an order of magnitude faster when only phasing is required. Applying Floria to a set of 109 deeply sequenced nanopore metagenomes took <20 min on average per sample and identified several species that have consistent strain heterogeneity. Applying Floria's short-read haplotyping to a longitudinal gut metagenomics dataset revealed a dynamic multi-strain Anaerostipes hadrus community with frequent strain loss and emergence events over 636 days. With Floria, accurate haplotyping of metagenomic datasets takes mere minutes on standard workstations, paving the way for extensive strain-level metagenomic analyses. AVAILABILITY AND IMPLEMENTATION: Floria is available at https://github.com/bluenote-1577/floria, and the Floria-PL pipeline is available at https://github.com/jsgounot/Floria_analysis_workflow along with code for reproducing the benchmarks.

Comparison of tet(X4)-containing contigs assembled from metagenomic sequencing data with plasmid sequences of isolates from a cohort of healthy subjects.

Recently discovered tet(X) gene variants have provided new insights into microbial antibiotic resistance mechanisms and their potential consequences for public health. This study focused on detection, analysis, and characterization of Tet(X4)-positive Enterobacterales from the gut microbiota of a healthy cohort of individuals in Singapore using cultivation-dependent and cultivation-independent approaches. Twelve Tet(X4)-positive Enterobacterales strains that were previously obtained from the cohort were fully genome-sequenced and comparatively analyzed. A metagenomic sequencing (MS) data set of the same samples was mined for contigs that harbored the tet(X4) resistance gene. The sequences of tet(X4)-containing contigs and plasmids sequences were compared. The presence of the resistance genes floR and estT (previously annotated as catD) was detected in the same cassette in 10 and 12 out of the 12 tet(X4)-carrying plasmids, respectively. MS detected tet(X4)-containing contigs in 2 out of the 109 subjects, while cultivation-dependent analysis previously reported a prevalence of 10.1%. The tet(X4)-containing sequences assembled from MS data are relatively short (~14 to 33 kb) but show high similarity to the respective plasmid sequences of the isolates. Our findings show that MS can complement efforts in the surveillance of antibiotic resistance genes for clinical samples, while it has a lower sensitivity than a cultivation-based method when the target organism has a low abundance. Further optimization is required if MS is to be utilized in antibiotic resistance surveillance.IMPORTANCEThe global rise in antibiotic resistance makes it necessary to develop and apply new approaches to detect and monitor the prevalence of antibiotic resistance genes in human populations. In this regard, of particular interest are resistances against last-resort antibiotics, such as tigecycline. In this study, we show that metagenomic sequencing can help to detect high abundance of the tigecycline resistance gene tet(X4) in fecal samples from a cohort of healthy human subjects. However, cultivation-based approaches currently remain the most reliable and cost-effective method for detection of antibiotic-resistant bacteria.

Contrasting Genomic Evolution Between Domesticated and Wild Kluyveromyces lactis Yeast Populations.

The process of domestication has variable consequences on genome evolution leading to different phenotypic signatures. Access to the complete genome sequences of a large number of individuals makes it possible to explore the different facets of this domestication process. Here, we sought to explore the genome evolution of Kluyveromyces lactis, a yeast species well known for its involvement in dairy processes and also present in natural environments. Using a combination of short- and long-read sequencing strategies, we investigated the genomic variability of 41 K. lactis isolates and found that the overall genetic diversity of this species is very high (θw = 3.3 × 10-2) compared with other species such as Saccharomyces cerevisiae (θw = 1.6 × 10-2). However, the domesticated dairy population shows a reduced level of diversity (θw = 1 × 10-3), probably due to a domestication bottleneck. In addition, this entire population is characterized by the introgression of the LAC4 and LAC12 genes, responsible for lactose fermentation and coming from the closely related species, Kluyveromyces marxianus, as previously described. Our results highlighted that the LAC4/LAC12 gene cluster was acquired through multiple and independent introgression events. Finally, we also identified several genes that could play a role in adaptation to dairy environments through copy number variation. These genes are involved in sugar consumption, flocculation, and drug resistance, and may play a role in dairy processes. Overall, our study illustrates contrasting genomic evolution and sheds new light on the impact of domestication processes on it.

Mutual Exclusion of Methanobrevibacter Species in the Human Gut Microbiota Facilitates Directed Cultivation of a Candidatus Methanobrevibacter Intestini Representative.

Methanogenic Archaea (methanogens) are a phylogenetically diverse group of microorganisms and are considered to be the most abundant archaeal representatives in the human gut. However, the gut methanogen diversity of human populations in many global regions remains poorly investigated. Here, we report the abundance and diversity of gut methanogenic Archaea in a multi-ethnic cohort of healthy Singaporeans by using a concerted approach of metagenomic sequencing, 16S rRNA gene amplicon sequencing, and quantitative PCR. Our results indicate a mutual exclusion of Methanobrevibacter species, i.e., the highly prevalent Methanobrevibacter smithii and the less prevalent Candidatus Methanobrevibacter intestini in more than 80% of the samples when using an amplicon sequencing-based approach. Leveraging on this finding, we were able to select a fecal sample to isolate a representative strain, TLL-48-HuF1, for Candidatus Methanobrevibacter intestini. The analyzed physiological parameters of M. smithii DSM 861T and strain TLL-48-HuF1 suggest high similarity of the two species. Comparative genome analysis and the mutual exclusion of the Methanobrevibacter species indicate potentially different niche adaptation strategies in the human host, which may support the designation of Candidatus M. intestini as a novel species. IMPORTANCE Methanogens are important hydrogen consumers in the gut and are associated with differing host health. Here, we determine the prevalence and abundance of archaeal species in the guts of a multi-ethnic cohort of healthy Singapore residents. While Methanobrevibacter smithii is the most prevalent and abundant methanogen in the human gut of local subjects, the recently proposed Candidatus Methanobrevibacter intestini is the abundant methanogen in a minority of individuals that harbor them. The observed potential mutual exclusion of M. smithii and Ca. M. intestini provides further support to the proposal that the two physiologically similar strains may belong to different Methanobrevibacter species.

