El cáncer colorrectal (CCR) es la segunda causa oncológica de muerte en Argentina. La asociación entre CCR y microbioma fecal alterada se reconoce cada vez más como potencial para la determinación de ...nuevos biomarcadores de valor diagnóstico, pronóstico y/o terapéutico. El objetivo de este trabajo es caracterizar y comparar resultados entre diferentes métodos de conservación y análisis del microbioma en muestras de voluntarios sanos y pacientes con CCR recolectadas en el Hospital Italiano de Buenos Aires. Localmente, las muestras se almacenaron a -80 °C y se secuenciaron las regiones V3-V4 del gen ARNr 16S en un IlluminamiSeq. En la Universidad de Leeds, las muestras se almacenaron a temperatura ambiente en gFBOT y se secuenció la región V4 del ARNr 16S en un IlluminaHiSeq. El análisis bioinformático se realizó utilizando Qiime2 (2020.2) y LefSe. Se compararon los taxones a nivel de phylums representativos de ambos grupos, no detectando diferencias en la abundancia relativa de Bacteroidetes, Firmicutes, Proteobacteria, Virrumicrobia y Actinobacterias. A nivel de género encontramos mayor riqueza en las muestras locales frente a las de referencias (370 vs. 239 OTUs), debido a la mayor cobertura obtenida por el análisis de una región adicional (V3-V4 vs. V4) que nos permitió obtener una mayor resolución de los niveles taxonómicos presentes en las muestras analizadas. En este estudio pudimos obtener resultados comparables, a pesar de modificar el método de almacenamiento y secuenciación. Para validar estos resultados preliminares se necesitan evaluar muestras adicionales.
Although Kraken's k-mer-based approach provides a fast taxonomic classification of metagenomic sequence data, its large memory requirements can be limiting for some applications. Kraken 2 improves ...upon Kraken 1 by reducing memory usage by 85%, allowing greater amounts of reference genomic data to be used, while maintaining high accuracy and increasing speed fivefold. Kraken 2 also introduces a translated search mode, providing increased sensitivity in viral metagenomics analysis.
Nearly all infectious agents contain DNA or RNA genomes, making sequencing an attractive approach for pathogen detection. The cost of high-throughput or next-generation sequencing has been reduced by ...several orders of magnitude since its advent in 2004, and it has emerged as an enabling technological platform for the detection and taxonomic characterization of microorganisms in clinical samples from patients. This review focuses on the application of untargeted metagenomic next-generation sequencing to the clinical diagnosis of infectious diseases, particularly in areas in which conventional diagnostic approaches have limitations. The review covers ( a) next-generation sequencing technologies and common platforms, ( b) next-generation sequencing assay workflows in the clinical microbiology laboratory, ( c) bioinformatics analysis of metagenomic next-generation sequencing data, ( d) validation and use of metagenomic next-generation sequencing for diagnosing infectious diseases, and ( e) significant case reports and studies in this area. Next-generation sequencing is a new technology that has the promise to enhance our ability to diagnose, interrogate, and track infectious diseases.
The number of microbial genomes sequenced each year is expanding rapidly, in part due to genome-resolved metagenomic studies that routinely recover hundreds of draft-quality genomes. Rapid algorithms ...have been developed to comprehensively compare large genome sets, but they are not accurate with draft-quality genomes. Here we present dRep, a program that reduces the computational time for pairwise genome comparisons by sequentially applying a fast, inaccurate estimation of genome distance, and a slow, accurate measure of average nucleotide identity. dRep achieves a 28 × increase in speed with perfect recall and precision when benchmarked against previously developed algorithms. We demonstrate the use of dRep for genome recovery from time-series datasets. Each metagenome was assembled separately, and dRep was used to identify groups of essentially identical genomes and select the best genome from each replicate set. This resulted in recovery of significantly more and higher-quality genomes compared to the set recovered using co-assembly.
Metagenomic strategy serves as the foundation for the ecological exploration of novel bioresources (e.g., industrial enzymes and bioactive molecules) and biohazards (e.g., pathogens and antibiotic ...resistance genes) in natural and engineered microbial systems across multiple disciplines. Recent advancements in sequencing technology have fostered rapid development in the field of microbiome research where an increasing number of studies have applied both illumina short reads (SRs) and nanopore long reads (LRs) sequencing in their metagenomic workflow. However, given the high complexity of an environmental microbiome data set and the bioinformatic challenges caused by the unique features of these sequencing technologies, integrating SRs and LRs is not as straightforward as one might assume. The fast renewal of existing tools and growing diversity of new algorithms make access to this field even more difficult. Therefore, here we systematically summarized the complete workflow from DNA extraction to data processing strategies for applying illumina and nanopore‐integrated metagenomics in the investigation in environmental microbiomes. Overall, this review aims to provide a timely knowledge framework for researchers that are interested in or are struggling with the SRs and LRs integration in their metagenomic analysis. The discussions presented will facilitate improved ecological understanding of community functionalities and assembly of natural, engineered, and human microbiomes, benefiting researchers from multiple disciplines.
Strategies and tools in illumina and nanopore‐integrated metagenomic analysis of microbiome data.
Highlights
A timely knowledge framework to integrate nanopore long reads and illumina short reads is provided.
Workflow of common strategies for illumina and nanopore integration is illustrated in Figure 1.
Algorithm basis and application properties of tools are summarized in Table 3.
ABSTRACT The adaptation of bacterial lineages to local environmental conditions creates the potential for broader genotypic diversity within a species, which can enable a species to dominate across ...ecological gradients because of niche flexibility. The genus Polynucleobacter maintains both free-living and symbiotic ecotypes and maintains an apparently ubiquitous distribution in freshwater ecosystems. Subspecies-level resolution supplemented with metagenome-derived genotype analysis revealed that differential functional constraints, not geographic distance, produce and maintain strain-level genetic conservation in Polynucleobacter populations across three geographically proximal riverine environments. Genes associated with cofactor biosynthesis and one-carbon metabolism showed habitat specificity, and protein-coding genes of unknown function and membrane transport proteins were under positive selection across each habitat. Characterized by different median ratios of nonsynonymous to synonymous evolutionary changes (dN/dS ratios) and a limited but statistically significant negative correlation between the dN/dS ratio and codon usage bias between habitats, the free-living and core genotypes were observed to be evolving under strong purifying selection pressure. Highlighting the potential role of genetic adaptation to the local environment, the two-component system protein-coding genes were highly stable (dN/dS ratio, < 0.03). These results suggest that despite the impact of the habitat on genetic diversity, and hence niche partition, strong environmental selection pressure maintains a conserved core genome for Polynucleobacter populations. IMPORTANCE Understanding the biological factors influencing habitat-wide genetic endemism is important for explaining observed biogeographic patterns. Polynucleobacter is a genus of bacteria that seems to have found a way to colonize myriad freshwater ecosystems and by doing so has become one of the most abundant bacteria in these environments. We sequenced metagenomes from locations across the Chicago River system and assembled Polynucleobacter genomes from different sites and compared how the nucleotide composition, gene codon usage, and the ratio of synonymous (codes for the same amino acid) to nonsynonymous (codes for a different amino acid) mutations varied across these population genomes at each site. The environmental pressures at each site drove purifying selection for functional traits that maintained a streamlined core genome across the Chicago River Polynucleobacter population while allowing for site-specific genomic adaptation. These adaptations enable Polynucleobacter to become dominant across different riverine environmental gradients.
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