High-throughput Sequencing Platforms Research Articles

Lysosome-related genes predict acute myeloid leukemia prognosis and response to immunotherapy

BackgroundAcute myeloid leukemia (AML) is a highly aggressive and pathogenic hematologic malignancy with consistently high mortality. Lysosomes are organelles involved in cell growth and metabolism that fuse to form specialized Auer rods in AML, and their role in AML has not been elucidated. This study aimed to identify AML subtypes centered on lysosome-related genes and to construct a prognostic model to guide individualized treatment of AML.MethodsGene expression data and clinical data from AML patients were downloaded from two high-throughput sequencing platforms. The 191 lysosomal signature genes were obtained from the database MsigDB. Lysosomal clusters were identified by unsupervised consensus clustering. The differences in molecular expression, biological processes, and the immune microenvironment among lysosomal clusters were subsequently analyzed. Based on the molecular expression differences between lysosomal clusters, lysosomal-related genes affecting AML prognosis were screened by univariate cox regression and multivariate cox regression analyses. Algorithms for LASSO regression analyses were employed to construct prognostic models. The risk factor distribution, KM survival curve, was applied to evaluate the survival distribution of the model. Time-dependent ROC curves, nomograms and calibration curves were used to evaluate the predictive performance of the prognostic models. TIDE scores and drug sensitivity analyses were used to explore the implication of the model for AML treatment.ResultsOur study identified two lysosomal clusters, cluster1 has longer survival time and stronger immune infiltration compared to cluster2. The differences in biological processes between the two lysosomal clusters are mainly manifested in the lysosomes, vesicles, immune cell function, and apoptosis. The prognostic model consisting of six prognosis-related genes was constructed. The prognostic model showed good predictive performance in all three data sets. Patients in the low-risk group survived significantly longer than those in the high-risk group and had higher immune infiltration and stronger response to immunotherapy. Patients in the high-risk group showed greater sensitivity to cytarabine, imatinib, and bortezomib, but lower sensitivity to ATRA compared to low -risk patients.ConclusionOur prognostic model based on lysosome-related genes can effectively predict the prognosis of AML patients and provide reference evidence for individualized immunotherapy and pharmacological chemotherapy for AML.

Integrative liquid chromatography-tandem mass spectrometry metabolomics and high-throughput sequencing technology reveal physicochemical characteristics and bacterial diversity of traditionally pickled mustard tuber from different regions of China

Abstract Objectives This work aimed to investigate and compare the physicochemical properties and bacterial diversity of traditionally pickled mustard tuber from different regions in China. Materials and Methods The pickles were obtained from Ningbo (NB), Nei Monggol (NMG), and Harbin (HEB). The pH was monitored by a pH meter. The titratable acidity and salinity were determined via titrimetric analysis. The nitrite content was measured using N-(1-naphthyl)-ethylenediamine dihydrochloride spectrophotometric method. The organic acids were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The bacterial diversity was conducted through Illumina high-throughput sequencing platform. Results There were significant differences in the physicochemical properties of pickled mustard tuber from various locations, with the NB pickles having the highest pH, salinity, and nitrite content and the lowest titratable acidity. Importantly, pickles with higher titratable acidity levels had relatively lower nitrite and salinity contents. A total of 63 organic acids were detected, with lactic acid and succinic acid being the most prevalent. Moreover, the organic acid compositions of the NMG and HEB samples were more similar than those of the NB pickles. Additionally, bioinformatic analysis revealed that Firmicutes and Proteobacteria appeared as the predominant bacterial phyla in all samples. Among these, Lactobacillus was the major genera. The most abundant bacteria were found in the HEB pickles. Further correlation analysis showed that there was a strong correlation between bacterial community and 11 internal physicochemical parameters. Conclusions This study could contribute to a better understanding of the physicochemical characteristics and bacterial diversity of pickled mustard tuber from different locations.

