Search

Motivation : Mobile genetic elements (MGEs) are as ubiquitous in nature as they are varied in type, ranging from viral insertions to transposons to incorporated plasmids. Horizontal transfer of MGEs across bacterial species may also pose a significant threat to global health due to their capability to harbor antibiotic resistance genes. However, despite cheap and rapid whole-genome sequencing, the varied nature of MGEs makes it difficult to fully characterize them, and existing methods for detecting MGEs often do not agree on what should count. In this manuscript, we first define and argue in favor of a divergence-based characterization of mobile-genetic elements. Results : Using that paradigm, we present skandiver , a tool designed to efficiently detect MGEs from whole-genome assemblies without the need for gene annotation or markers. skandiver determines mobile elements via genome fragmentation, average nucleotide identity (ANI), and divergence time. By building on the scalable skani software for ANI computation, skandiver can query hundreds of complete assemblies against >65 000 representative genomes in a few minutes and 19 GB memory, providing scalable and efficient method for elucidating mobile element profiles in incomplete, uncharacterized genomic sequences. For isolated and integrated large plasmids (>10 kb), skandiver's recall was 48% and 47%, MobileElementFinder was 59% and 17%, and geNomad was 86% and 32%, respectively. For isolated large plasmids, skandiver's recall (48%) is lower than state-of-the-art reference-based methods geNomad (86%) and MobileElementFinder (59%). However, skandiver achieves higher recall on integrated plasmids and, unlike other methods, without comparing against a curated database, making skandiver suitable for discovery of novel MGEs. Availability and implementation https://github.com/YoukaiFromAccounting/skandiver [ABSTRACT FROM AUTHOR]

With the rapid and significant cost reduction of next-generation sequencing, low-coverage whole-genome sequencing (lcWGS), followed by genotype imputation, is becoming a cost-effective alternative to single-nucleotide polymorphism (SNP)-array genotyping. The objectives of this study were 2-fold: (1) construct a haplotype reference panel for genotype imputation from lcWGS data in rainbow trout (Oncorhynchus mykiss); and (2) evaluate the concordance between imputed genotypes and SNP-array genotypes in 2 breeding populations. Medium-coverage (12×) whole-genome sequences were obtained from a total of 410 fish representing 5 breeding populations with various spawning dates. The short-read sequences were mapped to the rainbow trout reference genome, and genetic variants were identified using GATK. After data filtering, 20,434,612 biallelic SNPs were retained. The reference panel was phased with SHAPEIT5 and was used as a reference to impute genotypes from lcWGS data employing GLIMPSE2. A total of 90 fish from the Troutlodge November breeding population were sequenced with an average coverage of 1.3×, and these fish were also genotyped with the Axiom 57K rainbow trout SNP array. The concordance between array-based genotypes and imputed genotypes was 99.1%. After downsampling the coverage to 0.5×, 0.2×, and 0.1×, the concordance between array-based genotypes and imputed genotypes was 98.7, 97.8, and 96.7%, respectively. In the USDA odd-year breeding population, the concordance between array-based genotypes and imputed genotypes was 97.8% for 109 fish downsampled to 0.5× coverage. Therefore, the reference haplotype panel reported in this study can be used to accurately impute genotypes from lcWGS data in rainbow trout breeding populations. [ABSTRACT FROM AUTHOR]

Despite increasing in mass approximately 100-fold during larval life, the Drosophila CNS maintains its characteristic form. Dynamic interactions between the overlying basement membrane and underlying surface glia are known to regulate CNS structure in Drosophila , but the genes and pathways that establish and maintain CNS morphology during development remain poorly characterized. To identify genes that regulate CNS shape in Drosophila , we conducted an EMS-based, forward genetic screen of the second chromosome, uncovered 50 mutations that disrupt CNS structure, and mapped these alleles to 17 genes. Analysis of whole genome sequencing data wedded to genetic studies uncovered the affected gene for all but 1 mutation. Identified genes include well-characterized regulators of tissue shape, like LanB1 , viking , and Collagen type IV alpha1 , and previously characterized genes, such as Toll-2 and Rme-8 , with no known role in regulating CNS structure. We also uncovered that papilin and C1GalTA likely act in the same pathway to regulate CNS structure and found that the fly homolog of a glucuronosyltransferase, B4GAT1/LARGE1, that regulates Dystroglycan function in mammals is required to maintain CNS shape in Drosophila. Finally, we show that the senseless-2 transcription factor is expressed and functions specifically in surface glia found on peripheral nerves but not in the CNS to govern CNS structure, identifying a gene that functionally subdivides a glial subtype along the peripheral–central axis. Future work on these genes should clarify the genetic mechanisms that ensure the homeostasis of CNS form during development. [ABSTRACT FROM AUTHOR]

This study investigated the dominant blue eyes (DBE) trait linked to hearing impairment and variable white spotting in Maine Coon cats. Fifty-eight animals descending from 2 different DBE lineages, the Dutch and the Topaz lines, were sampled. They comprised 48 cats from the Dutch bloodline, including 9 green-eyed and 31 blue-eyed cats, with some individuals exhibiting signs of deafness, and 8 stillborn kittens. Samples from the Topaz lineage included 10 blue-eyed animals. A brainstem auditory evoked response test revealed a reduced to absent response to auditory stimuli and absent physiological waveforms in all of the 8 examined DBE animals. We sequenced the genome of 2 affected cats from the Dutch line and searched for variants in 19 candidate genes for the human Waardenburg syndrome and pigmentary disorders. This search yielded 9 private protein-changing candidate variants in the genes PAX3 , EDN3 , KIT , OCA2 , SLC24A5 , HERC2 , and TYRP1. The genotype–phenotype cosegregation was observed for the PAX3 variant within all animals from the Dutch lineage. The mutant allele was absent from 461 control genomes and 241 additionally genotyped green-eyed Maine Coons. We considered the PAX3 variant as the most plausible candidate—a heterozygous nonsense single base pair substitution in exon 6 of PAX3 (NC_051841.1:g.205,787,310G>A, XM_019838731.3:c.937C>T, XP_019694290.1:p.Gln313*), predicted to result in a premature stop codon. PAX3 variants cause auditory–pigmentary syndrome in humans, horses, and mice. Together with the comparative data from other species, our findings strongly suggest PAX3 :c.937C>T (OMIA:001688-9685) as the most likely candidate variant for the DBE, deafness, and minimal white spotting in the Maine Coon Dutch line. Finally, we propose the designation of DBERE (Rociri Elvis Dominant Blue Eyes) allele in the domestic cat. [ABSTRACT FROM AUTHOR]

The study of social parasitism faces numerous challenges arising from the intricate and intranidal host–parasite interactions and the rarity of parasites compared to their free-living counterparts. As a result, our understanding of the ecology and evolution of most social parasites remains limited. Using whole-genome and reduced-representation sequence data, we conducted a study to fill knowledge gaps on host use, colony social structure, and population genetics of the facultative dulotic ant Formica aserva Forel. Our study reveals the remarkable ability of F. aserva to exploit at least 20 different host species across its wide geographic distribution. In some cases, one social parasite colony exploits multiple hosts simultaneously, suggesting a high degree of generalization even at a local spatial scale. Approximately 80% of the colonies were monogyne (with a single queen), with many exhibiting higher rates of polyandry compared to most Formica ants. Although we identified a supergene on chromosome 3, its association with colony structure remains uncertain due to the rarity of polygyny in our sample. Population genetic analyses reveal substantial geographic population structure, with the greatest divergence between California populations and those from the rest of the range. Mitochondrial population structure differs from structure inferred from the nuclear genome on a broad geographic scale, suggesting a possible role of adaptive introgression or genetic drift. This study provides valuable insights into the ecology and evolution of F. aserva , underscoring the need for further research to decipher the complexities of host interactions and the genetic mechanisms that regulate social structure. [ABSTRACT FROM AUTHOR]

