High-throughput Sequencing Approach Research Articles

Awakening soil microbial utilization of nitrate by carbon regulation to lower nitrogen pollution

Soil microbial immobilization of nitrate (NO3−-N, INO3) and its role in mitigating NO3−-N pollution in agricultural ecosystems globally is often neglected because of the tenet that microbes preferentially use ammonium over NO3−-N. Soil INO3 driven by heterotrophic microorganisms is often carbon (C)-limited, and may therefore be stimulated by C sources with high C/nitrogen (N) ratios. Here, using 15NO3−-N labelling coupled with acetylene inhibition, quantitative PCR and Illumina high-throughput sequencing approaches, we demonstrated that INO3 was stimulated from zero to a substantial level by crop residue amendment without any environmental risk; the degree of stimulation depending on residue quality and soil type. The stimulation was predicted well by C mineralization of the added residue. Soil with a lower initial nutrient status had reduced INO3 efficiency due to stronger C constraint and lower return on energy investment. Fast-growing bacterial r-strategists were identified as the key driver of INO3. We estimate that, globally, residue amendment could cause a total NO3−-N loss reduction of about 33.6 Tg N yr−1 through INO3 stimulation. We suggest that soil INO3 driven by addition of exogenous C is an overlooked process that can help curb NO3−-N accumulation in soil and its subsequent release to the environment, the “nitrate time bomb”.

Revealing microbial patterns in the rhizosphere of pecan trees asymptomatic and symptomatic for Texas root rot using a high-throughput sequencing approach

The rhizosphere harbors diverse microbial communities that play crucial roles in nutrient cycling and plant health. Understanding the composition and dynamics of these communities is essential for optimizing agricultural productivity and disease management. In this study, bacterial and fungal communities in the rhizosphere of pecan trees (Carya illinoinensis) asymptomatic and symptomatic for Texas root rot were characterized using high-throughput sequencing of the 16S rRNA gene and ITS region. The results indicated that Proteobacteria and Actinobacteria were the predominant bacterial phyla in both conditions. The predominant fungal phyla comprised Basidiomycota and Ascomycota in symptomatic conditions and Ascomycota and Mortierellomycota in asymptomatic ones. At the genus level, Streptomyces and Pseudomonas were the most abundant bacterial genera; among fungi, Scleroderma and Psathyrella were predominant in symptomatic samples and Mortierella in asymptomatic samples. α-diversity analysis showed significant differences in the fungal community. Specific bacterial and fungal biomarkers for asymptomatic and symptomatic conditions were identified, highlighting their importance in the pecan tree rhizosphere. A total of six bacterial and seven fungal taxa were detected in symptomatic samples, while four bacterial and 16 fungal taxa were detected in asymptomatic ones. These included genera known for their roles in soil health, such as Pseudomonas, Kaistobacter, Streptomyces, Psathyrella, Scleroderma, and Mortierella. Co-occurrence network analysis revealed higher complexity and interaction levels among microbial communities in asymptomatic samples, suggesting a healthier network supporting microbial communities. This study provides comprehensive and valuable insights into microbiome dynamics and microbial resources for future studies aimed at improving agricultural production through the study of rhizosphere microorganisms.

Linking ecological niches to bacterial community structure and assembly in polluted urban aquatic ecosystems.

Bacterial communities play crucial roles in the functioning and resilience of aquatic ecosystems, and their responses to water pollution may be assessed from ecological niches. However, our understanding of such response patterns and the underlying ecological mechanisms remains limited. In this study, we comprehensively investigated the effects of water pollution on the bacterial structure and assembly within different ecological niches, including water, sediment, submerged plant leaf surfaces, and leaf surfaces, using a 16S high-throughput sequencing approach. Ecological niches had a greater impact on bacterial community diversity than pollution, with a distinct enrichment of unique dominant phyla in different niches. This disparity in diversity extends to the bacterial responses to water pollution, with a general reduction in α-diversity observed in the niches, excluding leaf surfaces. Additionally, the distinct changes in bacterial composition in response to pollution should be correlated with their predicted functions, given the enrichment of functions related to biogeochemical cycling in plant surface niches. Moreover, our study revealed diverse interaction patterns among bacterial communities in different niches, characterized by relatively simply associations in sediments and intricate or interconnected networks in water and plant surfaces. Furthermore, stochastic processes dominated bacterial community assembly in the water column, whereas selective screening of roots and pollution events increased the impact of deterministic processes. Overall, our study emphasizes the importance of ecological niches in shaping bacterial responses to water pollution. These findings improve our understanding of the complicated microbial response patterns to water pollution and have ecological implications for aquatic ecosystem health.

Diversity and plant growth promoting ability of rice root-associated bacteria in Burkina-Faso and cross-comparison with metabarcoding data.

