Characteristics of Corynespora cassiicola, the causal agent of tobacco Corynespora leaf spot, revealed by genomic and metabolic phenomic analysis.

Corynespora cassiicola is a highly diverse fungal pathogen that commonly occurs in tropical, subtropical, and greenhouse environments worldwide. In this study, the isolates were identified as C. cassiicola, and the optimum growth and sporulation were studied. The phenotypic characteristics of C. cassiicola, concerning 950 different growth conditions, were tested using Biolog PM plates 1-10. In addition, the strain of C. cassiicola DWZ from tobacco hosts was sequenced for the using Illumina PE150 and Pacbio technologies. The host resistance of tobacco Yunyan 87 with different maturity levels was investigated. In addition, the resistance evaluation of 10 common tobacco varieties was investigated. The results showed that C. cassiicola metabolized 89.47% of the tested carbon source, 100% of the nitrogen source, 100% of the phosphorus source, and 97.14% of the sulfur source. It can adapt to a variety of different osmotic pressure and pH environments, and has good decarboxylase and deaminase activities. The optimum conditions for pathogen growth and sporulation were 25-30 °C, and the growth was better on AEA and OA medium. The total length of the genome was 45.9 Mbp, the GC content was 51.23%, and a total of 13,061 protein-coding genes, 202 non-coding RNAs and 2801 and repeat sequences were predicted. Mature leaves were more susceptible than proper mature and immature leaves, and the average diameter of diseased spots reached 17.74 mm at 12 days. None of the tested ten cultivars exhibited obvious resistance to Corynespora leaf spot of tobacco, whereby all disease spot diameters reached > 10 mm and > 30 mm when at 5 and 10 days after inoculation, respectively. The phenotypic characteristics, genomic analysis of C. cassiicola and the cultivar resistance assessment of this pathogen have increased our understanding of Corynespora leaf spot of tobacco.

Delayed canine heartworm (Dirofilaria immitis) microfilarial reduction following Advocate™ for dogs (imidacloprid, moxidectin) treatment.

Macrocyclic lactone (ML) anthelmintics are currently the only class of drugs available for canine heartworm prevention. Recent reports of Dirofilaria immitis infection occurring in dogs reportedly receiving 'rigorous' prevention in Queensland, Australia, coupled with the confirmation of ML-resistant isolates in the USA, has led to speculation about the potential emergence of ML-resistance in Australia. In this study, we describe two cases (Dog 1 and 2) of asymptomatic canine heartworm disease in Townsville, Australia, that were reportedly receiving 'rigorous' heartworm prevention according to the owners' claims. We aimed to deploy currently available tools to assess the phenotypic and genotypic ML-resistance status of these two dogs. For phenotypic testing, we performed an in-vivo 7-day microfilariae suppression test using a dose of spot-on moxidectin (Advocate™ for Dogs, 100 g/L imidacloprid + 25 g/L moxidectin). This formulation is marketed as Advantage Multi® for Dogs in the USA, which claims a D. immitis microfilaricidal effect. For genetic testing, an Illumina amplicon metabarcoding approach was used to target single nucleotide polymorphisms (SNPs) previously associated with ML-resistance in D. immitis from the USA. Dog 1 and Dog 2 demonstrated <10 % and <40 % reductions in circulating microfilariae seven days after moxidectin treatment, respectively. These phenotypes were not corroborated by genetic SNP testing, as both dogs were classified as susceptible across all examined markers. To streamline testing of D. immitis SNPs, we developed a rhAmp™ SNP qPCR approach for rapidly genotyping suspect cases of ML-resistant infections at the two major loci (L15709_A and L30575). These findings illustrate a phenomenon shown in some heartworm cases outside the USA, whereby infected dogs are failing to see marked reductions in microfilaraemia after ML treatment but possess an ML-susceptible genotype.

Application of the Agilent 2100 Bioanalyzer instrument as quality control for next-generation sequencing.