Charcot-Marie-Tooth disease (CMT) is one of the most common and genetically heterogeneous inherited neurological diseases, with more than 130 disease-causing genes. Whole genome sequencing (WGS) has improved diagnosis across genetic diseases, but the diagnostic impact in CMT is yet to be fully reported. We present the diagnostic results from a single specialist inherited neuropathy centre, including the impact of WGS diagnostic testing. Patients were assessed at our specialist inherited neuropathy centre from 2009 to 2023. Genetic testing was performed using single gene testing, next-generation sequencing targeted panels, research whole exome sequencing and WGS and, latterly, WGS through the UK National Health Service. Variants were assessed using the American College of Medical Genetics and Genomics and Association for Clinical Genomic Science criteria. Excluding patients with hereditary ATTR amyloidosis, 1515 patients with a clinical diagnosis of CMT and related disorders were recruited. In summary, 621 patients had CMT1 (41.0%), 294 CMT2 (19.4%), 205 intermediate CMT (CMTi, 13.5%), 139 hereditary motor neuropathy (HMN, 9.2%), 93 hereditary sensory neuropathy (HSN, 6.1%), 38 sensory ataxic neuropathy (2.5%), 72 hereditary neuropathy with liability to pressure palsies (HNPP, 4.8%) and 53 'complex' neuropathy (3.5%). Overall, a genetic diagnosis was reached in 76.9% (1165/1515). A diagnosis was most likely in CMT1 (96.8%, 601/621), followed by CMTi (81.0%, 166/205) and then HSN (69.9%, 65/93). Diagnostic rates remained less than 50% in CMT2, HMN and complex neuropathies. The most common genetic diagnosis was PMP22 duplication (CMT1A; 505/1165, 43.3%), then GJB1 (CMTX1; 151/1165, 13.0%), PMP22 deletion (HNPP; 72/1165, 6.2%) and MFN2 (CMT2A; 46/1165, 3.9%). We recruited 233 cases to the UK 100 000 Genomes Project (100KGP), of which 74 (31.8%) achieved a diagnosis; 28 had been otherwise diagnosed since recruitment, leaving a true diagnostic rate of WGS through the 100KGP of 19.7% (46/233). However, almost half of the solved cases (35/74) received a negative report from the study, and the diagnosis was made through our research access to the WGS data. The overall diagnostic uplift of WGS for the entire cohort was 3.5%. Our diagnostic rate is the highest reported from a single centre and has benefitted from the use of WGS, particularly access to the raw data. However, almost one-quarter of all cases remain unsolved, and a new reference genome and novel technologies will be important to narrow the 'diagnostic gap'. [ABSTRACT FROM AUTHOR]

Objectives Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major nosocomial infectious pathogen with rapidly increasing prevalence. The genomic epidemiological characteristics of CRKP nationwide, especially the evolving trends within the predominant clones, should be evaluated clearly. Methods We collected 3415 K. pneumoniae strains from 28 hospitals across China. Antimicrobial susceptibility testing and WGS were performed. Subsequent genomic analyses, including sequence typing, K-locus (KL) identification, antimicrobial resistance gene screening, and virulence score assessment were performed. The phylogenetic relationship of clonal group 11 was determined based on core-genome analysis, and the presence of the pLVPK-like virulence plasmid in ST11 isolates was confirmed using plasmid core-gene analysis. Additionally, the trends of the ST11 lineage with different KL types on a global scale were investigated using Beast2. Results Of the K. pneumoniae strains, 708 were identified as CRKP isolates (20.7%), of which 97.7% were MDR. ST11 was the predominant clone, and KPC-2 was the prevalent carbapenemase in China, although the prevalence of specific clones and carbapenemases varied by geographic region. Among ST11 isolates, KL47 and KL64 were the predominant KL types, and KL64 gradually replaced KL47, with a higher percentage of KL64 isolates harbouring the pLVPK-like plasmid. Global genome data showed a significant increase in the effective population size of KL64 over the last 5 years. Conclusions The prevalence of CRKP was very high in certain regions in China. The increasing convergence of virulence and resistance, particularly in ST11-KL64 isolates, should be given more attention and further investigation. [ABSTRACT FROM AUTHOR]

Objectives This study aimed to explore the prevalence of macrolide resistance and the underlying resistance mechanisms in Haemophilus influenzae (n  = 2556) and Haemophilus parainfluenzae (n  = 510) collected between 2018 and 2021 from Bellvitge University Hospital, Spain. Methods Antimicrobial susceptibility was tested by microdilution. Whole-genome sequencing was performed using Illumina MiSeq and Oxford Nanopore technologies, and sequences were examined for macrolide resistance determinants and mobile genetic structures. Results Macrolide resistance was detected in 67 H. influenzae (2.6%) and 52 (10.2%) H. parainfluenzae strains and associated with resistance to other antimicrobials (co-trimoxazole, chloramphenicol, tetracycline). Differences in macrolide resistance existed between the two species. Acquired resistance genes were more prevalent in H. parainfluenzae (35/52; 67.3%) than in H. influenzae (12/67; 17.9%). Gene mutations and amino acid substitutions were more common in H. influenzae (57/67; 85%) than in H. parainfluenzae (16/52; 30.8%). Substitutions in L22 and in 23S rRNA were only detected in H. influenzae (34.3% and 29.0%, respectively), while substitutions in L4 and AcrAB/AcrR were observed in both species. The MEGA element was identified in 35 (67.3%) H. parainfluenzae strains, five located in an integrative and conjugative element (ICE); by contrast, 11 (16.4%) H. influenzae strains contained the MEGA element (all in an ICE). A new ICE HpaHUB8 was described in H. parainfluenzae. Conclusions Macrolide resistance was higher in H. parainfluenzae than in H. influenzae , with differences in the underlying mechanisms. H. parainfluenzae exhibits co-resistance to other antimicrobials, often leading to an extensively drug-resistant phenotype. This highlights the importance of conducting antimicrobial resistance surveillance. [ABSTRACT FROM AUTHOR]

Background In Santiago, Chile, where typhoid had been hyperendemic (1977–1991), we investigated whether residual chronic carriers could be detected among household contacts of non-travel-related typhoid cases occurring during 2017–2019. Methods Culture-confirmed cases were classified as autochthonous (domestically acquired) versus travel/immigration related. Household contacts of cases had stool cultures and serum Vi antibody measurements to detect chronic Salmonella Typhi carriers. Whole genome sequences of acute cases and their epidemiologically linked chronic carrier isolates were compared. Results Five of 16 autochthonous typhoid cases (31.3%) were linked to 4 chronic carriers in case households; 2 cases (onsets 23 months apart) were linked to the same carrier. Carriers were women aged 69–79 years with gallbladder dysfunction and Typhi fecal excretion; 3 had highly elevated serum anti-Vi titers. Genomic analyses revealed close identity (≤11 core genome single-nucleotide polymorphism [SNP] differences) between case and epidemiologically linked carrier isolates; all were genotypes prevalent in 1980s Santiago. A cluster of 4 additional autochthonous cases unlinked to a carrier was identified based on genomic identity (0-1 SNPs). Travel/immigration isolate genotypes were typical for the countries of travel/immigration. Conclusions Although autochthonous typhoid cases in Santiago are currently rare, 5 of 16 such cases (31.3%) were linked to elderly chronic carriers identified among household contacts of cases. [ABSTRACT FROM AUTHOR]

Background Genetic variants in SEC61A1 are associated with autosomal dominant tubulointerstitial kidney disease. SEC61A1 is a translocon in the endoplasmic reticulum membrane and variants affect biosynthesis of renin and uromodulin. Methods A patient is described that presented at 1 year of age with failure-to-thrive, kidney failure (glomerular filtration rate, GFR, 18 ml/min/1.73m2), hyperkalemia and acidosis. Genetic evaluation was performed by whole genome sequencing. Results The patient has a novel de novo heterozygous SEC61A1 variant, Phe458Val. Plasma renin was low or normal, aldosterone was low or undetectable and uromodulin was low. Kidney biopsy at 2 years exhibited subtle changes suggestive of tubular dysgenesis without tubulocystic or glomerulocystic lesions and with renin staining of the juxtaglomerular cells. The patient experienced extreme fatigue due to severe hypotension attributed to hypoaldosteronism and at 8 years of age fludrocortisone treatment was initiated with marked improvement in her well-being. Blood pressure and potassium normalized. Biopsy at 9 years showed extensive glomerulosclerosis and mild tubulointerstitial fibrosis, as well as tubular mitochondrial abnormalities, without specific diagnostic changes. Her GFR improved to 54 ml/min/1.73m2. Conclusions As the renin-angiotensin system promotes aldosterone release, and the patient had repeatedly undetectable aldosterone levels, the SEC61A1 variant presumably contributed to severe hypotension. Treatment with a mineralocorticoid had a beneficial effect and corrected the electrolyte and acid-base disorder. We suggest that the increased blood pressure hemodynamically improved the patient's kidney function. [ABSTRACT FROM AUTHOR]

We sequenced and genotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, adenovirus, and respiratory syncytial virus, among other pathogens, from residual anterior nasal swabs self-collected for rapid SARS-CoV-2 antigen testing at the US Naval Academy. This is a key proof-of-concept for an acute respiratory infection surveillance approach, which could leverage prevalent SARS-CoV-2 antigen self-testing. [ABSTRACT FROM AUTHOR]

