Search Results (79)

Increased Atlantic water transport and river discharge are more pronounced effects of global warming at high latitudes. Both phenomena may lead to changes in the species composition of small-celled algae populations in marine ecosystems, as well as to the emergence of new species. This study investigated the spatial distribution of picocyanobacterial (PC) abundance and the phylogenetic diversity of PC Synechococcus in the Kara Sea. PC abundance varied from 2 to 88 cells mL⁻¹ and increased with warming temperatures and decreasing salinity caused by river water influence. The contribution of Synechococcus to the total picophytoplankton biomass was low (<16%). The Synechococcus community was characterized at deep taxonomic level using amplicon sequencing targeting the petB gene. Diversity was low, revealing only Synechococcus subcluster 5.1 polar lineages I and IV, and euryhaline subcluster 5.2. Synechococcus subcluster 5.1.I represented on average 97% of the total reads assigned to cyanobacteria. For the first time, the presence of estuarine Synechococcus subcluster 5.2 was documented as far north as 82° N. Modified Atlantic water was the main source of cyanobacteria in the Kara Sea, followed by river discharge. Our study contributes to the understanding of PC sources in the Kara Sea and allows for the further monitoring of PC distribution and evolution. Full article

Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used in Australia for the control of blackleg disease (caused by Leptosphaeria maculans, also called Plenodomus lingam). Populations of L. maculans are highly variable and therefore at risk of evolving fungicide resistance. The baseline sensitivities of L. maculans isolates towards the SDHI fungicides pydiflumetofen and bixafen were determined through in vitro mycelial growth assays, and the mean EC₅₀s were found to be 4.89 and 2.71 ng mL⁻¹, respectively. L. maculans populations were also screened against three commercial SDHI fungicides, Saltro^®, ILeVO^®, and Aviator^®, using an in planta assay to reveal very low levels of resistance. Nineteen of these ascospore populations from 2022 were analysed in a deep amplicon sequencing (DAS) assay and showed no mutations in the genes likely to be associated with resistance to SDHI chemistries. This study establishes baseline sensitivities of L. maculans isolates towards commonly used SDHI fungicides, importantly before and during the introduction of these new chemistries for blackleg control, and outlines monitoring techniques to allow timely identification of resistance if it evolves. Full article

Soil microbial communities are essential for the natural attenuation of organic pollutants, yet their ecological responses under long-term contamination remain insufficiently understood. This study examined the bacterial community structure and the abundance of dechlorinating bacteria at a decommissioned pharmaceutical-chemical site in northern Jiangsu Province, China, where the primary pollutants were dichloromethane, 1,2-dichloroethane, and toluene. Eighteen soil samples from the surface (0.2 m) and deep (2.2 m) layers were collected using a Geoprobe-7822DT system and analyzed for physicochemical properties and microbial composition via 16S rRNA gene amplicon sequencing. The results showed that the bacterial community composition was significantly shaped by the soil pH, moisture content, pollutant type, and depth. Dechlorinating bacteria were detected at all sites but exhibited low relative abundance, with higher concentrations in the surface soils. Desulfuromonas, Desulfitobacterium, and Desulfovibrio were the dominant dechlorinators, while Dehalococcoides appeared only in the deep soils. A network analysis revealed positive correlations between the dechlorinators and BTEX-degrading and fermentative taxa, indicating potential cooperative interactions in pollutant degradation. However, the low abundance of dechlorinators suggests that the intrinsic bioremediation capacity is limited. These findings provide new insights into microbial ecology under complex organic pollution, and support the need for integrated remediation strategies that enhance microbial functional potential in legacy-contaminated soils. Full article