Genome-centric analysis of short and long read metagenomes reveals uncharacterized microbiome diversity in Southeast Asians.

Despite extensive efforts to address it, the vastness of uncharacterized 'dark matter' microbial genetic diversity can impact short-read sequencing based metagenomic studies. Population-specific biases in genomic reference databases can further compound this problem. Leveraging advances in hybrid assembly (using short and long reads) and Hi-C technologies in a cross-sectional survey, we deeply characterized 109 gut microbiomes from three ethnicities in Singapore to comprehensively reconstruct 4497 medium and high-quality metagenome assembled genomes, 1708 of which were missing in short-read only analysis and with >28× N50 improvement. Species-level clustering identified 70 (>10% of total) novel gut species out of 685, improved reference genomes for 363 species (53% of total), and discovered 3413 strains unique to these populations. Among the top 10 most abundant gut bacteria in our study, one of the species and >80% of strains were unrepresented in existing databases. Annotation of biosynthetic gene clusters (BGCs) uncovered more than 27,000 BGCs with a large fraction (36-88%) unrepresented in current databases, and with several unique clusters predicted to produce bacteriocins that could significantly alter microbiome community structure. These results reveal significant uncharacterized gut microbial diversity in Southeast Asian populations and highlight the utility of hybrid metagenomic references for bioprospecting and disease-focused studies.

High Complexity and Degree of Genetic Variation in Brettanomyces bruxellensis Population.

Genome-wide characterization of genetic variants of a large population of individuals within the same species is essential to have a deeper insight into its evolutionary history as well as the genotype-phenotype relationship. Population genomic surveys have been performed in multiple yeast species, including the two model organisms, Saccharomyces cerevisiae and Schizosaccharomyces pombe. In this context, we sought to characterize at the population level the Brettanomyces bruxellensis yeast species, which is a major cause of wine spoilage and can contribute to the specific flavor profile of some Belgium beers. We have completely sequenced the genome of 53 B. bruxellensis strains isolated worldwide. The annotation of the reference genome allowed us to define the gene content of this species. As previously suggested, our genomic data clearly highlighted that genetic diversity variation is related to ploidy level, which is variable in the B. bruxellensis species. Genomes are punctuated by multiple loss-of-heterozygosity regions, whereas aneuploidies as well as segmental duplications are uncommon. Interestingly, triploid genomes are more prone to gene copy number variation than diploids. Finally, the pangenome of the species was reconstructed and was found to be small with few accessory genes compared with S. cerevisiae. The pangenome is composed of 5,409 ORFs (open reading frames) among which 5,106 core ORFs and 303 ORFs that are variable within the population. All these results highlight the different trajectories of species evolution and consequently the interest of establishing population genomic surveys in more populations.

High-Quality de Novo Genome Assembly of the Dekkera bruxellensis Yeast Using Nanopore MinION Sequencing.

Genetic variation in natural populations represents the raw material for phenotypic diversity. Species-wide characterization of genetic variants is crucial to have a deeper insight into the genotype-phenotype relationship. With the advent of new sequencing strategies and more recently the release of long-read sequencing platforms, it is now possible to explore the genetic diversity of any nonmodel organisms, representing a fundamental resource for biological research. In the frame of population genomic surveys, a first step is to obtain the complete sequence and high-quality assembly of a reference genome. Here, we sequenced and assembled a reference genome of the nonconventional Dekkera bruxellensis yeast. While this species is a major cause of wine spoilage, it paradoxically contributes to the specific flavor profile of some Belgium beers. In addition, an extreme karyotype variability is observed across natural isolates, highlighting that D. bruxellensis genome is very dynamic. The whole genome of the D. bruxellensis UMY321 isolate was sequenced using a combination of Nanopore long-read and Illumina short-read sequencing data. We generated the most complete and contiguous de novo assembly of D. bruxellensis to date and obtained a first glimpse into the genomic variability within this species by comparing the sequences of several isolates. This genome sequence is therefore of high value for population genomic surveys and represents a reference to study genome dynamic in this yeast species.

Comprehensive survey of condition-specific reproductive isolation reveals genetic incompatibility in yeast.

Genetic variation within a species could cause negative epistasis leading to reduced hybrid fitness and post-zygotic reproductive isolation. Recent studies in yeasts revealed chromosomal rearrangements as a major mechanism dampening intraspecific hybrid fertility on rich media. Here, by analysing a large number of Saccharomyces cerevisiae crosses on different culture conditions, we show environment-specific genetic incompatibility segregates readily within yeast and contributes to reproductive isolation. Over 24% (117 out of 481) of cases tested show potential epistasis, among which 6.7% (32 out of 481) are severe, with at least 20% of progeny loss on tested conditions. Based on the segregation patterns, we further characterize a two-locus Dobzhansky-Müller incompatibility case leading to offspring respiratory deficiency caused by nonsense mutation in a nuclear-encoding mitochondrial gene and tRNA suppressor. We provide evidence that this precise configuration could be adaptive in fluctuating environments, highlighting the role of ecological selection in the onset of genetic incompatibility and reproductive isolation in yeast.