Background Isoniazid-resistant, rifampin-susceptible tuberculosis (Hr-TB) is associated with poor treatment outcomes and higher rates of acquisition of further drug resistance during treatment. Due to a lack of widespread diagnostics, Hr-TB is frequently undetected and its epidemiology is incompletely understood. Methods We studied the molecular epidemiology of Hr-TB among all patients diagnosed with culture-positive pulmonary tuberculosis between January 1 and June 30, 2017, at an urban referral tuberculosis clinic in Port-au-Prince, Haiti. Demographic and clinical data were extracted from the electronic medical record. Archived diagnostic Mycobacterium tuberculosis isolates were tested for genotypic and phenotypic isoniazid resistance using the Genotype MTBDR plus assay (Hain, Nehren, Germany) and culture-based testing, respectively. All isoniazid-resistant isolates and a randomly selected subset of isoniazid-susceptible isolates underwent whole-genome sequencing to confirm the presence of mutations associated with isoniazid resistance, to validate use of Genotype MTBDR plus in this population, and to identify potential transmission links between isoniazid-resistant isolates. Results and Conclusions Among 845 patients with culture-positive pulmonary tuberculosis in Haiti, 65 (7.7%) had Hr-TB based on the Genotype MTBDR plus molecular assay. Age < 20 years was significantly associated with Hr-TB (odds ratio, 2.39; 95% confidence interval, 1.14, 4.70; P =.015). Thirteen (20%) isoniazid-resistant isolates were found in 5 putative transmission clusters based on a single nucleotide polymorphism distance of ≤ 5. No patients in these transmission clusters were members of the same household. Adolescents are at higher risk for Hr-TB. Strains of isoniazid-resistant M tuberculosis are actively circulating in Haiti and transmission is likely occurring in community settings. [ABSTRACT FROM AUTHOR]

During routine clinical practice, infectious disease physicians encounter patients with difficult-to-diagnose clinical syndromes and may order advanced molecular testing to detect pathogens. These tests may identify potential infectious causes for illness and allow clinicians to adapt treatments or stop unnecessary antimicrobials. Cases of pathogen-agnostic disease testing also provide an important window into known, emerging, and reemerging pathogens and may be leveraged as part of national sentinel surveillance. A survey of Emerging Infections Network members, a group of infectious disease providers in North America, was conducted in May 2023. The objective of the survey was to gain insight into how and when infectious disease physicians use advanced molecular testing for patients with difficult-to-diagnose infectious diseases, as well as to explore the usefulness of advanced molecular testing and barriers to use. Overall, 643 providers answered at least some of the survey questions; 478 (74%) of those who completed the survey had ordered advanced molecular testing in the last two years, and formed the basis for this study. Respondents indicated that they most often ordered broad-range 16S rRNA gene sequencing, followed by metagenomic next-generation sequencing and whole genome sequencing; and commented that in clinical practice, some, but not all tests were useful. Many physicians also noted several barriers to use, including a lack of national guidelines and cost, while others commented that whole genome sequencing had potential for use in outbreak surveillance. Improving frontline physician access, availability, affordability, and developing clear national guidelines for interpretation and use of advanced molecular testing could potentially support clinical practice and public health surveillance. [ABSTRACT FROM AUTHOR]

Aims Adequately and accurately identifying carbapenemase-producing Enterobacterales (CPE) is vital for selecting appropriate antimicrobial therapy and implementing effective infection control measures. This study aims to optimize the phenotypic detection method of carbapenemase for routine diagnostics in clinical microbiology laboratories. Methods and Results Carbapenemase genes in 2665 non-duplicate CRE clinical strains collected from various regions of China were confirmed through whole-genome sequencing (WGS). The carbapenemase inhibition test (CIT) was conducted and interpreted using different methods and breakpoints, then compared with the NG-Test CARBA 5 for carbapenemase detection. The diagnostic performance of the CIT method was optimal when the carbapenemase types were determined by comparing the inhibition zone diameters of the imipenem disc with 3-aminophenylboronic acid (APB) plus ethylenediaminetetraacetic acid (EDTA) to those of the imipenem disc with either APB or EDTA alone, with a breakpoint of 4 mm. The overall sensitivities of the current CIT, the modified CIT, and NG-Test CARBA 5 were 91.4%, 94.9%, and 99.9%, respectively. For detecting isolates co-producing Klebsiella pneumoniae carbapenemase (KPC) and metallo-β-lactamases (MBLs), the modified CIT method had higher sensitivity than the current method (70.0% vs. 53.3%), though this difference was not statistically significant (P  = 0.063). The NG-Test CARBA 5 showed excellent performance for multi-carbapenemases diagnosis, with sensitivity and specificity of 97.1% and 100%, respectively. Conclusions Optimizing and standardizing the CIT method for clinical use is necessary. It has certain advantages in diagnosing multi-carbapenemase and rare carbapenemase production. However, for identifying common carbapenemase types, the NG-Test CARBA 5 demonstrated superior performance. [ABSTRACT FROM AUTHOR]

Aims To investigate enterococci carrying linezolid and vancomycin resistance genes from fecal samples recovered from wild boars Methods and results Florfenicol- and vancomycin-resistant enterococci, isolated on selective agar plates, were screened by PCR for the presence of linezolid and vancomycin resistance genes. Five isolates carried optrA or poxtA linezolid resistance genes; one strain was resistant to vancomycin for the presence of vanA gene. All isolates were tested for their antibiotic susceptibility and subjected to Whole Genome Sequencing (WGS) analysis. In Enterococcus faecalis (E. faecalis) V1344 and V1676, the optrA was located on the new pV1344-optrA and pV1676-optrA plasmids, respectively, whereas in Enterococcus faecium (E. faecium) V1339 this gene was on a 22 354-bp chromosomal genetic context identical to the one detected in a human E. faecium isolate. In both E. faecium V1682 and E. durans V1343, poxtA was on the p1818-c plasmid previously found in a human E. faecium isolate. In E. faecium V1328, the vanA gene was on the Tn 1546 transposon in turn located on a new pV1328-vanA plasmid. Only E. faecium V1682 successfully transferred the poxtA gene to an enterococcal recipient in filter mating assays. Conclusions The occurrence of genetic elements carrying linezolid and vancomycin resistance genes in enterococci from wild boars is a matter of concern, moreover, the sharing of plasmids and transposons between isolates from wild animals, human, and environment indicates an exchange of genetic material between these settings. [ABSTRACT FROM AUTHOR]

Motivation Somatic mosaicism has been implicated in several developmental disorders, cancers, and other diseases. Short tandem repeats (STRs) consist of repeated sequences of 1–6 bp and comprise >1 million loci in the human genome. Somatic mosaicism at STRs is known to play a key role in the pathogenicity of loci implicated in repeat expansion disorders and is highly prevalent in cancers exhibiting microsatellite instability. While a variety of tools have been developed to genotype germline variation at STRs, a method for systematically identifying mosaic STRs is lacking. Results We introduce prancSTR, a novel method for detecting mosaic STRs from individual high-throughput sequencing datasets. prancSTR is designed to detect loci characterized by a single high-frequency mosaic allele, but can also detect loci with multiple mosaic alleles. Unlike many existing mosaicism detection methods for other variant types, prancSTR does not require a matched control sample as input. We show that prancSTR accurately identifies mosaic STRs in simulated data, demonstrate its feasibility by identifying candidate mosaic STRs in Illumina whole genome sequencing data derived from lymphoblastoid cell lines for individuals sequenced by the 1000 Genomes Project, and evaluate the use of prancSTR on Element and PacBio data. In addition to prancSTR, we present simTR, a novel simulation framework which simulates raw sequencing reads with realistic error profiles at STRs. Availability and implementation prancSTR and simTR are freely available at https://github.com/gymrek-lab/trtools. Detailed documentation is available at https://trtools.readthedocs.io/. [ABSTRACT FROM AUTHOR]

It is known that some endangered species have persisted for thousands of years despite their very small effective population sizes and low levels of genetic polymorphisms. To understand the genetic mechanisms of long-term persistence in threatened species, we determined the whole genome sequences of akame (Lates japonicus), which has survived for a long time with extremely low genetic variations. Genome-wide heterozygosity in akame was estimated to be 3.3 to 3.4 × 10−4/bp, one of the smallest values in teleost fishes. Analysis of demographic history revealed that the effective population size in akame was around 1,000 from 30,000 years ago to the recent past. The relatively high ratio of nonsynonymous to synonymous heterozygosity in akame indicated an increased genetic load. However, a detailed analysis of genetic diversity in the akame genome revealed that multiple genomic regions, including genes involved in immunity, synaptic development, and olfactory sensory systems, have retained relatively high nucleotide polymorphisms. This implies that the akame genome has preserved the functional genetic variations by balancing selection, to avoid a reduction in viability and loss of adaptive potential. Analysis of synonymous and nonsynonymous nucleotide substitution rates has detected signs of positive selection in many akame genes, suggesting adaptive evolution to temperate waters after the speciation of akame and its close relative, barramundi (Lates calcarifer). Our results indicate that the functional genetic diversity likely contributed to the long-term persistence of this species by avoiding the harmful effects of the population size reduction. [ABSTRACT FROM AUTHOR]