RNA sequencing (RNA-Seq) is a powerful technique for the quantification of transcripts and the analysis of alternative splicing. Previously, our laboratory developed the targeted RNA long-amplicon sequencing (rLAS) method, which has the advantage of allowing deep analysis of targeted specific transcripts. The computational tools for analyzing RNA-Seq data have boosted alternative splicing research by detecting and quantifying splicing events. However, the performance of these splicing tools has not yet been investigated for rLAS. Here, we evaluated the performance of four splicing tools (MAJIQ, rMATS, MISO, and SplAdder) using samples with different types of known splicing events (exon-skipping, multiple-exon-skipping, alternative 5′ splicing, and alternative 3′ splicing). MAJIQ was able to detect all of the types of events, but it was unable to detect one of the exon-skipping events. On the other hand, rMATS was able to detect all of the exon-skipping events. However, rMATS failed to detect other types of events besides exon-skipping events. Both MISO and SplAdder were unable to detect any of the events. These results indicate that MAJIQ presents better performance for the different types of splicing events in rLAS and that rMATS shows better performance for exon-skipping splicing events. Full article

With the explosive growth of sequencing data, rapidly and accurately classifying and identifying species has become a critical challenge in amplicon analysis research. The internal transcribed spacer (ITS) region is widely used for fungal species classification and identification. However, most existing ITS databases cover limited fungal species diversity, and current classification methods struggle to efficiently handle such large-scale data. This study integrates multiple publicly available databases to construct an ITS sequence database encompassing 93,975 fungal species, making it a resource with broader species diversity for fungal taxonomy. In this study, a fungal classification model named FungiLT is proposed, integrating Transformer and BiLSTM architectures while incorporating a dual-channel feature fusion mechanism. On a dataset where each fungal species is represented by 100 ITS sequences, it achieves a species-level classification accuracy of 98.77%. Compared to BLAST, QIIME2, and the deep learning model CNN_FunBar, FungiLT demonstrates significant advantages in ITS species classification. This study provides a more efficient and accurate solution for large-scale fungal classification tasks and offers new technical support and insights for species annotation in amplicon analysis research. Full article

Epilepsy affects 50 million people worldwide and is drug-resistant in approximately one-third of cases. Even when a structural lesion is identified as the epileptogenic focus, understanding the underlying genetic causes is crucial to guide both counseling and treatment decisions. Both somatic and germline DNA variants may contribute to the lesion itself and/or influence the severity of symptoms. We therefore used whole exome sequencing (WES) to search for potentially pathogenic somatic DNA variants in brain samples from children with lesional epilepsy who underwent epilepsy surgery. WES was performed on 20 paired DNA samples extracted from both lesional brain tissue and reference tissue from the same patient, such as leukocytes or fibroblasts. The paired WES data were jointly analyzed using GATK Mutect2 to identify somatic single nucleotide variants (SNVs) or insertions/deletions (InDels), which were subsequently evaluated in silico for their disease-causing potential using MutationTaster2021. We identified known pathogenic somatic variants in five patients (25%) with variant allele frequencies (VAF) ranging from 3–35% in the genes MTOR, TSC2, PIK3CA, FGFR1, and PIK3R1 as potential causes of cortical malformations or central nervous system (CNS) tumors. Depending on the VAF, we used different methods such as Sanger sequencing, allele-specific qPCR, or targeted ultra-deep sequencing (amplicon sequencing) to confirm the variant. In contrast to the usually straightforward confirmation of germline variants, the validation of somatic variants is more challenging because current methods have limitations in sensitivity, specificity, and cost-effectiveness. In our study, WES identified additional somatic variant candidates in additional genes with VAFs ranging from 0.7–7.0% that could not be validated by an orthogonal method. This highlights the importance of variant validation, especially for those with very low allele frequencies. Full article

Classical preoperative skin antisepsis is insufficient in completely eliminating bacterial skin colonization for arthroplasty. In contrast, photodynamic therapy (PDT) with red light and methyl-aminolevulinate (MAL), combined with skin antisepsis, led to the absence of bacterial growth in healthy participants, though with local skin erythema, posing an obstacle for orthopedic surgery. Therefore, we explored whether artificial daylight PDT (PDT-DL) was superior to red light. Twenty healthy participants were allocated to either 5-aminolevulinic acid-(5-ALA) PDT-DL (n = 10) or MAL-PDT-DL (n = 10) before antisepsis with povidone-iodine/alcohol. Skin swabs from the groin were taken to cultivate bacteria at baseline, after PDT-DL, and after the subsequent antisepsis. Additional swabs were taken on day 4 before and after antisepsis without PDT. The contralateral groin of each participant and of ten additional healthy volunteers served as the control (n = 30). In selected participants, 16S rRNA-based amplicon deep sequencing was performed. All participants showed a baseline bacterial colonization. After a PDT-DL with skin antisepsis, bacterial growth occurred in three (30%) and in one (10%) participants with 5-ALA and MAL, respectively, compared to the sixteen (55%) participants in the control group. On day 4, three (30%) participants per group showed positive cultures post antisepsis. Adverse effects were reported in six (60%) and zero (0%) participants for 5-ALA- and MAL-PDT-DL, respectively. The skin bacteriome changes correlated with the bacterial culture results. The MAL-PDT-DL with skin antisepsis significantly increased bacterial reduction on the skin without adverse effects. This offers an opportunity to prevent infections in arthroplasty patients and reduce antibiotic use, thus contributing to antibiotic stewardship goals emphasized in the One Health approach. Full article