Diversity and novelty of venom peptides from Conus (Asprella) rolani revealed by analysis of its venom duct transcriptome

Conus species in the sub-genus Asprella are poorly studied because they inhabit deep-water habitats. To date, only a few peptides have been characterized from this clade. In this study, the venom duct transcriptome of a member of this clade, Conus rolani, was mined for potential conopeptides. Using a high-throughput RNA sequencing platform (Illumina) and a multiple k-mer de novo assembly, we found 103 putative conopeptide precursor amino acid sequences, including the few peptides previously reported for this species. The sequences, predominantly novel based on amino acid sequence, were diverse, comprising 36 gene superfamilies (including the “unassigned” superfamilies). As observed in other Conus species, the O1 gene superfamily was the most diverse (12 distinct sequences) but interestingly none of the sequences were found to contain the conserved amino acids associated with certain bioactivities in peptides found in piscivorous Conus species. The O2 superfamily was also highly diverse but con-ikot-ikot and an unassigned superfamily (MMSRMG) were more diverse than the rest of the superfamilies. In terms of gene expression levels, the understudied MEFRR paralog of the ancestral divergent M---L-LTVA superfamily was found to be the most highly expressed in the transcriptome, suggesting a novel role. Additionally, a conopeptide with high sequence similarity to A2 secretory group XII phospholipases is the first reported member of this phospholipase group in Conus and potentially represents a novel superfamily, expanding the catalog of known phospholipases present in cone snail venoms. The discovery of these putative conopeptides provides the first but early glimpse of the diversity and novelty of the peptides in the Asprella group and sets the stage for their functional characterization.

Differences in the response of normal oral mucosa, oral leukoplakia, oral squamous cell carcinoma-derived mesenchymal stem cells, and epithelial cells to photodynamic therapy

ObjectiveThe objective of this study is to investigate the variances in transcriptome gene expression of normal oral mucosa-derived mesenchymal stem cell (OM-MSC), oral leukoplakia-derived MSC (OLK-MSC) and oral squamous cell carcinoma-derived MSC(OSCC-MSC). as Additionally, the study aims to compare the in vitro proliferation, migration, invasion ability, and response to photodynamic therapy (PDT) of these three MSC, HOK, DOK, leuk1, and Cal27 cell lines. MethodsHOK, DOK, leuk1, Cal27 cells were cultured in vitro. 3 MSC cells were obtained from OM, OLK, OSCC tissue (n = 3) and identified through flow cytometry. They were also cultured in vitro for osteogenic and lipogenic-induced differentiation. Based on the Illumina HiSeq high-throughput sequencing platform, OM-MSC, OLK-MSC, OSCC-MSC (n = 3) were subjected to transcriptome sequencing, functional annotation, and enrichment analysis of differentially expressed genes and related genes. CCK8 assay, wound healing assay, and transwell assay were performed to compare the proliferation, migration, and invasion of the seven types of cells. The 7 cells were incubated with 0, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM of the photosensitizer (5-aminolevulinic acid, 5-ALA) in vitro. Subsequently, they were irradiated with a 150 mM, 635 nm laser for 1 min, and the cell activity was detected using the CCK8 assay after 24 h. The mitochondrial changes in the 7 cells before and after the treatment of PDT were detected using the JC-10 probe, and the changes in ATP content were measured before and after the PDT treatment. ResultsOM-MSC, OLK-MSC, and OSCC-MSC expressed positive MSC surface markers. After osteogenic and lipogenic-induced differentiation culture, stained calcium nodules and lipid droplets were visible, meeting the identification criteria of MSC. Pathway enrichment analysis revealed that the differentially expressed genes (DEGs) of OSCC-MSC compared to OLK-MSC were primarily associated with the PI3K-Akt signaling pathway and tumor-related pathways. OSCC-MSC exhibited stronger migratory and invasive abilities compared to Cal27. The IC50 values required for OM, OLK, and OSCC-derived MSC were lower than those required for epithelial cells treated with PDT, which were 1.396 mM, 0.9063 mM, and 2.924 mM, respectively. Cell membrane and mitochondrial disruption were observed in seven types of cells after 24 h of PDT treatment. However, HOK, DOK, leuk1, and Cal27 cells had an ATP content increased. ConclusionsOLK, OSCC epithelial cells require higher concentrations of 5-ALA for PDT treatment than MSC of the same tissue origin. The concentration of 5-ALA required increases with increasing cell malignancy. Differences in the response of epithelial cells and MSC to PDT treatment may have varying impacts on OLK recurrence and malignancy.