Plant-associated bacteria are essential partners in plant health and development. In addition to taking advantage of the rapid advances recently achieved in high-throughput sequencing approaches, studies on plant-microbiome interactions require experiments with culturable bacteria. A study on the rice root microbiome was recently initiated in Burkina Faso. As a follow up, the aim of the present study was to develop a collection of corresponding rice root-associated bacteria covering maximum diversity, to assess the diversity of the obtained isolates based on the culture medium used, and to describe the taxonomy, phenotype and abundance of selected isolates in the rice microbiome. More than 3,000 isolates were obtained using five culture media (TSA, NGN, NFb, PCAT, Baz). The 16S rRNA fragment sequencing of 1,013 selected isolates showed that our working collection covered four bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes) and represented 33% of the previously described diversity of the rice root microbiome at the order level. Phenotypic in vitro analysis of the plant growth promoting capacity of the isolates revealed an overall ammonium production and auxin biosynthesis capacity, while siderophore production and phosphate solubilisation were enriched in Burkholderia, Ralstonia, Acinetobacter and Pseudomonas species. Of 45 representative isolates screened for growth promotion on seedlings of two rice cultivars, five showed an ability to improve the growth of both cultivars, while five others were effective on only one cultivar. The best results were obtained with Pseudomonas taiwanensis ABIP 2315 and Azorhizobium caulinodans ABIP 1219, which increased seedling growth by 158% and 47%, respectively. Among the 14 best performing isolates, eight appeared to be abundant in the rice root microbiome dataset from previous study. The findings of this research contribute to the in vitro and in planta PGP capacities description of rice root-associated bacteria and their potential importance for plants by providing, for the first time, insight into their prevalence in the rice root microbiome.

Large Deletion in the Factor VIII Gene Is a Predictor of Immune Tolerance Induction Failure in People with Severe and Moderately-Severe Hemophilia a and High-Responding Inhibitors

Introduction Immune tolerance induction (ITI) is a therapeutic approach to eradicate inhibitors against factor VIII (FVIII) in people with inherited hemophilia A (PwHA). The success of ITI is highly variable, ranging from 60% to 80% across studies. Beyond inhibitor titers, other predictors of ITI outcome are unknown. Deleterious variants in FVIII gene ( F8) are well established risk factors for alloantibodies development in PwHA, although few studies have investigated the role of F8 variants on ITI outcome.Therefore we aimed to comprehensively analyze the association of F8 pathogenic variants on ITI outcome in severe PwHA with high-responding inhibitors. Methods We included severe (FVIII < 1 international units [IU]/dL) and moderately-severe (FVIII 1-2 IU/dL) unrelated PwHA and high-responding inhibitors who completed ITI from a large, admixed population of two well-characterized cohorts - the HEMFIL and the Brazilian Immune Tolerance (BrazIT) studies. We collected socio-demographic, clinical and laboratory data. ITI outcomes were defined according to previous definitions as failure, partial and total successes. Inversions of intron 1 and 22 (Inv22) were detected by polymerase chain reaction (PCR), and high-throughput sequencing approaches were used to unveil the additional F8 variants. The association between F8 pathogenic variants and ITI outcome was adjusted for inhibitor levels. To investigate whether F8 variants associated with inhibitor development were also related to ITI outcome, variants were categorized as “high-risk”, “intermediate-risk”, and “low-risk” categories. These categories for inhibitor development were then compared with the outcomes of ITI (total and partial successes and failure) using data from this study. Results We included158 PwHA, median age 6.6 years at ITI start, 90.5% were severe (Table 1). Inv22 was the most prevalent variant (55.1%) (Table 1). In comparison with Inv22-1, the risk of ITI failure was about 9 times higher (adjusted odds ratio [adjOR] 9.29; 95% confidence interval [95% CI] 1.95-53.70) among carriers of large deletions (Table 2). Conversely, Inv22-2 was associated with favorable ITI outcome in a univariate analysis (OR 0.15; 0.01-0.84), and after adjustment (adjOR 0.32; 95% CI 0.02-1.96) (Table 2). F8 deleterious variants sorted as high-risk and intermediate-risk according to a previously published classification on inhibitor development were associated with failure and successful outcomes, respectively. Conclusion Our study showed that F8 large deletions are independent predictors of ITI failure, and Inv22-2 is likely to be a predictor of successful ITI. We found a correspondence between variants classified as high-risk and intermediate-risk to inhibitor development with ITI failure and success, respectively. We suggest that F8 genotyping should be considered before indication of ITI, as ITI outcome can vary according to individual variant burden.

Genotyping and traceability analysis of norovirus in Yantai between 2017 and 2019.

To investigate the diversity and evolution of noroviruses in Yantai in recent years, this study focused on the coat protein regions of norovirus-positive samples with nucleic acid detection (cycle threshold) values below 30 between 2017 and 2019. A total of 81 sequences were obtained for genotyping. Initially, a high-throughput sequencing approach was established to perform the whole-genome sequencing of multiple typical diarrheal strains. Using bioinformatics software such as BEAST, recombinant variant analysis was performed for each genotype of the norovirus strains, and genetic evolutionary analysis was conducted for the dominant strain GII.4, as well as the rare variant GII.21. The results showed that there were multiple genotypes such as GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.6, GII.13, GII.17, GII.21, and GIX.1 in the positive samples of norovirus from 2017 to 2019. GII.4 is characterized by diverse genotypes, with new changes in antigenic epitopes occurring during the course of the epidemic. This may have led to the emergence of a new pandemic. This suggests a need to strengthen surveillance. The results of this study suggest that attention should be paid to the predominant genotypes prevalent in neighboring countries and regions, and the safety supervision of imported food should be strengthened to aid in the prevention and control of related viruses.