Due to their involvement in pathogen-mediated immune responses, the hypervariable genes of the Major Histocompatibility Complex (MHC) have become a paradigm for investigating the evolution and maintenance of genetic (adaptive) diversity, contextually providing insight into the viability of wild populations, which is meaningful for conservation. Here, we provide the first preliminary characterization of MHC polymorphism and evolution in trouts from Albania, a known hotspot of Salmonid diversity harboring ecologically and phylogenetically distinct native (threatened) taxa. Overall, 36 trout—including Lake Ohrid-endemic Salmo ohridanus and S. letnica, and both riverine and lacustrine native brown trout (the S. trutta complex) from the Drin-Skadar drainage—were genotyped at the MHC-DAB locus through next-generation amplicon sequencing. We identified 34 alleles (including 30 novel alleles), unveiling remarkable population/taxon MHC-DAB distinctiveness. Despite apparent functional (supertype) similarity, S. letnica and the S. trutta complex showed MHC-typical high sequence/allele diversity and evidence of global/codon-specific positive selection, particularly at antigen-binding sites. Conversely, deep-water-adapted S. ohridanus revealed unexpectedly reduced allelic/supertype diversity and relaxed selection. Evolution by reticulation and signals of trans-species polymorphism emerged from sequence genealogies. Further investigations and increased sampling will provide a deeper understanding of the evolutionary mechanisms yielding the observed pattern of MHC diversity across Albanian trout taxa and populations. Full article

The emergence of SARS-CoV-2 variants has heightened concerns about vaccine efficacy, posing challenges in controlling the spread of COVID-19. As part of the COVID-19 Vaccine Effectiveness and Variants (COVVAR) study in Uganda, this study aimed to genotype and characterize SARS-CoV-2 variants in patients with COVID-19-like symptoms who tested positive on a real-time PCR. Amplicon deep sequencing was performed on 163 oropharyngeal/nasopharyngeal swabs collected from symptomatic patients. Genome assembly, lineage classification and phylogenetic analysis was performed using the Edge Bioinformatics pipeline version 2.4.0, Pangolin version 4.3.1 and iqtree version 2.3.6 software respectively. Of the 163 deep sequences analyzed between April 2023 and March 2024, the most common were XBB.1 lineages and sublineages (113, 69.3%), followed by JN.1* (12, 7.4%), XBB.2* (11, 6.7%) and FL* (11, 6.7%), EG* (7, 4.3%), others (BQ.1.1, FY.4.1, FY.4.1.2, GY.2.1, HK.27.1) (5, 3.1%) and CM* (4, 2.5%). XBB.1* dominated from April to July 2023; thereafter, other variants, including JN.1* were increasingly detected. There was no statistically significant association between vaccine status and lineage assignment (Fisher’s exact test, p-value = 0.994). Our findings showed that the Omicron variant, specifically the XBB.1* lineage, was the dominant circulating virus. However, the emergence of the JN.1 variant that exhibits a significant spike protein mutation profile could impact COVID-19 transmission in Uganda. Full article