Next-generation sequencing (NGS) technologies have expanded the spectrum of forensic DNA analysis by facilitating efficient and precise genotyping of a large number of genetic markers. Yet, challenges persist regarding complex sample processing and assurance of equal molar concentrations across pooled samples. Since optimal cluster density is crucial for sequencing performance, the determination of both quantity and quality is indispensable for library preparation. In this study, we investigated the application of the Agilent 2100 Bioanalyzer for library quality control, as studies for forensic approaches, particularly for highly degraded postmortem samples, are rare. Our analysis encompassed assessing total DNA concentrations, fluorescence unit (FU) values, and adapter dimer concentrations in purified DNA libraries derived from buccal swabs and tissue samples of decomposed corpses. The sensitivity study tested a serial dilution derived from buccal swabs and revealed a decrease in FU values and an increase in adapter dimers with declining DNA input concentrations. Deviations in total DNA concentrations and average peak heights between the Agilent 2100 Bioanalyzer runs indicated a lack of repeatability in data and presented challenges in accurate quantification, which was also observed in previous studies. Yet, the analysis of degraded samples from decomposed human remains has shown the ability to detect adapter dimer concentrations, which can be crucial for the quality of subsequent NGS library preparation and sequencing success. Therefore, the Agilent 2100 Bioanalyzer proves to be a valuable tool for NGS quality control.

Complete genome sequence of the OXA-48-producing Klebsiella pneumoniae strain 11978.

We announce the complete genome sequence of Klebsiella pneumoniae strain 11978 isolated from a patient hospitalized in Turkey in 2001. The genome belongs to sequence type 14 and includes three plasmids. Notably, it presents an IncL plasmid carrying blaOXA-48, which demonstrated global success in terms of dissemination.

The complete mitochondrial genome of the basidiomycetous fungus, Tinctoporellus epimiltinus strain RS1.

Tinctoporellus epimiltinus is widely known as a wood-decaying fungus. In the present study, we identified the complete mitochondrial genome of this species using next-generation sequencing technology. Our findings revealed that the genomic structure is a circular molecule with a size of 51,878 bp. Consistent with most Basidiomycota species, it consists of 14 core protein-coding genes, one ribosomal protein gene (rps3), 26 transfer RNA genes, and small and large ribosomal RNA (rns and rnl) genes. Seven additional open reading frames were identified. These included two sequences similar to DNA polymerases, an endonuclease-like sequence, and four hypothetical proteins. The mitochondrial genome exhibited a nucleotide composition of A (36.24%), C (12.04%), G (13.18%), and T (38.55%), resulting in a 25.21% GC content. A phylogenetic tree constructed using the combined mitochondrial gene dataset provided insight into the phylogenetic relationships of this species within the context of Basidiomycota and its members.

Genomic characterization of Volzhskoe tick virus (Bunyaviricetes) from a Hyalomma marginatum tick, Hungary.

Hyalomma marginatum, a vector for the high-consequence pathogen, the Crimean-Congo hemorrhagic fever virus (CCHFV), needs particular attention due to its impact on public health. Although it is a known vector for CCHFV, its general virome is largely unexplored. Here, we report findings from a citizen science monitoring program aimed to understand the prevalence and diversity of tick-borne pathogens, particularly focusing on Hyalomma ticks in Hungary. In 2021, we identified one adult specimen of Hyalomma marginatum and subjected it to Illumina-based viral metagenomic sequencing. Our analysis revealed sequences of the uncharacterized Volzhskoe tick virus, an unclassified member of the class Bunyaviricetes. The in silico analysis uncovered key genetic regions, including the glycoprotein and the RNA-dependent RNA polymerase (RdRp) coding regions. Phylogenetic analysis indicated a close relationship between our Volzhskoe tick virus sequences and other unclassified Bunyaviricetes species. These related species of unclassified Bunyaviricetes were detected in vastly different geolocations. These findings highlight the remarkable diversity of tick specific viruses and emphasize the need for further research to understand the transmissibility, seroreactivity or the potential pathogenicity of Volzhskoe tick virus and related species.

Corrigendum: Clinical value of metagenomic next-generation sequencing by Illumina and Nanopore for the detection of pathogens in bronchoalveolar lavage fluid in suspected community-acquired pneumonia patients.

[This corrects the article DOI: 10.3389/fcimb.2022.1021320.].

Disseminated Echovirus 11 infection in a newborn in the Province of Bolzano, Italy.

