Biology subjects

Top, J.

Publications and source records attributed to Top, J..

7 recordsLinked to original sources

bioRxiv2025

Limited value of Nanopore adaptive sampling in a long-read metagenomic profiling workflow of clinical sputum samples

Oxford Nanopore adaptive sampling (NAS) is a method by which the long-read sequencing flowcell accepts or rejects DNA molecules that are actively being sequenced based on their initial [~]500bp sequences, selectively increasing target data output. NAS promises up to 5-10x enrichment of target sequencing yield without additional sample preparation, but this optimal performance is dependent on ideal sample parameters which may be difficult to achieve under many real-world use-cases. We evaluated the use of NAS with the current R10.4.1 flowcell chemistry for profiling clinical sputum metagenomes, achieving at best 3.1x enrichment of bacterial sequence output due to the shorter read lengths ([~]2.5kb) from the PCR amplification necessary to compensate for low DNA extraction yields. More critically, we encountered rapid pore loss during our runs that reduced total sequencing yield by an estimated 80%. We were unable to mitigate the pore loss despite extensive attempts to reduce contaminant carry-over, and we could not determine its cause but ruled out NAS and pore underloading as contributing factors. We conclude that the utility of NAS is often limited by the characteristics of the metagenomic sample studied, and that the factors contributing to pore loss need to be resolved before ONT sequencing can be reliably applied to long-read metagenomics.

microbiology↗

bioRxiv2023

Dietary cystine restriction increases the proliferative capacity of the small intestine of mice

Over 88 million people are currently estimated to have adopted towards a vegan or vegetarian diet. Cysteine is a semi-essential amino acid, which availability is largely dependent on dietary intake of meat, eggs and whole grains. Vegan/vegetarian diets are therefore inherently low in cysteine concentrations. Sufficient uptake of cysteine is crucial, as it serves as substrate for protein synthesis and conversion to taurine and glutathione. In this study, we therefore investigate the effect of low dietary cystine, the oxidized derivative of cysteine, on intestinal epithelial layer function. Mice (8/group) received a high fat diet with normal or low cystine concentration for 2 weeks. We observed no changes in plasma methionine, cysteine, taurine or glutathione levels after 2 weeks. Stem cell markers as well as the proliferation marker Ki67 were increased upon cystine restriction in the small intestine. In line with this, gene set enrichment analysis indicated enrichment of Wnt signaling in the small intestine of mice on the low cystine diet, indicative of proliferative cells. Increased proliferation was absent in the colon. In the colon, dietary cystine restriction results in an increase in goblet cells, but no significant changes in the thickness of the mucus barrier or in its protective capacity. Also the microbiome was not changed upon dietary restriction. In conclusion, we show that cystine restriction for two weeks does not seem to induce any systemic effects. The increased proliferative capacity and number of goblet cells observed in the intestine may be the effect of starting epithelial damage or a reaction of the epithelium to start enlarging the absorptive capacity.

molecular biology↗

bioRxiv2023

Detecting co-selection through excess linkage disequilibrium in bacterial genomes

Population genomics has revolutionised our ability to study bacterial evolution by enabling data-driven discovery of the genetic architecture of trait variation. Genome-wide association studies (GWAS) have more recently become accompanied by genome-wide epistasis and co-selection (GWES) analysis, which offers a phenotype-free approach to generating hypotheses about selective processes that simultaneously impact multiple loci across the genome. However, existing GWES methods only consider associations between distant pairs of loci within the genome due to the strong impact of linkage-disequilibrium (LD) over short distances. Based on the general functional organisation of genomes it is nevertheless expected that the majority of co-selection and epistasis will act within relatively short genomic proximity, on co-variation occurring within genes and their promoter regions, and within operons. Here we introduce LDWeaver, which enables an exhaustive GWES across both short- and long-range LD, to disentangle likely neutral co-variation from selection. We demonstrate the ability of LDWeaver to efficiently generate hypotheses about co-selection using large genomic surveys of multiple major human bacterial pathogen species and validate several findings using functional annotation and phenotypic measurements. Our approach will facilitate the study of bacterial evolution in the light of rapidly expanding population genomic data.

bioinformatics↗

bioRxiv2022

The population-level impact of Enterococcus faecalis genetics on intestinal colonisation and extraintestinal infection

Enterococcus faecalis is a commensal pathogenic bacterium commonly found in the human gastrointestinal tract and a cause of opportunistic infections typically associated with multidrug resistance. The E. faecalis genetic changes associated with pathogenicity and extraintestinal infection, particularly through gut-to-bloodstream translocation, are poorly understood. Here, we investigate the E. faecalis genetic signatures associated with intestinal colonisation and extraintestinal infection and infection of hospitalised and non-hospitalised individuals using heritability estimation and a genome-wide association study (GWAS). We analysed 750 whole-genome sequences of faecal and bloodstream E. faecalis isolates from hospitalised patients and non-hospitalised individuals, respectively, predominantly in Europe. We found that E. faecalis infection of individuals depending on their hospitalisation status and extraintestinal infection are heritable traits and that [~]24% and [~]34% of their variation is explained by the considered genetic effects, respectively. Further, a GWAS using linear mixed models did not pinpoint any clear enrichment of individual genetic changes in isolates from different isolation sites and individuals with varying hospitalisation statuses, suggesting that these traits are highly polygenic. Altogether, our findings indicate that E. faecalis infection and extraintestinal infection are influenced by variation in genetic, host, and environmental factors, and ultimately the opportunistic pathogenic lifestyle of this versatile host generalist bacterium.

microbiology↗

bioRxiv2020

Genomic rearrangements uncovered by genome-wide co-evolution analysis of a major nosocomial pathogen Enterococcus faecium