To date, only a few microbial community studies of cold seeps at the South China Sea (SCS) have been reported. The cold seep dominated by tubeworms was discovered at South Yungan East Ridge (SYER) offshore southwestern Taiwan by miniROV. The tubeworms were identified and proposed as Paraescarpia formosa sp. nov. through morphological and phylogenetic analyses. The endosymbionts in the trunk of P. formosa analyzed by a 16S rRNA gene clone library represented only one phylotype, which belonged to the family Sedimenticolaceae in Gammaproteobacteria. In addition, the archaeal and bacterial communities in the habitat of tubeworm P. formosa were investigated by using high-phylogenetic-resolution full-length 16S rRNA gene amplicon sequencing. The results showed that anerobic methane-oxidizing archaea (ANME)-1b was most abundant and ANME-2ab was minor in a consortia of the anerobic oxidation of methane (AOM). The known sulfate-reducing bacteria (SRB) partners in AOM consortia, such as SEEP-SRB1, -SRB2, and -SRB4, Desulfococcus and Desulfobulbus, occurred in a small population (0–5.7%) at the SYER cold seep, and it was suggested that ANME-1b and ANME-2ab might be coupled with multiple SRB in AOM consortia. Besides AOM consortia, various methanogenic archaea, including Bathyarchaeota (Subgroup-8), Methanocellales, Methanomicrobiales, Methanosarcinales, Methanofastidiosales and Methanomassiliicoccales, were identified, and sulfur-oxidizing bacteria Sulfurovum and Sulfurimonas in phylum Epsilonbacteraeota were dominant. This study revealed the first investigation of microbiota in and around tubeworm P. formosa discovered at the SYER cold seep offshore southwestern Taiwan. We could gain insights into the chemosynthetic communities in the deep sea, especially regarding the cold seep ecosystems at the SCS. Full article

Burkholderia (sensu lato) is a diverse group of β-Proteobacteria that exists worldwide in various environments. The SBE clade of this group was thought to be mutualistic with stinkbugs. Riptortus–Burkholderia was suggested as an ideal model system for studying insect–microbe symbiosis. To explore the strain-level diversity of Burkholderia at the individual and population levels of Riptortus stinkbugs (Hemiptera: Alydidae), and to uncover the factors affecting the Burkholderia community, large-scale sampling of two Riptortus species and deep sequencing data (16S amplicon) were used in the present study. Our results showed that: (1) the proportions of facultative symbiotic bacteria Burkholderia were very high, with an average proportion of 87.1% in the samples; (2) only six out of 1373 Burkholderia amplicon sequence variants (ASVs) did not belong to the SBE clade, accounting for only 0.03% of Burkholderia; (3) a relatively small number of Burkholderia ASVs had a large number of sequences, with 22, 54, and 107 ASVs accounting for more than 1.0%, 0.1%, and 0.01% of the total Burkholderia sequences, respectively; (4) multiple Burkholderia ASVs were present in most Riptortus individuals, but there was one dominant or two codominant ASVs, and codominance was more likely to occur when the genetic distance between the two codominant ASVs was small; and (5) the beta diversity of Burkholderia was significantly different between the two host species (PerMANOVA: both Jaccard and Bray–Curtis, p < 0.001) and among localities (PerMANOVA: both Jaccard and Bray–Curtis, p < 0.001). Two-way PerMANOVA also indicated that both the host (Bray–Curtis, p = 0.020; Jaccard, p = 0.001) and geographical location (Bray–Curtis, p = 0.041; Jaccard, p = 0.045) influence Burkholderia communities; furthermore, Mantel tests showed that the Burkholderia communities were significantly correlated with the geographical distance of sample locations (R = 0.056, p = 0.001). Together, our findings demonstrate the fine-scale diversity of Burkholderia symbionts and suggest a region- and host-dependent pattern of Burkholderia in Riptortus stinkbugs. Full article

Aquifers are rich in microbial diversity. However, there is a lack of information about sessile communities in these environments because of the difficulty in sampling fresh in situ rock surfaces. Thus, this study’s objective was to better understand the sessile community in a fractured aquifer. Additionally, the impact of the rock mineral composition on microbial community composition during colonization was explored. Using a system of bioreactors, we recreated the environmental conditions of a 1.5 m deep aquifer in Covey Hill (QC, Canada) using groundwater samples collected from the site. We carried out 16S/18S rRNA amplicon sequencing of the water and sessile communities after 24 days of incubation. Our data showed that many microbial taxa overlapped between the sessile and planktonic communities, indicating colonization of the solid surfaces. Quartz and feldspar had a significant impact on bacterial community structure. Sessile communities were dominated by Gaillonella, Alkanindiges, unclassified Acetobacteraceae, Apoikiales, Glissomonadida, and Synurales. We could not detect any Archaea in the sessile community. The sessile communities contained bacterial genera involved in iron cycling and adapted to acidic and low-carbon-concentration environments. Eukaryotic predators dominated the sessile community. Full article