PURPOSE: Recently, cases of serious illness in newborns infected with Echovirus 11 have been reported in Europe, including Italy. Here, we report the case of a newborn diagnosed with disseminated Echovirus 11 infection, which occurred in October 2023 in the Province of Bolzano, Italy. METHODS: A molecular screening, by Real-Time RT-PCR, was employed to analyse the cerebrospinal fluid, blood and stool samples, and nasal swabs. The entire viral genome was sequenced using both Illumina and Nanopore technologies. RESULTS: The patient was admitted to hospital due to fever. Molecular testing revealed the presence of enterovirus RNA. Typing confirmed the presence of Echovirus 11. The patient was initially treated with antibiotic therapy and, following the diagnosis of enterovirus infection, also with human immunoglobulins. Over the following days, the patient remained afebrile, with decreasing inflammation indices and in excellent general condition. Genomic and phylogenetic characterization suggested that the strain was similar to strains from severe cases reported in Europe. CONCLUSIONS: Despite the low overall risk for the neonatal population in Europe, recent cases of Echovirus 11 have highlighted the importance of surveillance and complete genome sequencing is fundamental to understanding the phylogenetic relationships of Echovirus 11 variants.

The complete genomic sequence of the type strain Brochothrix thermosphacta DSM 20171 highlights a diversity of prophages in this species.

The bacterium Brochothrix thermosphacta is a known muscle food spoiler. Here, the complete genome sequence of the B. thermosphacta type strain, DSM 20171, is reported. Prediction of prophages and genomic islands reveals an unsuspected diversity in this bacterial species that deserves further investigation.

Genome drafts of Pseudogracilibacillus spp. from soil and sediment of Mount Makiling, a dormant volcano in the Philippines.

We present the draft whole-genome sequences of Pseudogracilibacillus spp. isolated from the soils and sediments of Sipit Creek located at Mount Makiling, a dormant volcano in the southern part of Luzon Island, Philippines. This Pseudogracilibacillus spp. genome report extends the body of knowledge on a lesser-known genus of Bacillaceae.

Draft genome sequence of ectomycorrhizal edible mushroom Phlebopus portentosus strain YAF023.

Phlebopus portentosus is a favorite ectomycorrhizal edible mushroom in tropical China. Here, we present a draft genome sequence of P. portentosus strain YAF023. The genome resource will support subsequent research into the relationship between ectomycorrhizal fungi and trees.

Identification of the new allele HLA-A*24:632 in a Greek individual using next generation sequencing.

The HLA-Α*24:632 allele differs from HLA-A*24:02:01:01 by a single nucleotide substitution in exon 2.

Indigenous metal-tolerant mine water bacterial populations under varying metal stresses.

The present study investigated the indigenous metal-tolerant bacterial populations in the mine-water microbiome. Our intention was to assess the effects of the metal concentrations in mine water on the bacterial community of mine waters. The bacterial communities in Vanadium and Gold mine-water samples were exposed to different heavy-metal Arsenic, Cadmium, Chromium, Nickel, Mercury and Vanadium at two different concentrations (5 and 25 mM). The 16S rRNA amplicon from mine waters were sequenced using the Illumina's NGS MiSeq platform. Data analysis revealed a high diversity in the bacterial populations associated with the different heavy metals at different concentrations. The taxonomic profiles obtained after the exposure were different in different salts, but mostly dominated by Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Firmicutes at variable relative abundance. Principal Component Analysis (PCoA) predicts the clear community shift after exposure with heavy metals salts and emergence of tolerant community depending upon the specific community present in the original mine water.

Identification of the new allele HLA-C*04:01:01:186 in a Greek individual using next generation sequencing.

The newly discovered HLA-C*04:01:01:186 allele differs from HLA-C*04:01:01:01 by a single nucleotide substitution in intron 3.

Introduction of Avian metapneumovirus subtype A to the United States: molecular insights and implications.