Enterococcus faecium is a gut commensal of the gastro-digestive tract, but also known as nosocomial pathogen among hospitalized patients. Population genetics based on whole-genome sequencing has revealed that E. faecium strains from hospitalized patients form a distinct clade, designated as clade A1 and that plasmids are major contributors to the emergence of nosocomial E. faecium. Here we further explored the adaptive evolution of E. faecium using a genome-wide co-evolution study (GWES) to identify co-evolving SNPs. We identified three genomic regions harboring large numbers of SNPs in tight linkage which are not proximal to each other based on the completely assembled chromosome of clade A1 reference hospital isolate AUS0004. Close examination of these regions revealed that they are located at the borders of four different types of large-scale genomic rearrangements, insertion sites of two different genomic islands and an IS30-like transposon. In non-clade A1 isolates, these regions are adjacent to each other and they lack the insertions of the genomic islands and IS30-like transposon. Additionally, among the clade A1 isolates there is one group of pet isolates lacking the genomic rearrangement and insertion of the genomic islands, suggesting a distinct evolutionary trajectory. In silico analysis of the biological functions of the genes encoded in three regions revealed a common link to a stress response. This suggests that these rearrangements may reflect adaptation to the stringent conditions in the hospital environment, such as antibiotics and detergents, to which bacteria are exposed. In conclusion, to our knowledge, this is the first study using GWES to identify genomic rearrangements, suggesting that there is considerable untapped potential to unravel hidden evolutionary signals from population genomic data. Impact statementEnterococcus faecium has emerged as an important nosocomial pathogen around the world. Population genetics revealed that clinical E. faecium strains form a distinct clade, designated as clade A1 and that plasmids are major contributors to the emergence of nosocomial E. faecium. Here, the adaptive evolution of E. faecium was further explored using an unsupervised machine learning method (SuperDCA) to identify genome-wide co-evolving SNPs. We identified three genomic regions harboring large numbers of SNPs in tight linkage which are separated by a large chromosomal distance in a clinical clade A1 reference isolate, but appeared adjacent to each other in non-clade A1 isolates. We identified four different types of large-scale genomic rearrangements and in all cases, we found insertion of two different genomic islands and an insertion element at the border. In contrast, no genomic rearrangement and insertions were identified among a group of clade A1 pet isolates, suggesting a distinct evolutionary trajectory. Based on the in silico predicted biological functions, we found a common link to a stress response for the genes encoded in three regions. This suggests that these rearrangements may reflect adaptation to the stringent conditions in the hospital environment, such as antibiotics and detergents, to which bacteria are exposed. Data summaryRaw core-genome alignment (1.1 MB, Harvest suite v1.1.2), including the 1,644 Clade A isolates and the complete E. faecium AUS0004 (accession number CP003351) as a reference is available under the following gitlab repository https://gitlab.com/sirarredondo/efm_gwes.

genomics↗

bioRxiv2019

Genomes of a major nosocomial pathogen Enterococcus faecium are shaped by adaptive evolution of the chromosome and plasmidome

Enterococcus faecium is a gut commensal of many mammals but is also recognized as a major nosocomial human pathogen, as it is listed on the WHO global priority list of multi-drug resistant organisms. Previous research has suggested that nosocomial strains have multiple zoonotic origins and are only distantly related to those involved in human commensal colonization. Here we present the first comprehensive population-wide joint genomic analysis of hospital, commensal and animal isolates using both short- and long-read sequencing techniques. This enabled us to investigate the population plasmidome, core genome variation and genome architecture in detail, using a combination of machine learning, population genomics and genome-wide co-evolution analysis. We observed a high level of genome plasticity with large-scale inversions and heterogeneous chromosome sizes, collectively painting a high-resolution picture of the adaptive landscape of E. faecium, and identified plasmids as the main indicator for host-specificity. Given the increasing availability of long-read sequencing technologies, our approach could be widely applied to other human and animal pathogen populations to unravel fine-scale mechanisms of their evolution.

genomics↗

bioRxiv2018

mlplasmids: a user-friendly tool to predict plasmid- and chromosome-derived sequences for single species

Assembly of bacterial short-read whole genome sequencing (WGS) data frequently results in hundreds of contigs for which the origin, plasmid or chromosome, is unclear. Long-read sequencing has emerged as a solution to resolve plasmid structures and to obtain complete genomes for most bacterial species. This information can be used to generate and label datasets from short-read based contigs as plasmid- or chromosome-derived. We investigated the use of several popular machine learning methods to classify short-read contigs with known plasmid- or chromosome-origin from Enterococcus faecium, Klebsiella pneumoniae and Escherichia coli using pentamer frequencies. Based on resulting F1-scores we selected support-vector machine (SVM) models as best classifier for all three bacterial species (F1-score E. faecium = 0.94, F1-score K. pneumoniae = 0.90, F1-score E. coli = 0.76), which outperformed other existing plasmid tools using an independent set of isolates (precision E. faecium = 0.92, precision K. pneumoniae = 0.86, precision E. coli = 0.82). We demonstrated the scalability of our model by accurately predicting the plasmidome of a large collection of 1,644 E. faecium isolates with only short-read WGS available using a standard laptop with a single core. A low number of false positive predicted sequences suggests that the assignment of a particular gene of interest as plasmid- or chromosome-encoded by the models is plausible. The SVM classifiers are publicly available as a new R package called mlplasmids at https://gitlab.com/sirarredondo/mlplasmids under the GNU General Public License v3.0. We additionally developed a graphical-user interface using the Shiny package which can be accessed at https://sarredondo.shinyapps.io/mlplasmids/. Single genomes can easily be predicted by uploading genome assemblies. We anticipate that this tool may significantly facilitate research on the dissemination of plasmids encoding antibiotic resistance and/or contributing to host adaptation.

microbiology↗