Background: Detecting Helicobacter pylori in fecal samples is easier and more comfortable than invasive techniques, especially in children. Thus, the objective of the present work was to detect H. pylori in feces from children by molecular methods as an alternative for diagnostic and epidemiological studies. Methods: Forty-five fecal samples were taken from pediatric patients who presented symptoms compatible with H. pylori infection. HpSA test, culture, real-time quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), direct viable count associated with FISH (DVC-FISH), and Illumina-based deep-amplicon sequencing (DAS) were applied. Results: No H. pylori colonies were isolated from the samples. qPCR analysis detected H. pylori in the feces of 24.4% of the patients. In comparison, DVC-FISH analysis showed the presence of viable H. pylori cells in 53.3% of the samples, 37% of which carried 23S rRNA mutations that confer resistance to clarithromycin. After DAS, H. pylori-specific 16S rDNA sequences were detected in 26 samples. In addition, DNA from H. hepaticus was identified in 10 samples, and H. pullorum DNA was detected in one sample. Conclusion: The results of this study show the presence of H. pylori, H. hepaticus, and H. pullorum in children’s stools, demonstrating the coexistence of more than one Helicobacter species in the same patient. The DVC-FISH method showed the presence of viable, potentially infective H. pylori cells in a high percentage of the children’s stools. These results support the idea that fecal–oral transmission is probably a common route for H. pylori and suggest possible fecal–oral transmission of other pathogenic Helicobacter species. Full article

This study investigates the nitrate gradients within the deep biosphere of karst carbonate rocks and their resident microbiota. Samples were taken from borehole cores at depths down to 350 m below the surface, collected during geological site investigations for proposed railway tunnels and analysed using 16S rRNA amplicon sequencing. 16S rRNA amplicon sequencing analysis revealed relatively low microbial diversity, which can serve as a reliable indicator of the pristine nature of deep karst. However, some local hotspots of diversity are independent of depth. Pseudomonadota dominated the samples, with Gammaproteobacteria dominating at the class level. The low nitrate content in deep karst, in contrast to higher values closer to the surface, serves as an additional marker of its undisturbed and unpolluted status. Based on the prediction of functional profiles from 16S rRNA sequencing data, nitrates remain low due to indigenous microbial denitrification and assimilatory nitrate reduction. Pathways related to nitrogen fixation, ammonia assimilation, and nitrification were not confirmed. When elevated nitrate levels are observed in karst, they are most probably related to anthropogenic activities. Environmental factors other than depth and nitrate content play an important role in shaping bacterial communities. Full article

Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of diseases which cause visual loss due to Mendelian mutations in over 250 genes, making genetic diagnosis challenging and time-consuming. Here, we developed a new tool, CDIP (Cost-effective Deep-sequencing IRD Panel) in which a simultaneous sequencing of common mutations is performed. CDIP is based on simultaneous amplification of 47 amplicons harboring common mutations followed by next-generation sequencing (NGS). Following five rounds of calibration of NGS-based steps, CDIP was used in 740 IRD samples. The analysis revealed 151 mutations in 131 index cases. In 54 (7%) of these cases, CDIP identified the genetic cause of disease (the remaining were single-heterozygous recessive mutations). These include a patient that was clinically diagnosed with retinoschisis and found to be homozygous for NR2E3-c.932G>A (p.R311Q), and a patient with RP who is hemizygous for an RPGR variant, c.292C>A (p.H98N), which was not included in the analysis but is located in proximity to one of these mutations. CDIP is a cost-effective deep sequencing panel for simultaneous detection of common founder mutations. This protocol can be implemented for additional populations as well as additional inherited diseases, and mainly in populations with strong founder effects. Full article