Opportunistic pathogens are environmental microbes that are generally harmless and only occasionally cause disease. Unlike obligate pathogens, the growth and survival of opportunistic pathogens do not rely on host infection or transmission. Their versatile lifestyles make it challenging to decipher how and why virulence has evolved in opportunistic pathogens. The coincidental evolution hypothesis postulates that virulence results from exaptation or pleiotropy, i.e. traits evolved for adaptation to living in one environment that have a different function in another. In particular, adaptation to avoid or survive protist predation has been suggested to contribute to the evolution of bacterial virulence (the training ground hypothesis). Here, we used experimental evolution to determine how the selective pressure imposed by a protist predator impacts the virulence and fitness of a ubiquitous environmental opportunistic bacterial pathogen that has acquired multidrug resistance: Serratia marcescens. To this aim, we evolved S. marcescens in the presence or absence of generalist protist predator, Tetrahymena thermophila. After 60 d of evolution, we evaluated genotypic and phenotypic changes by comparing evolved S. marcescens with the ancestral strain. Whole-genome shotgun sequencing of the entire evolved populations and individual isolates revealed numerous cases of parallel evolution, many more than statistically expected by chance, in genes associated with virulence. Our phenotypic assays suggested that evolution in the presence of a predator maintained virulence, whereas evolution in the absence of a predator resulted in attenuated virulence. We also found a significant correlation between virulence, biofilm formation, growth, and grazing resistance. Overall, our results provide evidence that bacterial virulence and virulence-related traits are maintained by selective pressures imposed by protist predation. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a severe motor neuron disease with uncertain genetic predisposition in most sporadic cases. The spatial architecture of cell types and gene expression are the basis of cell–cell interactions, biological function and disease pathology, but are not well investigated in the human motor cortex, a key ALS-relevant brain region. Recent studies indicated single nucleus transcriptomic features of motor neuron vulnerability in ALS motor cortex. However, the brain regional vulnerability of ALS-associated genes and the genetic link between region-specific genes and ALS risk remain largely unclear. Here, we developed an entropy-weighted differential gene expression matrix-based tool (SpatialE) to identify the spatial enrichment of gene sets in spatial transcriptomics. We benchmarked SpatialE against another enrichment tool (multimodal intersection analysis) using spatial transcriptomics data from both human and mouse brain tissues. To investigate regional vulnerability, we analysed three human motor cortex and two dorsolateral prefrontal cortex tissues for spatial enrichment of ALS-associated genes. We also used Cell2location to estimate the abundance of cell types in ALS-related cortex layers. To dissect the link of regionally expressed genes and ALS risk, we performed burden analyses of rare loss-of-function variants detected by whole-genome sequencing in ALS patients and controls, then analysed differential gene expression in the TargetALS RNA-sequencing dataset. SpatialE showed more accurate and specific spatial enrichment of regional cell type markers than multimodal intersection analysis in both mouse brain and human dorsolateral prefrontal cortex. Spatial transcriptomic analyses of human motor cortex showed heterogeneous cell types and spatial gene expression profiles. We found that 260 manually curated ALS-associated genes are significantly enriched in layer 5 of the motor cortex, with abundant expression of upper motor neurons and layer 5 excitatory neurons. Burden analyses of rare loss-of-function variants in Layer 5-associated genes nominated NOMO1 as a novel ALS-associated gene in a combined sample set of 6814 ALS patients and 3324 controls (P = 0.029). Gene expression analyses in CNS tissues revealed downregulation of NOMO1 in ALS, which is consistent with a loss-of-function disease mechanism. In conclusion, our integrated spatial transcriptomics and genomic analyses identified regional brain vulnerability in ALS and the association of a layer 5 gene (NOMO1) with ALS risk. [ABSTRACT FROM AUTHOR]

Chromosomal rearrangements can lead to the coupling of reproductive barriers, but whether and how they contribute to the completion of speciation remains unclear. Marine snails of the genus Littorina repeatedly form hybrid zones between populations segregating for multiple inversion arrangements, providing opportunities to study their barrier effects. Here, we analyzed 2 adjacent transects across hybrid zones between 2 ecotypes of Littorina fabalis ("large" and "dwarf") adapted to different wave exposure conditions on a Swedish island. Applying whole-genome sequencing, we found 12 putative inversions on 9 of 17 chromosomes. Nine of the putative inversions reached near differential fixation between the 2 ecotypes, and all were in strong linkage disequilibrium. These inversions cover 20% of the genome and carry 93% of divergent single nucleotide polymorphisms (SNPs). Bimodal hybrid zones in both transects indicated that the 2 ecotypes of Littorina fabalis maintain their genetic and phenotypic integrity following contact. The bimodality reflects the strong coupling between inversion clines and the extension of the barrier effect across the whole genome. Demographic inference suggests that coupling arose during a period of allopatry and has been maintained for > 1,000 generations after secondary contact. Overall, this study shows that the coupling of multiple chromosomal inversions contributes to strong reproductive isolation. Notably, 2 of the putative inversions overlap with inverted genomic regions associated with ecotype differences in a closely related species (Littorina saxatilis), suggesting the same regions, with similar structural variants, repeatedly contribute to ecotype evolution in distinct species. [ABSTRACT FROM AUTHOR]

A study conducted in Djibouti, East Africa, has found the presence of a dangerous strain of bacteria called hypervirulent carbapenem-resistant Klebsiella pneumoniae (CR-hvKP) in both humans and fish. This strain is resistant to last-resort antibiotics and exhibits multidrug-resistance phenotypes. The study suggests that these bacteria may be spreading in the environment, emphasizing the need for further research and measures to prevent their spread. The study also found that the CR-hvKP isolates carried hybrid resistance/virulence plasmids, containing both antimicrobial resistance genes and virulence genes. This indicates the emergence and possible dissemination of CR-hvKP in Africa and raises concerns about the contamination of surface waters by these bacteria. [Extracted from the article]

Soybean is an important agricultural crop known for its high protein and oil content, contributing to essential nutritional and health benefits for humans. Domesticated in China over 5,000 years ago, soybean has since adapted to diverse environments and spread worldwide. This study aimed to investigate the genomic characteristics and population structures of 2,317 publicly available soybean whole-genome sequences from diverse geographical regions, including China, Korea, Japan, Europe, North America, and South America. We used large-scale whole-genome sequencing data to perform high-resolution analyses to reveal the genetic characteristics of soybean accessions. Soybean accessions from China and Korea exhibited landrace characteristics, indicating higher genetic diversity and adaptation to local environments. On the other hand, soybean accessions from Japan, the European Union, and South America were found to have low genetic diversity due to artificial selection and breeding for agronomic traits. We also identified key variants and genes associated with the ability to adapt to different environments. In Korean soybean accessions, we observed strong selection signals for isoflavone synthesis, an adaptive trait critical for improving soybean adaptability, survival, and reproductive success by mitigating environmental stress. Identifying specific genomic regions showing unique patterns of selective sweeps for genes such as HIDH, CYP73A11, IFS1, and CYP81E11 associated with isoflavone synthesis provided valuable insights into potential adaptation mechanisms. Our research has significantly improved our understanding of soybean diversity at the genetic level. We have identified key genetic variants and genes influencing adaptability, laying the foundation for future advances in genomics-based breeding programs and crop improvement efforts. [ABSTRACT FROM AUTHOR]

Background Grip strength is a robust indicator of overall health, is moderately heritable, and predicts longevity in older adults. Methods Using genome-wide linkage analysis, we identified a novel locus on chromosome 18p (mega-basepair region: 3.4–4.0) linked to grip strength in 3 755 individuals from 582 families aged 64 ± 12 years (range 30–110 years; 55% women). There were 26 families that contributed to the linkage peak (cumulative logarithm of the odds [LOD] score = 10.94), with 6 families (119 individuals) accounting for most of the linkage signal (LOD = 6.4). In these 6 families, using whole genome sequencing data, we performed association analyses between the 7 312 single nucleotide (SNVs) and insertion deletion (INDELs) variants in the linkage region and grip strength. Models were adjusted for age, age2, sex, height, field center, and population substructure. Results We found significant associations between genetic variants (8 SNVs and 4 INDELs, p  < 5 × 10−5) in the Disks Large-associated Protein 1 (DLGAP1) gene and grip strength. Haplotypes constructed using these variants explained up to 98.1% of the LOD score. Finally, RNAseq data showed that these variants were significantly associated with the expression of nearby Myosin Light Chain 12A (MYL12A), Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1), Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3) genes (p  < .0004). Conclusions The DLGAP1 gene plays an important role in the postsynaptic density of neurons; thus, it is both a novel positional and biological candidate gene for follow-up studies aimed at uncovering genetic determinants of muscle strength. [ABSTRACT FROM AUTHOR]