Abstract 5481: Spatial multiplex proteins profiling of tumor microenvironment to decipher de novo metastasis in nasopharyngeal carcinoma

Abstract Background: Nasopharyngeal carcinoma (NPC) has a high incidence rate in Southeast Asia, where more than 90% of NPC cases are associated with Epstein-Barr virus (EBV). Although NPC patients are generally sensitive to radiotherapy and chemotherapy, about 20-30% of patients have treatment failure due to the development of distant metastasis, i.e. de novo metastasis. In this study, we utilized GeoMx Digital Spatial Profiler (DSP), a multiplex and high-throughput sequencing platform designed for spatially- and morphologically resolved protein expression profiling to investigate the intra-tumoral and inter-tumoral heterogeneity of tumor microenvironment (TME) and identify the mechanisms underlying distant failure in NPC. Methods: We utilized GeoMx Protein Assays consisting of 147 proteins from 15 functional modules to compare the difference between NPC cases developing distant failure (DF) (n=21) and stage-, age-, gender- and treatment-matched NPC cases without developing distant failure (noDF) (n=15) within five years of treatment. Two fluorescent morphological markers including PanCK and CD45, were used for delineating the structural architecture of the tissues, identifying cell types, and selecting regions of interest (ROIs), followed by sequencing for protein expression quantification. NanoString GeoMx DSP Analysis Suite was used for quality control and data normalization. The NPC TME heterogeneity was further investigated by the 10X Visum platform for spatial transcriptome. The public single-cell RNA sequencing (scRNA-Seq) and bulk RNA sequencing datasets were obtained for the downstream analyses. Results: By analyzing protein expression profiles, the study revealed the intratumoral and intertumoral heterogeneity associated with EBV latent infection in the TME of NPC. Regions with high EBV latent gene expression exhibited a noticeable reduction in T cell infiltration, indicating an "immune cold" phenotype, whereas regions with low EBV latent gene expression displayed heavy T cell infiltration, indicating an "immune hot" phenotype. Moreover, the study identified a panel of autophagic and anti-apoptotic proteins that were significantly upregulated in the cancer cells of NPC cases developing distant failure after treatment. This observation, supported by the single-cell RNA sequencing datasets, further implicated EBV latent infection for the upregulation of autophagic and anti-apoptotic genes. Notably, the study also demonstrated that the genes upregulated in the immune cells in NPC cases with distant failure were associated with poor progression-free survival in an independent NPC cohort. Conclusions: These findings shed light on the crucial role of EBV latent infection in TME heterogeneity and highlight the potential involvement of autophagy and anti-apoptosis in de novo metastasis in NPC. Citation Format: Larry Ka-Yue Chow, Ying Wang, Lanqi Gong, Siqi Xu, Zhaozheng Hou, Xinyuan Guan, Dora Lai-Wan Kwong, Victor Ho-Fun Lee, Wai To ng, Anne Wing-Mui Lee, Zhonghua Liu, Yunfei Xia, Wei Dai. Spatial multiplex proteins profiling of tumor microenvironment to decipher de novo metastasis in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5481.

Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing*

ObjectiveViral encephalitis is an infectious disease severely affecting human health. It is caused by a wide variety of viral pathogens, including herpes viruses, flaviviruses, enteroviruses, and other viruses. The laboratory diagnosis of viral encephalitis is a worldwide challenge. Recently, high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections. Thus, In this study, we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing. MethodsWe designed nine pairs of specific polymerase chain reaction (PCR) primers for the 12 viruses by reviewing the relevant literature. The detection ability of the primers was verified by software simulation and the detection of known positive samples. Amplicon sequencing was used to validate the samples, and consistency was compared with Sanger sequencing. ResultsThe results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×, and the sequence lengths were consistent with the sizes of the predicted amplicons. The sequences were verified using the National Center for Biotechnology Information BLAST, and all results were consistent with the results of Sanger sequencing. ConclusionAmplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis. It is also a useful tool for the high-volume screening of clinical samples.