Avian metapneumovirus (aMPV) poses a significant threat to the poultry industry worldwide, primarily affecting turkeys and chickens. The recent detection of aMPV-A and -B subtypes in the United States marks a significant shift after a prolonged period free of aMPV following the eradication of the previously circulating subtype C. Hence, the demand for molecular diagnostic tests for aMPV has arisen due to their limited availability in the US market. In this study, we present the molecular characterization based on the complete genome sequence of aMPV subtype A, which was detected in the US for the first time. Four RT-qPCR positive samples were subjected to next-generation sequencing analysis, resulting in the assembly of one complete and one near-complete genome sequences. Phylogenetic analysis revealed that the isolated strains clustered within the aMPV-A subtype and were most closely related to recent Mexican strains. A detailed amino acid analysis identified unique mutations in the G gene of the US isolates compared to Mexican strains. Additionally, we compared the performance, cross-reactivity, and limit of detection of our revised aMPV subtype-specific RT-qPCR test with two commercial kits, demonstrating similar detection and subtyping capabilities. These findings highlight the importance of accurate diagnostic methods for disease management in the poultry industry, provide valuable insights into the epidemiology of aMPV, and underscore the need for continued vigilance and surveillance to mitigate its impact on poultry production.

An updated LSU database and pipeline for environmental DNA identification of arbuscular mycorrhizal fungi.

Recent work established a backbone reference tree and phylogenetic placement pipeline for identification of arbuscular mycorrhizal fungal (AMF) large subunit (LSU) rDNA environmental sequences. Our previously published pipeline allowed any environmental sequence to be identified as putative AMF or within one of the major families. Despite this contribution, difficulties in implementation of the pipeline remain. Here, we present an updated database and pipeline with (1) an expanded backbone tree to include four newly described genera and (2) several changes to improve ease and consistency of implementation. In particular, packages required for the pipeline are now installed as a single folder (conda environment) and the pipeline has been tested across three university computing clusters. This updated backbone tree and pipeline will enable broadened adoption by the community, advancing our understanding of these ubiquitous and ecologically important fungi.

Towards genomic medicine: a tailored next-generation sequencing panel for hydroxyurea pharmacogenomics in Tanzania.

BACKGROUND: Pharmacogenomics of hydroxyurea is an important aspect in the management of sickle cell disease (SCD), especially in the era of genomic medicine. Genetic variations in loci associated with HbF induction and drug metabolism are prime targets for hydroxyurea (HU) pharmacogenomics, as these can significantly impact the therapeutic efficacy and safety of HU in SCD patients. METHODS: This study involved designing of a custom panel targeting BCL11A, ARG2, HBB, HBG1, WAC, HBG2, HAO2, MYB, SAR1A, KLF10, CYP2C9, CYP2E1 and NOS1 as potential HU pharmacogenomics targets. These genes were selected based on their known roles in HbF induction and HU metabolism. The panel was designed using the Illumina Design Studio (Illumina, San Diego, CA, USA) and achieved a total coverage of 96% of all genomic targets over a span of 51.6 kilobases (kb). This custom panel was then sequenced using the Illumina MiSeq platform to ensure high coverage and accuracy. RESULTS: We are reporting a successfully designed Illumina (MiSeq) HU pharmacogenomics custom panel encompassing 51.6 kilobases. The designed panel achieved greater than 1000x amplicon coverage which is sufficient for genomic analysis. CONCLUSIONS: This study provides a valuable tool for research in HU pharmacogenomics, especially in Africa where SCD is highly prevalent, and personalized medicine approaches are crucial for improving patient outcomes. The custom-designed Illumina (MiSeq) panel, with its extensive coverage and high sequencing depth, provides a robust platform for studying genetic variations associated with HU response. This panel can contribute to the development of tailored therapeutic strategies, ultimately enhancing the management of SCD through more effective and safer use of hydroxyurea.

Comparison of Metabarcoding and Microscopy Methodologies to Analyze Diatom Communities in Five Estuaries Along the Southern Coast of the Korean Peninsula.

