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.

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.

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.

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.

Biostatistical Aspects of Whole Genome Sequencing Studies: Preprocessing and Quality Control.

Rapid advances in high-throughput DNA sequencing technologies have enabled large-scale whole genome sequencing (WGS) studies. Before performing association analysis between phenotypes and genotypes, preprocessing and quality control (QC) of the raw sequence data need to be performed. Because many biostatisticians have not been working with WGS data so far, we first sketch Illumina's short-read sequencing technology. Second, we explain the general preprocessing pipeline for WGS studies. Third, we provide an overview of important QC metrics, which are applied to WGS data: on the raw data, after mapping and alignment, after variant calling, and after multisample variant calling. Fourth, we illustrate the QC with the data from the GENEtic SequencIng Study Hamburg-Davos (GENESIS-HD), a study involving more than 9000 human whole genomes. All samples were sequenced on an Illumina NovaSeq 6000 with an average coverage of 35× using a PCR-free protocol. For QC, one genome in a bottle (GIAB) trio was sequenced in four replicates, and one GIAB sample was successfully sequenced 70 times in different runs. Fifth, we provide empirical data on the compression of raw data using the DRAGEN original read archive (ORA). The most important quality metrics in the application were genetic similarity, sample cross-contamination, deviations from the expected Het/Hom ratio, relatedness, and coverage. The compression ratio of the raw files using DRAGEN ORA was 5.6:1, and compression time was linear by genome coverage. In summary, the preprocessing, joint calling, and QC of large WGS studies are feasible within a reasonable time, and efficient QC procedures are readily available.

The novel HLA-C*04:01:01:182 allele identified in a candidate bone marrow donor by next-generation sequencing.

HLA-C*04:01:01:182 differs from the HLA-C*04:01:01:06 allele by one nucleotide substitution in the 5'UTR.

The novel HLA-C*14:02:01:31 allele identified in a candidate bone marrow donor by next-generation sequencing.

Characterisation of the novel HLA-C*14:02:01:31 allele in a 21-year-old Greek bone marrow donor.

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.

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.

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.

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.

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.

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.

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.

Beyond the rootzone: Unveiling soil property and biota gradients around plants.

Although the effects of plants on soil properties are well known, the effects of distance from plant roots to root-free soil on soil properties and associated soil organisms are much less studied. Previous research on the effects of distance from a plant explored specific soil organisms and properties, however, comparative studies across a wide range of plant-associated organisms and multiple model systems are lacking. We conducted a controlled greenhouse experiment using soil from two contrasting habitats. Within each soil type, we cultivated two plant species, individually and in combination, studying soil organisms and properties in the root centre, the root periphery, and the root-free zones. We showed that the distance from the cultivated plant (representing decreasing amount of plant roots) had a significant impact on the abiotic properties of the soil (pH and available P and N) and also on the composition of the fungal, bacterial, and nematode communities. The specific patterns, however, did not always match our expectations. For example, there was no significant relationship between the abundance of fungal pathogens and the distance from the cultivated plant compared to a strong decrease in the abundance of arbuscular mycorrhizal fungi. Changes in soil chemistry along the distance from the cultivated plant were probably one of the important drivers that affected bacterial communities. The abundance of nematodes also decreased with distance from the cultivated plant, and the rate of their responses reflected the distribution of their food sources. The patterns of soil changes along the gradient from plant to root-free soil were largely similar in two contrasting soil types and four plant species or their mixtures. This suggests that our results can be generalised to other systems and contribute to a better understanding of the mechanisms of soil legacy formation.

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.

Illumina short read-based shotgun metagenomic data sets of simulated bacterial communities derived from fresh spinach and surface water.

Paired-end short reads of Illumina HiSeq, MiSeq, and NovaSeq of simulated bacterial communities from fresh spinach and surface water were generated in silico at various sequencing depths. Multidrug-resistant Salmonella enterica serotype Indiana was included in the spinach community, while the water community contained multidrug-resistant Pseudomonas aeruginosa.

A Combinatorial Single-Molecule Real-Time and Illumina Sequencing Analysis of Postembryonic Gene Expression in the Asian Citrus Psyllid Diaphorina citri.

Huanglongbing (HLB) is a systemic plant disease caused by 'Candidatus Liberibacter asiaticus (CLas)' and transmitted by Diaphorina citri. D. citri acquires the CLas bacteria in the nymph stage and transmits it in the adult stage, indicating that molting from the nymph to adult stages is crucial for HLB transmission. However, the available D. citri reference genomes are incomplete, and gene function studies have been limited to date. In the current research, PacBio single-molecule real-time (SMRT) and Illumina sequencing were performed to investigate the transcriptome of D. citri nymphs and adults. In total, 10,641 full-length, non-redundant transcripts (FLNRTs), 594 alternative splicing (AS) events, 4522 simple sequence repeats (SSRs), 1086 long-coding RNAs (lncRNAs), 281 transcription factors (TFs), and 4459 APA sites were identified. Furthermore, 3746 differentially expressed genes (DEGs) between nymphs and adults were identified, among which 30 DEGs involved in the Hippo signaling pathway were found. Reverse transcription-quantitative PCR (RT-qPCR) further validated the expression levels of 12 DEGs and showed a positive correlation with transcriptome data. Finally, the spatiotemporal expression pattern of genes involved in the Hippo signaling pathway exhibited high expression in the D. citri testis, ovary, and egg. Silencing of the D. citri transcriptional co-activator (DcYki) gene significantly increased D. citri mortality and decreased the cumulative molting. Our results provide useful information and a reliable data resource for gene function research of D. citri.