Control-FREEC viewer: a tool for the visualization and exploration of copy number variation data

BackgroundCopy number alterations (CNAs) are genetic changes commonly found in cancer that involve different regions of the genome and impact cancer progression by affecting gene expression and genomic stability. Computational techniques can analyze copy number data obtained from high-throughput sequencing platforms, and various tools visualize and analyze CNAs in cancer genomes, providing insights into genetic mechanisms driving cancer development and progression. However, tools for visualizing copy number data in cancer research have some limitations. In fact, they can be complex to use and require expertise in bioinformatics or computational biology. While copy number data analysis and visualization provide insights into cancer biology, interpreting results can be challenging, and there may be multiple explanations for observed patterns of copy number alterations.ResultsWe created Control-FREEC Viewer, a tool that facilitates effective visualization and exploration of copy number data. With Control-FREEC Viewer, experimental data can be easily loaded by the user. After choosing the reference genome, copy number data are displayed in whole genome or single chromosome view. Gain or loss on a specific gene can be found and visualized on each chromosome. Analysis parameters for subsequent sessions can be stored and images can be exported in raster and vector formats.ConclusionsControl-FREEC Viewer enables users to import and visualize data analyzed by the Control-FREEC tool, as well as by other tools sharing a similar tabular output, providing a comprehensive and intuitive graphical user interface for data visualization.

An ultra high-throughput, massively multiplexable, single-cell RNA-seq platform in yeasts.

Yeasts are naturally diverse, genetically tractable, and easy to grow such that researchers can investigate any number of genotypes, environments, or interactions thereof. However, studies of yeast transcriptomes have been limited by the processing capabilities of traditional RNA sequencing techniques. Here we optimize a powerful, high-throughput single-cell RNA sequencing (scRNAseq) platform, SPLiT-seq (Split Pool Ligation-based Transcriptome sequencing), for yeasts and apply it to 43,388 cells of multiple species and ploidies. This platform utilizes a combinatorial barcoding strategy to enable massively parallel RNA sequencing of hundreds of yeast genotypes or growth conditions at once. This method can be applied to most species or strains of yeast for a fraction of the cost of traditional scRNAseq approaches. Thus, our technology permits researchers to leverage "the awesome power of yeast" by allowing us to survey the transcriptome of hundreds of strains and environments in a short period of time and with no specialized equipment. The key to this method is that sequential barcodes are probabilistically appended to cDNA copies of RNA while the molecules remain trapped inside of each cell. Thus, the transcriptome of each cell is labeled with a unique combination of barcodes. Since SPLiT-seq uses the cell membrane as a container for this reaction, many cells can be processed together without the need to physically isolate them from one another in separate wells or droplets. Further, the first barcode in the sequence can be chosen intentionally to identify samples from different environments or genetic backgrounds, enabling multiplexing of hundreds of unique perturbations in a single experiment. In addition to greater multiplexing capabilities, our method also facilitates a deeper investigation of biological heterogeneity, given its single-cell nature. For example, in the data presented here, we detect transcriptionally distinct cell states related to cell cycle, ploidy, metabolic strategies, and so forth, all within clonal yeast populations grown in the same environment. Hence, our technology has two obvious and impactful applications for yeast research: the first is the general study of transcriptional phenotypes across many strains and environments, and the second is investigating cell-to-cell heterogeneity across the entire transcriptome.

Advanced sequencing-based high-throughput and long-read single-cell transcriptome analysis.

Cells are the fundamental building blocks of living systems, exhibiting significant heterogeneity. The transcriptome connects the cellular genotype and phenotype, and profiling single-cell transcriptomes is critical for uncovering distinct cell types, states, and the interplay between cells in development, health, and disease. Nevertheless, single-cell transcriptome analysis faces daunting challenges due to the low abundance and diverse nature of RNAs in individual cells, as well as their heterogeneous expression. The advent and continuous advancements of next-generation sequencing (NGS) and third-generation sequencing (TGS) technologies have solved these problems and facilitated the high-throughput, sensitive, full-length, and rapid profiling of single-cell RNAs. In this review, we provide a broad introduction to current methodologies for single-cell transcriptome sequencing. First, state-of-the-art advancements in high-throughput and full-length single-cell RNA sequencing (scRNA-seq) platforms using NGS are reviewed. Next, TGS-based long-read scRNA-seq methods are summarized. Finally, a brief conclusion and perspectives for comprehensive single-cell transcriptome analysis are discussed.