The study of microalgal communities is critical for understanding aquatic ecosystems. These communities primarily comprise diatoms (Heterokontophyta), with two methods commonly used to study them: Microscopy and metabarcoding. However, these two methods often deliver different results; thus, their suitability for analyzing diatom communities is frequently debated and evaluated. This study used these two methods to analyze the diatom communities in identical water samples and compare the results. The taxonomy of the species constituting the diatom communities was confirmed, and both methods showed that species belonging to the orders Bacillariales and Naviculales (class Bacillariophyceae) are the most diverse. In the lower taxonomic levels (family, genus, and species), microscopy tended to show a bias toward detecting diatom species (Nitzschia frustulum, Nitzschia inconspicua, Nitzschia intermedia, Navicula gregaria, Navicula perminuta, Navicula recens, Navicula sp.) belonging to the Bacillariaceae and Naviculaceae families. The results of the two methods differed in identifying diatom species in the communities and analyzing their structural characteristics. These results are consistent with the fact that diatoms belonging to the genera Nitzschia and Navicula are abundant in the communities; furthermore, only the Illumina MiSeq data showed the abundance of the Melosira and Entomoneis genera. The results obtained from microscopy were superior to those of Illumina MiSeq regarding species-level identification. Based on the results obtained via microscopy and Illumina MiSeq, it was revealed that neither method is perfect and that each has clear strengths and weaknesses. Therefore, to analyze diatom communities effectively and accurately, these two methods should be combined.

Comparison of Infinium MethylationEPIC v2.0 to v1.0 for human population epigenetics: considerations for addressing EPIC version differences in DNA methylation-based tools.

Background: The recently launched DNA methylation profiling platform, Illumina MethylationEPIC BeadChip Infinium microarray v2.0 (EPICv2), is highly correlated with measurements obtained from its predecessor MethylationEPIC BeadChip Infinium microarray v1.0 (EPICv1). However, the concordance between the two versions in the context of DNA methylation-based tools, including cell type deconvolution algorithms, epigenetic clocks, and inflammation and lifestyle biomarkers has not yet been investigated. Findings: We profiled DNA methylation on both EPIC versions using matched venous blood samples from individuals spanning early to late adulthood across three cohorts. On combining the DNA methylomes of the cohorts, we observed that samples primarily clustered by the EPIC version they were measured on. Within each cohort, when we calculated cell type proportions, epigenetic age acceleration (EAA), rate of aging estimates, and biomarker scores for the matched samples on each version, we noted significant differences between EPICv1 and EPICv2 in the majority of these estimates. These differences were not significant, however, when estimates were adjusted for EPIC version or when EAAs were calculated separately for each EPIC version. Conclusions: Our findings indicate that EPIC version differences predominantly explain DNA methylation variation and influence estimates of DNA methylation-based tools, and therefore we recommend caution when combining cohorts run on different versions. We demonstrate the importance of calculating DNA methylation-based estimates separately for each EPIC version or accounting for EPIC version either as a covariate in statistical models or by using version correction algorithms.

A Comparison of Structural Variant Calling from Short-Read and Nanopore-Based Whole-Genome Sequencing Using Optical Genome Mapping as a Benchmark.

The identification of structural variants (SVs) in genomic data represents an ongoing challenge because of difficulties in reliable SV calling leading to reduced sensitivity and specificity. We prepared high-quality DNA from 9 parent-child trios, who had previously undergone short-read whole-genome sequencing (Illumina platform) as part of the Genomics England 100,000 Genomes Project. We reanalysed the genomes using both Bionano optical genome mapping (OGM; 8 probands and one trio) and Nanopore long-read sequencing (Oxford Nanopore Technologies [ONT] platform; all samples). To establish a "truth" dataset, we asked whether rare proband SV calls (n = 234) made by the Bionano Access (version 1.6.1)/Solve software (version 3.6.1_11162020) could be verified by individual visualisation using the Integrative Genomics Viewer with either or both of the Illumina and ONT raw sequence. Of these, 222 calls were verified, indicating that Bionano OGM calls have high precision (positive predictive value 95%). We then asked what proportion of the 222 true Bionano SVs had been identified by SV callers in the other two datasets. In the Illumina dataset, sensitivity varied according to variant type, being high for deletions (115/134; 86%) but poor for insertions (13/58; 22%). In the ONT dataset, sensitivity was generally poor using the original Sniffles variant caller (48% overall) but improved substantially with use of Sniffles2 (36/40; 90% and 17/23; 74% for deletions and insertions, respectively). In summary, we show that the precision of OGM is very high. In addition, when applying the Sniffles2 caller, the sensitivity of SV calling using ONT long-read sequence data outperforms Illumina sequencing for most SV types.