Background Hypertrophic cardiomyopathy in the neonate has a diverse genetic background, and non-sarcomeric variants may not be identified on commercial genetic testing panels. NDUFB11 is an X-linked mitochondrial Complex I protein and is known to cause histiocytoid cardiomyopathy but has not been described in female infants with hypertrophic cardiomyopathy. We present this first reported case of obstructive hypertrophic cardiomyopathy in a female neonate secondary to a pathogenic variant in NDUFB11. Case summary A term female neonate presented following a prenatal diagnosis of biventricular hypertrophy and growth restriction. She developed lactic acidosis after birth and whole-genome sequencing identified a de novo variant in the mitochondrial Complex I gene, NDUFB11 (c.391G>A, p.Glu131Lys). There was progression of left ventricular hypertrophy and obstruction, with rapid development of heart failure symptoms. She was unresponsive to beta-blocker medical therapy and was not suitable for advanced mechanical support. There was subsequent clinical deterioration resulting in death by 3 months of age. Discussion Hemizygous variants in NDUFB11 have been associated with hypertrophic cardiomyopathy in male infants previously, and skewed X-linked inactivation likely resulted in the presentation described here in a female infant. This variant was not identifiable by commercial cardiomyopathy panels. We highlight the importance of rapid whole-genome sequencing in cases of infantile hypertrophic cardiomyopathy and the importance of genetic diagnosis in guiding prognosis and care for these individuals. [ABSTRACT FROM AUTHOR]

Obesity, characterized by the accumulation of excess fat, is a complex condition resulting from the combination of genetic and epigenetic factors. Recent studies have found correspondence between DNA methylation and cell differentiation, suggesting a role of the former in cell fate determination. There is a lack of comprehensive understanding concerning the underpinnings of preadipocyte differentiation, specifically when cells are undergoing terminal differentiation (TD). To gain insight into dynamic genome-wide methylation, 3T3 L1 preadipocyte cells were differentiated by a hormone cocktail. The genomic DNA was isolated from undifferentiated cells and 4 hours, 2 days postdifferentiated cells, and 15 days TD cells. We employed whole-genome bisulfite sequencing (WGBS) to ascertain global genomic DNA methylation alterations at single base resolution as preadipocyte cells differentiate. The genome-wide distribution of DNA methylation showed similar overall patterns in pre-, post-, and terminally differentiated adipocytes, according to WGBS analysis. DNA methylation decreases at 4 hours after differentiation initiation, followed by methylation gain as cells approach TD. Studies revealed novel differentially methylated regions (DMRs) associated with adipogenesis. DMR analysis suggested that though DNA methylation is global, noticeable changes are observed at specific sites known as "hotspots." Hotspots are genomic regions rich in transcription factor (TF) binding sites and exhibit methylation-dependent TF binding. Subsequent analysis indicated hotspots as part of DMRs. The gene expression profile of key adipogenic genes in differentiating adipocytes is context-dependent, as we found a direct and inverse relationship between promoter DNA methylation and gene expression. [ABSTRACT FROM AUTHOR]

In Mycobacterium tuberculosis (MTB) control, whole genome sequencing-based molecular drug susceptibility testing (molDST-WGS) has emerged as a pivotal tool. However, the current reliance on a single-strain reference limits molDST-WGS's true potential. To address this, we introduce a new pan-lineage reference genome, 'MtbRf'. We assembled 'unmapped' reads from 3,614 MTB genomes (751 L1; 881 L2; 1,700 L3; and 282 L4) into 35 shared, annotated contigs (54 coding sequences [CDSs]). We constructed MtbRf through: (1) searching for contig homologues among genome database that precipitate results uniquely within Mycobacteria genus; (2) comparing genomes with H37Rv ('lift-over') to define 18 insertions; and (3) filling gaps in H37Rv with insertions. MtbRf adds 1.18% sequences to H37rv, salvaging >60% of previously unmapped reads. Transcriptomics confirmed gene expression of new CDSs. The new variants provided a moderate DST predictive value (AUROC 0.60–0.75). MtbRf thus unveils previously hidden genomic information and lays the foundation for lineage-specific molDST-WGS. [ABSTRACT FROM AUTHOR]

The mouse serves as a mammalian model for understanding the nature of variation from new mutations, a question that has both evolutionary and medical significance. Previous studies suggest that the rate of single-nucleotide mutations (SNMs) in mice is ∼50% of that in humans. However, information largely comes from studies involving the C57BL/6 strain, and there is little information from other mouse strains. Here, we study the mutations that accumulated in 59 mouse lines derived from four inbred strains that are commonly used in genetics and clinical research (BALB/cAnNRj, C57BL/6JRj, C3H/HeNRj, and FVB/NRj), maintained for eight to nine generations by brother–sister mating. By analyzing Illumina whole-genome sequencing data, we estimate that the average rate of new SNMs in mice is ∼ μ = 6.7 × 10−9. However, there is substantial variation in the spectrum of SNMs among strains, so the burden from new mutations also varies among strains. For example, the FVB strain has a spectrum that is markedly skewed toward C→A transversions and is likely to experience a higher deleterious load than other strains, due to an increased frequency of nonsense mutations in glutamic acid codons. Finally, we observe substantial variation in the rate of new SNMs among DNA sequence contexts, CpG sites, and their adjacent nucleotides playing an important role. [ABSTRACT FROM AUTHOR]

Background Streptococcus pneumoniae serotype 3 remains a problem globally. Malawi introduced 13-valent pneumococcal conjugate vaccine (PCV13) in 2011, but there has been no direct protection against serotype 3 carriage. We explored whether vaccine escape by serotype 3 is due to clonal expansion of a lineage with a competitive advantage. Methods The distribution of serotype 3 Global Pneumococcal Sequence Clusters (GPSCs) and sequence types (STs) globally was assessed using sequences from the Global Pneumococcal Sequencing Project. Whole-genome sequences of 135 serotype 3 carriage isolates from Blantyre, Malawi (2015–2019) were analyzed. Comparative analysis of the capsule locus, entire genomes, antimicrobial resistance, and phylogenetic reconstructions were undertaken. Opsonophagocytosis was evaluated using serum samples from vaccinated adults and children. Results Serotype 3 GPSC10-ST700 isolates were most prominent in Malawi. Compared with the prototypical serotype 3 capsular polysaccharide locus sequence, 6 genes are absent, with retention of capsule polysaccharide biosynthesis. This lineage is characterized by increased antimicrobial resistance and lower susceptibility to opsonophagocytic killing. Conclusions A serotype 3 variant in Malawi has genotypic and phenotypic characteristics that could enhance vaccine escape and clonal expansion after post-PCV13 introduction. Genomic surveillance among high-burden populations is essential to improve the effectiveness of next-generation pneumococcal vaccines. [ABSTRACT FROM AUTHOR]

Background Klebsiella pneumoniae carbapenemase–producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. Methods We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. Results We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. Conclusions Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence. [ABSTRACT FROM AUTHOR]

Background Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background. Methods We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform. Results Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%–28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P <.001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%–94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF. Conclusions We demonstrate the feasibility of magnetic bead–based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance. [ABSTRACT FROM AUTHOR]

Motivation World Health Organization estimates that there were over 10 million cases of tuberculosis (TB) worldwide in 2019, resulting in over 1.4 million deaths, with a worrisome increasing trend yearly. The disease is caused by Mycobacterium tuberculosis (MTB) through airborne transmission. Treatment of TB is estimated to be 85% successful, however, this drops to 57% if MTB exhibits multiple antimicrobial resistance (AMR), for which fewer treatment options are available. Results We develop a robust machine-learning classifier using both linear and nonlinear models (i.e. LASSO logistic regression (LR) and random forests (RF)) to predict the phenotypic resistance of Mycobacterium tuberculosis (MTB) for a broad range of antibiotic drugs. We use data from the CRyPTIC consortium to train our classifier, which consists of whole genome sequencing and antibiotic susceptibility testing (AST) phenotypic data for 13 different antibiotics. To train our model, we assemble the sequence data into genomic contigs, identify all unique 31-mers in the set of contigs, and build a feature matrix M , where M [ i , j ] is equal to the number of times the i th 31-mer occurs in the j th genome. Due to the size of this feature matrix (over 350 million unique 31-mers), we build and use a sparse matrix representation. Our method, which we refer to as MTB++, leverages compact data structures and iterative methods to allow for the screening of all the 31-mers in the development of both LASSO LR and RF. MTB++ is able to achieve high discrimination (F -1 >80%) for the first-line antibiotics. Moreover, MTB++ had the highest F -1 score in all but three classes and was the most comprehensive since it had an F -1 score >75% in all but four (rare) antibiotic drugs. We use our feature selection to contextualize the 31-mers that are used for the prediction of phenotypic resistance, leading to some insights about sequence similarity to genes in MEGARes. Lastly, we give an estimate of the amount of data that is needed in order to provide accurate predictions. Availability The models and source code are publicly available on Github at https://github.com/M-Serajian/MTB-Pipeline. [ABSTRACT FROM AUTHOR]