Comparative Transcriptomics Data Profiling Reveals E2F Targets as an Important Biological Pathway Overexpressed in Intellectual Disability Disorder.

Intellectual disability (ID) is an early childhood neurodevelopmental disorder that is characterized by impaired intellectual functioning and adaptive behavior. It is one of the major concerns in the field of neurodevelopmental disorders across the globe. Diversified approaches have been put forward to overcome this problem. Among all these approaches, high throughput transcriptomic analysis has taken an important dimension. The identification of genes causing ID rapidly increased over the past 3 to 5 years owing to the use of sophisticated high throughput sequencing platforms. Early monitoring and preventions are much important for such disorder as their progression occurs during fetal development. This study is an attempt to identify differentially expressed genes (DEGs) and upregulated biological processes involved in development of ID patients through comparative analysis of available transcriptomics data. A total of 7 transcriptomic studies were retrieved from National Center for Biotechnology Information (NCBI) and were subjected to quality check and trimming prior to alignment. The normalization and differential expression analysis were carried out using DESeq2 and EdgeR packages of Rstudio to identify DEGs in ID. In progression of the study, functional enrichment analysis of the results obtained from both DESeq2 and EdgeR was done using gene set enrichment analysis (GSEA) tool to identify major upregulated biological processes involved in ID. Our findings concluded that monitoring the level of E2F targets, estrogen, and genes related to oxidative phosphorylation, DNA repair, and glycolysis during the developmental stage of an individual can help in the early detection of ID disorder.

Effects of Different Proportions of Organic Fertilizer in Place of Chemical Fertilizer on Microbial Diversity and Community Structure of Pineapple Rhizosphere Soil

With the development of sustainable agriculture, the application of organic fertilizers to crops instead of chemical fertilizers has become an inevitable trend. However, little is known about the proportion of organic fertilizers replacing chemical fertilizers and how it affects the underlying microbial mechanisms of continuous pineapple soil. In this study, we used the Illumina Miseq high-throughput sequencing platform to study the rhizosphere soil of continuously cropped pineapples to study the diversity and community structure of pineapple rhizosphere microorganisms. The results showed that, with 97% similarity, the number of OTUs of all samples obtained using hierarchical clustering analysis is 3645. Both conventional fertilization (CF) and optimal fertilization (YF) increased the relative abundance of Proteobacteria, Actinomycetes, and Sclericutes, and decreased the relative abundance of Acidobacteria, Bacteroidetes, Blastomonas, and Verrucobacteria. The replacement of organic fertilizers increased the relative abundance of Bacteroidetes, among which the relative abundance of chlorocurvus treated with the replacement of organic fertilizers of 80% was the highest. Different fertilization methods also changed the diversity and abundance of bacteria in the soil of the pineapple rhizosphere; the diversity of the species was E > D > C > CK > B > YF > CF. Based on the analysis of the PCoA and NMDS of soil bacterial communities, treatment E was similar to treatment D, treatment CK was similar to treatment YF, and treatment C and treatment D had little structural difference. On the basis of an analysis of the composition and function of the flora, it can be found that different fertilization methods have significant differences in the bacterial groups of the rhizosphere bacterial community of pineapple soil. The relative abundance of beneficial bacteria was increased. When organic fertilizer replaces chemical fertilizer, it promotes the role of bacteria related to the carbon cycle in the rhizosphere.

Individual genotypes from environmental DNA: Fingerprinting snow tracks of three large carnivore species.