Motivation Eukaryotic cells contain organelles called mitochondria that have their own genome. Most cells contain thousands of mitochondria which replicate, even in nondividing cells, by means of a relatively error-prone process resulting in somatic mutations in their genome. Because of the higher mutation rate compared to the nuclear genome, mitochondrial mutations have been used to track cellular lineage, particularly using single-cell sequencing that measures mitochondrial mutations in individual cells. However, existing methods to infer the cell lineage tree from mitochondrial mutations do not model "heteroplasmy," which is the presence of multiple mitochondrial clones with distinct sets of mutations in an individual cell. Single-cell sequencing data thus provide a mixture of the mitochondrial clones in individual cells, with the ancestral relationships between these clones described by a mitochondrial clone tree. While deconvolution of somatic mutations from a mixture of evolutionarily related genomes has been extensively studied in the context of bulk sequencing of cancer tumor samples, the problem of mitochondrial deconvolution has the additional constraint that the mitochondrial clone tree must be concordant with the cell lineage tree. Results We formalize the problem of inferring a concordant pair of a mitochondrial clone tree and a cell lineage tree from single-cell sequencing data as the Nested Perfect Phylogeny Mixture (NPPM) problem. We derive a combinatorial characterization of the solutions to the NPPM problem, and formulate an algorithm, MERLIN, to solve this problem exactly using a mixed integer linear program. We show on simulated data that MERLIN outperforms existing methods that do not model mitochondrial heteroplasmy nor the concordance between the mitochondrial clone tree and the cell lineage tree. We use MERLIN to analyze single-cell whole-genome sequencing data of 5220 cells of a gastric cancer cell line and show that MERLIN infers a more biologically plausible cell lineage tree and mitochondrial clone tree compared to existing methods. Availability and implementation https://github.com/raphael-group/MERLIN. [ABSTRACT FROM AUTHOR]

Summary The reliable and timely recognition of outbreaks is a key component of public health surveillance for foodborne diseases. Whole genome sequencing (WGS) offers high resolution typing of foodborne bacterial pathogens and facilitates the accurate detection of outbreaks. This detection relies on grouping WGS data into clusters at an appropriate genetic threshold. However, methods and tools for selecting and adjusting such thresholds according to the required resolution of surveillance and epidemiological context are lacking. Here we present DODGE (Dynamic Outbreak Detection for Genomic Epidemiology), an algorithm to dynamically select and compare these genetic thresholds. DODGE can analyse expanding datasets over time and clusters that are predicted to correspond to outbreaks (or "investigation clusters") can be named with established genomic nomenclature systems to facilitate integrated analysis across jurisdictions. DODGE was tested in two real-world Salmonella genomic surveillance datasets of different duration, 2 months from Australia and 9 years from the United Kingdom. In both cases only a minority of isolates were identified as investigation clusters. Two known outbreaks in the United Kingdom dataset were detected by DODGE and were recognized at an earlier timepoint than the outbreaks were reported. These findings demonstrated the potential of the DODGE approach to improve the effectiveness and timeliness of genomic surveillance for foodborne diseases and the effectiveness of the algorithm developed. Availability and implementation DODGE is freely available at https://github.com/LanLab/dodge and can easily be installed using Conda. [ABSTRACT FROM AUTHOR]

Variants in cis -regulatory elements link the noncoding genome to human pathology; however, detailed analytic tools for understanding the association between cell-level brain pathology and noncoding variants are lacking. CWAS-Plus, adapted from a Python package for category-wide association testing (CWAS), enhances noncoding variant analysis by integrating both whole-genome sequencing (WGS) and user-provided functional data. With simplified parameter settings and an efficient multiple testing correction method, CWAS-Plus conducts the CWAS workflow 50 times faster than CWAS, making it more accessible and user-friendly for researchers. Here, we used a single-nuclei assay for transposase-accessible chromatin with sequencing to facilitate CWAS-guided noncoding variant analysis at cell-type-specific enhancers and promoters. Examining autism spectrum disorder WGS data (n  = 7280), CWAS-Plus identified noncoding de novo variant associations in transcription factor binding sites within conserved loci. Independently, in Alzheimer's disease WGS data (n  = 1087), CWAS-Plus detected rare noncoding variant associations in microglia-specific regulatory elements. These findings highlight CWAS-Plus's utility in genomic disorders and scalability for processing large-scale WGS data and in multiple-testing corrections. CWAS-Plus and its user manual are available at https://github.com/joonan-lab/cwas/ and https://cwas-plus.readthedocs.io/en/latest/ , respectively. [ABSTRACT FROM AUTHOR]

The emergence and rapid spread of SARS-CoV-2 prompted the global community to identify innovative approaches to diagnose infection and sequence the viral genome because at several points in the pandemic positive case numbers exceeded the laboratory capacity to characterize sufficient samples to adequately respond to the spread of emerging variants. From week 10, 2020, to week 13, 2023, Slovenian routine complete genome sequencing (CGS) surveillance network yielded 41 537 complete genomes and revealed a typical molecular epidemiology with early lineages gradually being replaced by Alpha, Delta, and finally Omicron. We developed a targeted next-generation sequencing based variant surveillance strategy dubbed Spike Screen through sample pooling and selective SARS-CoV-2 spike gene amplification in conjunction with CGS of individual cases to increase throughput and cost-effectiveness. Spike Screen identifies variant of concern (VOC) and variant of interest (VOI) signature mutations, analyses their frequencies in sample pools, and calculates the number of VOCs/VOIs at the population level. The strategy was successfully applied for detection of specific VOC/VOI mutations prior to their confirmation by CGS. Spike Screen complemented CGS efforts with an additional 22 897 samples sequenced in two time periods: between week 42, 2020, and week 24, 2021, and between week 37, 2021, and week 2, 2022. The results showed that Spike Screen can be applied to monitor VOC/VOI mutations among large volumes of samples in settings with limited sequencing capacity through reliable and rapid detection of novel variants at the population level and can serve as a basis for public health policy planning. [ABSTRACT FROM AUTHOR]

Tumor mutational signatures have gained prominence in cancer research, yet the lack of standardized methods hinders reproducibility and robustness. Leveraging colorectal cancer (CRC) as a model, we explored the influence of computational parameters on mutational signature analyses across 230 CRC cell lines and 152 CRC patients. Results were validated in three independent datasets: 483 endometrial cancer patients stratified by mismatch repair (MMR) status, 35 lung cancer patients by smoking status and 12 patient-derived organoids (PDOs) annotated for colibactin exposure. Assessing various bioinformatic tools, reference datasets and input data sizes including whole genome sequencing, whole exome sequencing and a pan-cancer gene panel, we demonstrated significant variability in the results. We report that the use of distinct algorithms and references led to statistically different results, highlighting how arbitrary choices may induce variability in the mutational signature contributions. Furthermore, we found a differential contribution of mutational signatures between coding and intergenic regions and defined the minimum number of somatic variants required for reliable mutational signature assignment. To facilitate the identification of the most suitable workflows, we developed Comparative Mutational Signature analysis on Coding and Extragenic Regions (CoMSCER), a bioinformatic tool which allows researchers to easily perform comparative mutational signature analysis by coupling the results from several tools and public reference datasets and to assess mutational signature contributions in coding and non-coding genomic regions. In conclusion, our study provides a comparative framework to elucidate the impact of distinct computational workflows on mutational signatures. [ABSTRACT FROM AUTHOR]

Aim To investigate the possible contamination of raw flour and raw flour-based products, such as pancake/batter mixes, with Salmonella , generic Escherichia coli , and Shiga-toxin-producing E. coli (STEC). Samples included flours available for sale in the UK over a period of four months (January to April 2020). The Bread and Flour regulations, 1998 state the permitted ingredients in flour and bread but it does not specify the regular monitoring of the microbiological quality of flour and flour-based products. Methods and results Samples of raw flour were collected by local authority sampling officers in accordance with current guidance on microbiological food sampling then transported to the laboratory for examination. Microbiological testing was performed to detect Salmonella spp. generic E. coli , and STEC characterized for the presence of STEC virulence genes: stx 1, stx 2, and subtypes, eae, ipah, aggR, lt, sth , and stp , using molecular methods Polymerase Chain Reaction (PCR). Of the 882 flours sampled, the incidence of Salmonella was 0.1% (a single positive sample that contained multiple ingredients such as flour, dried egg, and dried milk, milled in the UK), and 68 samples (7.7%) contained generic E. coli at a level of >20 CFU/g. Molecular characterization of flour samples revealed the presence of the Shiga-toxin (stx) gene in 10 samples (5 imported and 5 from the UK) (1.1%), from which STEC was isolated from 7 samples (0.8%). Salmonella and STEC isolates were sequenced to provide further characterization of genotypes and to compare to sequences of human clinical isolates held in the UKHSA archive. Using our interpretive criteria based on genetic similarity, none of the STEC flour isolates correlated with previously observed human cases, while the singular Salmonella serotype Newport isolate from the mixed ingredient product was similar to a human case in 2019, from the UK, of S. Newport. Although there have been no reported human cases of STEC matching the isolates from these flour samples, some of the same serotypes and stx subtypes detected are known to have caused illness in other contexts. Conclusion Results indicate that while the incidence was low, there is a potential for the presence of Salmonella and STEC in flour, and a genetic link was demonstrated between a Salmonella isolate from a flour-based product and a human case of salmonellosis. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a grave public health concern and is considered the foremost contributor to human mortality resulting from infectious disease. Due to the stringent clonality and extremely restricted genomic diversity, conventional methods prove inefficient for in-depth exploration of minor genomic variations and the evolutionary dynamics operating in Mycobacterium tuberculosis (M.tb) populations. Until now, the majority of reviews have primarily focused on delineating the application of whole-genome sequencing (WGS) in predicting antibiotic resistant genes, surveillance of drug resistance strains, and M.tb lineage classifications. Despite the growing use of next generation sequencing (NGS) and WGS analysis in TB research, there are limited studies that provide a comprehensive summary of there role in studying macroevolution, minor genetic variations, assessing mixed TB infections, and tracking transmission networks at an individual level. This highlights the need for systematic effort to fully explore the potential of WGS and its associated tools in advancing our understanding of TB epidemiology and disease transmission. We delve into the recent bioinformatics pipelines and NGS strategies that leverage various genetic features and simultaneous exploration of host-pathogen protein expression profile to decipher the genetic heterogeneity and host-pathogen interaction dynamics of the M.tb infections. This review highlights the potential benefits and limitations of NGS and bioinformatics tools and discusses their role in TB detection and epidemiology. Overall, this review could be a valuable resource for researchers and clinicians interested in NGS-based approaches in TB research. [ABSTRACT FROM AUTHOR]

Aims Urinary tract infections are the most common hospital-acquired infection, 80% of which are associated with catheterization. Diagnostic methods may influence the reported identities of these pathogens, and phenotypic testing under laboratory conditions may not reflect infection phenotypes. This study aimed to evaluate the efficacy of diagnostic methods and whether medium composition alters phenotypes by characterizing catheter-associated urinary tract infection isolates from a UK hospital. Methods and results We compared five bacterial identification methods, including biochemical testing, matrix-assisted laser desorption/ionization biotyping, and genome sequencing, finding differences in genus- or species-level identifications. Antibiotic susceptibility comparisons between phenotypic assays and genomic predictions showed high agreement only in multidrug-resistant strains. To determine whether growth rate and biofilm formation were affected by medium composition, strains were grown in both planktonic and biofilm states. Low planktonic growth and significant biofilm formation were observed in artificial urine compared to rich laboratory media, underscoring the importance of assay design. Conclusions This study highlights the risks of relying on a single diagnostic method for species identification, advocating for whole-genome sequencing for accuracy. It emphasizes the continued importance of phenotypic methods in understanding antibiotic resistance in clinical settings and the need for characterization conditions that mirror those encountered by pathogens in the body. [ABSTRACT FROM AUTHOR]

Aims The purpose of this work was to study extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) in freshwaters, hospital effluents, and wastewaters during two sampling campaigns in 2021. Methods and results Water sampling was performed at 24 stations in the Ourthe watershed in Belgium. A total of 644 ESBL (n  = 642) and AmpC (n  = 2) E. coli strains were isolated. Disk-diffusion assays were performed following the EUCAST's recommendations. All strains were tested for the presence of bla CTX-M-1, bla CTX-M-2, and bla CTX-M-9 gene groups by PCR. Genes belonging to bla CTX-M-1 and bla CTX-M-9 groups were detected, respectively, in 73.6% and 14.9% of the strains. No bla CTX-M-2 group's gene was found. A subset of strains (n  = 40) was selected for whole genome sequencing. Escherichia coli serotype O18: H7 ST 1463 was predominant (n  = 14) in the sequenced strains and showed pathogenicity in the Galleria mellonella larvae model. β-lactamase genes identified were bla CTX-M (n  = 21), with bla CTX-M-15 mostly represented (n  = 15), as well as bla TEM (n  = 11), bla OXA (n  = 7), bla SHV (n  = 9), and carbapenemase (CP) genes were observed in several strains— bla KPC-3 (n  = 19), bla NDM-1 (n  = 1), bla VIM-1 (n  = 2), and bla OXA-244 (n  = 2)—even from freshwaters. Conclusions ESBL-EC are widely distributed in the aquatic environment in Belgium and contain a variety of ESBL and CP genes. [ABSTRACT FROM AUTHOR]

The Mojave poppy bee, Perdita meconis Griswold (Hymenoptera: Anthophila: Andrenidae), is a species of conservation concern that is restricted to the eastern Mojave Desert of North America. It is a specialist pollinator of two poppy genera, Arctomecon and Argemone (Papaveraceae), and is being considered for listing under the US Endangered Species Act along with one of its pollinator hosts, the Las Vegas bearpoppy (Arctomecon californica). Here, we present a near chromosome-level genome of the Mojave poppy bee to provide a genomic resource that will aid conservation efforts and future research. We isolated DNA from a single, small (<7 mm), male specimen collected using non-ideal preservation methods and then performed whole-genome sequencing using PacBio HiFi technology. After quality and contaminant filtering, the final draft genome assembly is 327 Mb, with an N50 length of 17.5 Mb. Annotated repetitive elements compose 37.3% of the genome, although a large proportion (24.87%) of those are unclassified repeats. Additionally, we annotated 18,245 protein-coding genes and 19,433 transcripts. This genome represents one of only a few genomes from the large bee family Andrenidae and one of only a few genomes for pollinator specialists. We highlight both the potential of this genome as a resource for future research, and how high-quality genomes generated from small, non-ideal (in terms of preservation) specimens could facilitate biodiversity genomics. [ABSTRACT FROM AUTHOR]

African cichlid fishes are known for their high rates of phenotypic evolution. A rapid rate of diversification is apparent also in the diversity of their sex chromosomes. To date, sex determiners have been identified on 18 of 22 chromosomes in the standard karyotype. Here, we use whole-genome sequencing to characterize the sex chromosomes of seven populations of basal haplochromines, focusing on the genus Pseudocrenilabrus. We identify six new sex chromosome systems, including the first report of a cichlid sex–determining system on linkage group 12. We then quantify the rates and patterns of sex chromosome turnover in this clade. Finally, we test whether some autosomes become sex chromosomes in East African cichlids more often than expected by chance. [ABSTRACT FROM AUTHOR]

The European sprat is a small plankton-feeding clupeid present in the northeastern Atlantic Ocean, in the Mediterranean Sea, and in the brackish Baltic Sea and Black Sea. This species is the target of a major fishery and, therefore, an accurate characterization of its genetic population structure is crucial to delineate proper stock assessments that aid ensuring the fishery's sustainability. Here, we present (i) a draft genome assembly, (ii) pooled whole genome sequencing of 19 population samples covering most of the species' distribution range, and (iii) the design and test of a single nucleotide polymorphism (SNP)-chip resource and use this to validate the population structure inferred from pooled sequencing. These approaches revealed, using the populations sampled here, three major groups of European sprat: Oceanic, Coastal, and Brackish with limited differentiation within groups even over wide geographical stretches. Genetic structure is largely driven by six large putative inversions that differentiate Oceanic and Brackish sprats, while Coastal populations display intermediate frequencies of haplotypes at each locus. Interestingly, populations from the Baltic and the Black Seas share similar frequencies of haplotypes at these putative inversions despite their distant geographic location. The closely related clupeids European sprat and Atlantic herring both show genetic adaptation to the brackish Baltic Sea, providing an opportunity to explore the extent of genetic parallelism. This analysis revealed limited parallelism because out of 125 independent loci detected in the Atlantic herring, three showed sharp signals of selection that overlapped between the two species and contained single genes such as PRLRA , which encodes the receptor for prolactin, a freshwater-adapting hormone in euryhaline species, and THRB , a receptor for thyroid hormones, important both for metabolic regulation and the development of red cone photoreceptors. [ABSTRACT FROM AUTHOR]