Continued advancements in environmental DNA (eDNA) research have made it possible to access intraspecific variation from eDNA samples, opening new opportunities to expand non-invasive genetic studies of wildlife populations. However, the use of eDNA samples for individual genotyping, as typically performed in non-invasive genetics, still remains elusive. We present successful individual genotyping of eDNA obtained from snow tracks of three large carnivores: brown bear (Ursus arctos), European lynx (Lynx lynx) and wolf (Canis lupus). DNA was extracted using a protocol for isolating water eDNA and genotyped using amplicon sequencing of short tandem repeats (STR), and for brown bear a sex marker, on a high-throughput sequencing platform. Individual genotypes were obtained for all species, but genotyping performance differed among samples and species. The proportion of samples genotyped to individuals was higher for brown bear (5/7) and wolf (7/10) than for lynx (4/9), and locus genotyping success was greater for brown bear (0.88). The sex marker was typed in six out of seven brown bear samples. Results for three species show that reliable individual genotyping, including sex identification, is now possible from eDNA in snow tracks, underlining its vast potential to complement the non-invasive genetic methods used for wildlife. To fully leverage the application of snow track eDNA, improved understanding of the ideal species- and site-specific sampling conditions, as well as laboratory methods promoting genotyping success, is needed. This will also inform efforts to retrieve and type nuclear DNA from other eDNA samples, thereby advancing eDNA-based individual and population-level studies.

Changes in the soil fungal communities of steppe grasslands at varying degradation levels in North China.

The grasslands in North China are rich in fungal resources. However, the knowledge of the structure and function of fungal communities and the role of microbial communities in vegetation restoration and succession are limited. Thus, we used an Illumina HiSeq PE250 high-throughput sequencing platform to study the changing characteristics of soil fungal communities in degraded grasslands, which were categorized as non-degraded (ND), lightly degraded, moderately degraded, and severely degraded (SD). Moreover, a correlation analysis between soil physical and chemical properties and fungal communities was completed. The results showed that the number of plant species, vegetation coverage, aboveground biomass, and diversity index decreased significantly with increasing degradation, and there were significant differences in the physical and chemical properties of the soil among the different degraded grasslands. The dominant fungal phyla in the degraded grassland were as follows: Ascomycota, 44.88%-65.03%; Basidiomycota, 12.68%-29.91%; and unclassified, 5.51%-16.91%. The dominant fungi were as follows: Mortierella, 6.50%-11.41%; Chaetomium, 6.71%-11.58%; others, 25.95%-36.14%; and unclassified, 25.56%-53.0%. There were significant differences in the microbial Shannon-Wiener and Chao1 indices between the ND and degraded meadows, and the composition and diversity of the soil fungal community differed significantly as the meadows continued to deteriorate. The results showed that pH was the most critical factor affecting soil microbial and fungal communities in SD grasslands, whereas soil microbial and fungal communities in ND grasslands were mainly affected by water content and other environmental factors.

Targeted Whole Genome Sequencing of the Capripoxvirus Genome from Clinical Tissue Samples and Lyophilized Vaccine Batches.

Diseases caused by Capripoxviruses (CaPVs) are of great economic importance in sheep, goats, and cattle. Since CaPV strains are serologically indistinguishable and genetically highly homologous, typing of closely related strains can only be achieved by whole-genome sequencing. In this chapter, we describe a robust, cost-effective, and widely applicable protocol for reconstructing (nearly) complete CaPV genomes directly from clinical samples or commercial vaccine batches in less than a week. Taking advantage of the genetic similarity of CaPVs, a set of pan-CaPVs long-range PCRs was developed that covers the entire genome with only a limited number of tiled amplicons. The resulting amplicons can be sequenced on all currently available high-throughput sequencing platforms. As an example, we have included a detailed protocol for performing nanopore sequencing and a pipeline for assembling the resulting tiled amplicon data.

Differences in Soil Fungal Communities between Forested Reclamation and Forestry Sites in the Alberta Oil Sands Region.

Fungi play key roles in forest soils and provide benefits to trees via mycorrhizal symbioses. After severe disturbance, forest regrowth can be impeded because of changes in fungal communities. In 2013-2014, soil fungi in forest floor and mineral soil were examined by Roche 454 pyrosequencing in undisturbed, harvested, and burned jack pine stands in a forested area near Fort Chipewyan, Alberta. These fungal communities were compared with jack pine, white spruce, and larch stands in Gateway Hill, a nearby certified reclaimed area. In 2014, a more detailed sampling of forestry and reclamation jack pine sites examined fungi in soil fractions using two high-throughput sequencing platforms and a sporocarp survey. The significances of compositional and functional differences in fungal communities between the forested and reclamation sites were assessed using permutation tests of partially constrained ordinations, accounting for confounding factors by variance partitioning. Taxa associated with the forestry area were primarily ectomycorrhizal. Fungal richness and diversity were greater in soils from the reclamation sites and included significantly more pathogenic taxa and taxa with unknown functional properties. Fungal community dissimilarities may have been artefacts of historical legacies or, alternatively, may have resulted from contrasting niche differentiation between forestry and reclamation sites.