Dingoes come from an ancient canid lineage that originated in East Asia around 8,000 to 11,000 years BP. As Australia's largest terrestrial predator, dingoes play an important ecological role. A small, protected population exists on a world heritage listed offshore island, K'gari (formerly Fraser Island). Concern regarding the persistence of dingoes on K'gari has risen due to their low genetic diversity and elevated inbreeding levels. However, whole-genome sequence data is lacking from this population. Here, we include five new whole-genome sequences of K'gari dingoes. We analyze a total of 18 whole-genome sequences of dingoes sampled from mainland Australia and K'gari to assess the genomic consequences of their demographic histories. Long (>1 Mb) runs of homozygosity (ROHs)—indicators of inbreeding—are elevated in all sampled dingoes. However, K'gari dingoes showed significantly higher levels of very long ROH (>5 Mb), providing genomic evidence for small population size, isolation, inbreeding, and a strong founder effect. Our results suggest that, despite current levels of inbreeding, the K'gari population is purging strongly deleterious mutations, which, in the absence of further reductions in population size, may facilitate the persistence of small populations despite low genetic diversity and isolation. However, there may be little to no purging of mildly deleterious alleles, which may have important long-term consequences, and should be considered by conservation and management programs. [ABSTRACT FROM AUTHOR]

Background Bacteroides fragilis , an anaerobic gut bacterium and opportunistic pathogen, comprises two genetically divergent groups (or divisions) at the species level. Differences exist both in the core and accessory genomes and the beta-lactamase genes, with the cephalosporinase gene cep A represented only in division I and the carbapenemase gene cfi A only in division II. Methods Multidrug resistance in a clinical B. fragilis strain was examined by whole-genome sequencing. Results Strain CNM20200260 carried the antimicrobial resistance genes cep A, cfi A2, ant (6'), erm (F), mef (En2), est (T), tet (Q) and cat (A), along with 82-Phe mutation in gyr A (together with 47 amino acid changes in gyr A/B and par C/parE). bex A/B and other efflux pump genes were also observed. None of the detected insertion sequences was located upstream of cfi A2. The genome-based taxonomy coefficients (average nucleotide identity, DNA–DNA hybridization similarity and difference in genomic G + C%) with respect to genomes of the strains of B. fragilis division II and the novel species Bacteroides hominis (both cfi A-positive) met the criteria for CNM20200260 to belong to either species (>95%, >70% and <1%, respectively). No such similarity was seen with type strain NCTC 9343 or the representative genome FDAARGOS 1225 of B. fragilis (division I, cfi A-negative). Strain CNM20200260 harboured four out of nine Kyoto Encyclopedia of Genes and Genomes orthologues defined for division I and one of two defined for division II. Conclusions This is the first description of the co-occurrence of cep A and cfi A in a Bacteroides strain, confirming the complexity of the taxonomy of this species. [ABSTRACT FROM AUTHOR]

Background Carbapenem-resistant Pseudomonas aeruginosa are being increasingly described worldwide. Here, we investigated the molecular mechanisms underlying carbapenem resistance in an extremely drug-resistant P. aeruginosa isolate from a neonatal intensive care unit in Morocco. Materials and methods P. aeruginosa strain O82J1 was identified using MALDI-TOF-MS. Carba NP, immunochromatographic assay NG Carba5 and antimicrobial susceptibility testing using disc diffusion and microbroth were performed. Whole-genome sequencing using the Illumina and MinION technologies and different software packages available at the Center of Genomic Epidemiology were used to predict the resistome, sequence type and plasmid types. Results P. aeruginosa O82J1 co-expressed two metallo-β-lactamases, bla NDM-1 and bla VIM-2, and was susceptible to colistin and apramycin only. It belonged to ST773 that is frequently reported worldwide as a high-risk P. aeruginosa clone. The bla VIM-2 gene was integron-borne on a IncP-2 465-kb plasmid, whereas the bla NDM-1 gene was chromosomally encoded and embedded in an integrative conjugative element, probably at the origin of its acquisition. A total of 23 antimicrobial resistance genes were detected including a bla PER-1 ESBL gene, and an 16S-rRNA methyltransferase gene rmtB. Conclusions The isolation of XDR P. aeruginosa isolates expressing several carbapenemases in a neonatal intensive care unit is of great concern due to the reduced treatment options, relying only on colistin, but not recommended in neonates, and apramycin, not yet approved for human therapy. Concerns were further elevated due to the resistance to cefiderocol and ATM/AVI, two novel and last-resort antibiotics recommended to treat infections caused by Gram-negative bacteria, particularly XDR P. aeruginosa in adults. [ABSTRACT FROM AUTHOR]

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in Caenorhabditis elegans , Escherichia coli , Saccharomyces cerevisiae , and Chlamydomonas reinhardtii. This method, demands merely 20 min of hands-on time for a single-worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the B-Raf Proto-oncogene, Serine/Threonine Kinase (BRAF) V600E mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species. [ABSTRACT FROM AUTHOR]

Ribosomal DNA (rDNA) has a vital role in ribosome biogenesis as it contains the genes that encode ribosomal RNA (rRNA) separated by intergenic spacers (IGSs). The rRNA genes occur in hundreds to tens of thousands of copies per haploid genome in eukaryotes and are generally highly conserved with low variation within species. Due to the repetitive nature and large size of rDNA arrays, detecting intraindividual variation can be difficult. In this study, we use whole-genome sequences of 169 Daphnia pulex individuals from 10 natural populations to measure the copy number and sequence variation in rDNA. This revealed that variation in rDNA copy number between individuals spans an order of magnitude. We further observed a substantial level of sequence variation within individual genomes. As expected, single-nucleotide polymorphisms occurred in regions of lower functional constraint such as the IGS and expansion segments of the rRNA genes. The presence of strong linkage disequilibrium among variants facilitated identification of haplotypes within each population. Although there was evidence of recombination among haplotypes from different populations, it is insufficient to eliminate linkage disequilibrium within populations. Estimating copy number and haplotype diversity within individuals revealed that the level of intraindividual sequence variation is not strongly correlated with copy number. The observed patterns of variation highlight a complex evolutionary history of rDNA in D. pulex. Future research should explore the functional implications of rDNA copy number and sequence variation on organismal phenotypes. [ABSTRACT FROM AUTHOR]

The accessibility of genomic tools in evolutionary biology has allowed for a thorough exploration of various evolutionary processes associated with adaptation and speciation. However, genomic studies in natural systems present numerous challenges, reflecting the inherent complexities of studying organisms in their native habitats. The utilization of museum specimens for genomics research has received increased attention in recent times, facilitated by advancements in ancient DNA techniques. In this study, we have utilized a museum genomics approach to analyze historic specimens of Woolly-necked storks (Ciconia spp.) and examine their genetic composition and taxonomic status and explore the evolutionary and adaptive trajectories of populations over the years. The Woolly-necked storks are distributed in Asia and Africa with a taxonomic classification that has been a matter of ambiguity. Asian and African Woollynecks were recently recognized as different species based on their morphological differences; however, their genomic validation was lacking. In this study, we have used ∼70-year-old museum samples for whole-genome population-scale sequencing. Our study has revealed that Asian and African Woollynecks are genetically distinct, consistent with the current taxonomic classification based on morphological features. However, we also found a high genetic divergence between the Asian subspecies Ciconia episcopus neglecta and Ciconia episcopus episcopus , suggesting this classification requires a detailed examination to explore processes of ongoing speciation. Because taxonomic classification directly impacts conservation efforts, and there is evidence of declining populations of Asian Woollynecks in Southeast Asia, our results highlight that population-scale studies are urgent to determine the genetic, ecological, and phylogenetic diversity of these birds. [ABSTRACT FROM AUTHOR]

Background Genomic disorders caused by copy number variations (CNVs) are prevalent in patients with kidney disease; however, their contribution to chronic kidney failure (KF) of undetermined aetiology (uKF) is unclear. We screened patients with uKF aged 50 years or younger to establish the prevalence of causative CNVs. Methods We enrolled patients with an onset of KF ≤50 years from suspected undetermined aetiology for initial review of medical records to exclude patients with clear-cut clinical or histopathological kidney diagnoses or patients with already established genetic kidney diseases. Next, we performed single nucleotide polymorphism (SNP) array–based CNV screening. All the detected CNVs were systematically classified and evaluated as possible causes of the patient's kidney disease. Patients with CNVs not explaining the kidney phenotype were additionally screened for causal variants in 540 genes using whole-genome sequencing. Results We enrolled 172 patients, of whom 123 underwent SNP-array. Pathogenic CNVs corresponding to known genomic disorders were identified in 12 patients (9.8%). The identified genomic disorders provided a causative kidney diagnosis in three patients, all of whom had reached KF by age 18 years. The remaining nine patients had CNVs with unclear kidney disease causality. Subsequently, whole-genome sequencing provided a causative genetic diagnosis in an additional four patients, including two diagnostic sequence variants unrelated to the detected CNVs. Conclusions Genomic disorders were prevalent in this cohort with uKF, and causative CNVs were identified in 5 of 123 patients. Further studies combining the analysis of CNVs and sequence variants are needed to clarify the causal role of genomic disorders in kidney disease. [ABSTRACT FROM AUTHOR]