CLT-seq as a universal homopolymer-sequencing concept reveals poly(A)-tail-tuned ncRNA regulation.

Dynamic tuning of the poly(A) tail is a crucial mechanism for controlling translation and stability of eukaryotic mRNA. Achieving a comprehensive understanding of how this regulation occurs requires unbiased abundance quantification of poly(A)-tail transcripts and simple poly(A)-length measurement using high-throughput sequencing platforms. Current methods have limitations due to complicated setups and elaborate library preparation plans. To address this, we introduce central limit theorem (CLT)-managed RNA-seq (CLT-seq), a simple and straightforward homopolymer-sequencing method. In CLT-seq, an anchor-free oligo(dT) primer rapidly binds to and unbinds from anywhere along the poly(A) tail string, leading to position-directed reverse transcription with equal probability. The CLT mechanism enables the synthesized poly(T) lengths, which correspond to the templated segment of the poly(A) tail, to distribute normally. Based on a well-fitted pseudogaussian-derived poly(A)-poly(T) conversion model, the actual poly(A)-tail profile is reconstructed from the acquired poly(T)-length profile through matrix operations. CLT-seq follows a simple procedure without requiring RNA-related pre-treatment, enrichment or selection, and the CLT-shortened poly(T) stretches are more compatible with existing sequencing platforms. This proof-of-concept approach facilitates direct homopolymer base-calling and features unbiased RNA-seq. Therefore, CLT-seq provides unbiased, robust and cost-efficient transcriptome-wide poly(A)-tail profiling. We demonstrate that CLT-seq on the most common Illumina platform delivers reliable poly(A)-tail profiling at a transcriptome-wide scale in human cellular contexts. We find that the poly(A)-tail-tuned ncRNA regulation undergoes a dynamic, complex process similar to mRNA regulation. Overall, CLT-seq offers a simplified, effective and economical approach to investigate poly(A)-tail regulation, with potential implications for understanding gene expression and identifying therapeutic targets.

Aptamers selected and identified as a colorimetric dual-mode sensing platform for selective and sensitive detection of capsaicin

Capsaicin (CAP), a prominent indicator, has been identified for detecting the gutter oil in adulterated edible oils. In view of the limited availability of efficient and reliable methods for detecting gutter oil, the present study employed the Capture-SELEX technology, which utilizes a high-throughput sequencing (HTS) platform, to facilitate the precise tracking of screening rounds and facilitate the isolation of aptamers with specific affinity for CAP. Isothermal titration calorimetry (ITC) determined the highest affinity and Kd value of 15.5 ± 4.69 nM for aptamer Cap-1. A colorimetric dual-mode aptasensor was designed for CAP detection based on graphene oxide (GO)@gold nanoparticles (AuNPs) composite. A high-sensitivity assay can be achieved using a Synergy™ H1 multimode zymography instrument and a smartphone. Under optimal conditions, the colorimetric dual-mode aptasensor has a detection range of 0.5 ∼ 120 ng mL−1 and a low limit of detection (LOD) of 0.14 ng mL−1. The quantitative recovery of the sensor for CAP in edible oil was 86.25∼98.12 %. Compared with the conventional single-mode aptasensor, the dual-mode aptasensor has independent signal conversion, which provides suitable detection solutions for different scenarios and improves detection accuracy and reliability.

Phosphorus Coupled with High Nitrogen Addition Exerts a Great Influence on Soil Bacterial Community in a Semiarid Grassland.

Nitrogen (N) and phosphorus (P) addition, either individually or in combination, has been demonstrated to enhance plant productivity in grassland ecosystems. Soil bacterial community, which is the driver of litter decomposition and nutrient cycling, is assumed to control responses of terrestrial ecosystem structure and function to N and P addition. Using a high-throughput Illumina MiSeq sequencing platform, we conducted a 9-year field experiment of N (0, 5, 10, and 20 g N m-2 yr-1) and P (0 and 10 g P m-2 yr-1) additions in the Inner Mongolian steppes to elucidate long-term effects of N and P addition on soil bacterial richness, diversity and composition. We found that N addition reduced the relative abundance of Acidobacteria, Chloroflexi, and Nitrospirae, while increased that of Bacteroides. The results showed that the bacterial biomarker was enriched in P addition treatments, either individually or combined with N addition. Both N and P addition altered the bacterial community structure, while only N addition greatly decreased bacterial richness and diversity. More importantly, we showed that all of these effects were most significant in N3P treatment (20 g N m-2 yr-1 and 10 g P m-2 yr-1), implying that P coupled with a high-level N addition exerted a great influence on soil bacterial community. Structural equation models revealed that N and P addition had a great direct effect on soil bacterial community and an indirect effect on it mainly by changing the litter biomass. Our findings highlighted that severe niche differentiation was induced by P along with a high-level N, further emphasizing the importance of simultaneously evaluating response of soil bacterial community to N and P addition, especially in the context of increasing anthropogenic nutrient additions.

Oomycete Soil Diversity Associated with Betula and Alnus in Forests and Urban Settings in the Nordic-Baltic Region.

This study aimed to determine the differences and drivers of oomycete diversity and community composition in alder- and birch-dominated park and natural forest soils of the Fennoscandian and Baltic countries of Estonia, Finland, Lithuania, Norway, and Sweden. For this, we sequenced libraries of PCR products generated from the DNA of 111 soil samples collected across a climate gradient using oomycete-specific primers on a PacBio high-throughput sequencing platform. We found that oomycete communities are most affected by temperature seasonality, annual mean temperature, and mean temperature of the warmest quarter. Differences in composition were partly explained by the higher diversity of Saprolegniales in Sweden and Norway, as both total oomycete and Saprolegniales richness decreased significantly at higher longitudes, potentially indicating the preference of this group of oomycetes for a more temperate maritime climate. None of the evaluated climatic variables significantly affected the richness of Pythiales or Peronosporales. Interestingly, the relative abundance and richness of Pythiales was higher at urban sites compared to forest sites, whereas the opposite was true for Saprolegniales. Additionally, this is the first report of Phytophthora gallica and P. plurivora in Estonia. Our results indicate that the composition of oomycetes in soils is strongly influenced by climatic factors, and, therefore, changes in climate conditions associated with global warming may have the potential to significantly alter the distribution range of these microbes, which comprise many important pathogens of plants.

Epidemiological characteristics of mpox epidemic in Guangzhou

Objective: To understand the epidemiological characteristics of mpox epidemic in Guangzhou and provide scientific evidence for the prevention and control of the disease. Methods: Based on the mpox surveillance system in Guangzhou, suspected mpox cases with fever and rash were reported by local hospitals at all levels to centers for disease control and prevention in Guangzhou for sampling, investigation and diagnosis. Descriptive epidemiological analysis was conducted on the clinical characteristics and treatment of the mpox cases and positive detection rate reported in Guangzhou as of 24:00 on June 23. Whole genome sequencing of the virus isolates was performed using Illumina Miniseq high-throughput sequencing platform. Results: The first mpox case in Guangzhou was reported on June 10 in 2023. As of 24:00 on June 23, a total of 25 confirmed mpox cases were reported. All the mpox cases were men with a M(Q1,Q3) of 32 (26, 36) years, the majority of the cases were MSM (96.0%). The main clinical features were rash (100.0%, 25/25), lymphadenectasis (100.0%, 25/25) and fever (52.0%, 13/25). Rash usually occurred near the genitals (88.0%, 22/25). The close contacts, mainly family members (40.4%, 23/57), showed no similar symptoms, such as fever or rash. The positive rate of mpox virus in household environment samples was 30.5%. The analyses on 3 complete gene sequences of mpox virus indicated that the strains belonged to West African type Ⅱb clade, B.1.3 lineage. Conclusions: Hidden transmission of mpox virus had occurred in MSM in Guangzhou. However, the size of affected population is relatively limited, and the possibility of wide spread of the virus is low.