TGFB1 induces fetal reprogramming and enhances intestinal regeneration.

The gut epithelium has a remarkable ability to recover from damage. We employed a combination of high-throughput sequencing approaches, mouse genetics, and murine and human organoids and identified a role for TGFB signaling during intestinal regeneration following injury. At 2days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. The depletion of macrophages or genetic disruption of TGFB signaling significantly impaired the regenerative response. Intestinal regeneration is characterized by the induction of a fetal-like transcriptional signature during repair. In organoid culture, TGFB1 treatment was necessary and sufficient to induce the fetal-like/regenerative state. Mesenchymal cells were also responsive to TGFB1 and enhanced the regenerative response. Mechanistically, pro-regenerative factors, YAP/TEAD and SOX9, are activated in the epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for cellular therapy.

Five new F10 variants in hereditary factor x deficiency detected by high-throughput sequencing.

Factor X deficiency is a rare inherited bleeding disorder. To date, 181 variants are reported in the recently updated F10-gene variant database. This study aimed to describe new F10 variants. The F10 gene was analysed in 16 consecutive families with FX deficiency by a targeted high-throughput sequencing approach, including F10, F9, F8 genes, and 78 genes dedicated to haematological malignancies. We identified 19 variants (17 missense, one nonsense and one frameshift) and two copy number variations. Two patients presenting a combined FVII-FX deficiency showed a loss of one F10 gene copy (del13q34) associated with a missense variant on the remaining allele, leading to a FX:C significantly lower than the FVII:C level and explaining their unusual bleeding history. We reported five novel variants. Three missense variants (p.Glu22Val affecting the signal peptide cleavage site, p.Cys342Tyr removing the disulphide bond between the FX heavy and light chains, and p.Val385Met located in FX peptidase S1 domain) were detected at compound heterozygosis status in three patients with severe bleeding symptoms and FX:C level below 10 IU/dL. Two truncating variants p.Tyr279* and p.Thr434Aspfs*13 leading to an altered FX protein were found at heterozygous state in two patients with mild bleeding history. This study showed the feasibility and the interest of high-throughput sequencing approach for rare bleeding disorders, enabling the report of F10 gene screening in a 3-weeks delay, suitable for clinical use. The description of five new variants may contribute to a better understanding of the phenotype-genotype correlation in FX deficiency.

Enhancing biosecurity against virus disease threats to Australian grain crops: current situation and future prospects

Australia is a major grain exporter, and this trade makes an important contribution to its economy. Fortunately, it remains free of many damaging virus diseases and virus vectors found elsewhere. However, its crop biosecurity is under increasing pressure from global ecological, climatic, and demographic challenges. Stringent biosecurity and plant health programs safeguard Australian grain production from damaging virus and virus vector incursions entering via different pathways. These programs formerly relied upon traditional testing procedures (indicator hosts, serology, PCRs) to intercept incoming virus-contaminated plant material. Recently, the integration of rapid genomic diagnostics innovation involving High Throughput Sequencing (HTS) smart tools into sample testing schedules is under exploration to improve virus testing accuracy, efficiency, and cost effectiveness under diverse circumstances. This process includes evaluating deployment of Illumina and Oxford Nanopore Technology shotgun sequencing. It also includes evaluating targeted viral genome HTS and virus vector metabarcoding approaches. In addition, using machine learning and deep learning capacities for big data analyses and remote sensing technologies will improve virus surveillance. Tracking damaging virus variants will be improved by surveillance networks which combine virus genomic-surveillance systems with an interoperable virus database. Sequencing Australian virus specimen collections will help ensure the accuracy of virus identifications based solely on genetic information. Enhancing routine diagnosis and data collection using these innovations will improve post entry virus interception and background virus and vector surveillance. This will help reduce the frequency of new incursions, improve virus management during eradication, containment and other plant health activities, and achieve more profitable Australian grain production.

High-volume hybridoma sequencing on the NeuroMabSeq platform enables efficient generation of recombinant monoclonal antibodies and scFvs for neuroscience research

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

Integrative omics approaches to advance rare disease diagnostics.

Over the past decade high-throughput DNA sequencing approaches, namely whole exome and whole genome sequencing became a standard procedure in Mendelian disease diagnostics. Implementation of these technologies greatly facilitated diagnostics and shifted the analysis paradigm from variant identification to prioritisation and evaluation. The diagnostic rates vary widely depending on the cohort size, heterogeneity and disease and range from around 30% to 50% leaving the majority of patients undiagnosed. Advances in omics technologies and computational analysis provide an opportunity to increase these unfavourable rates by providing evidence for disease-causing variant validation and prioritisation. This review aims to provide an overview of the current application of several omics technologies including RNA-sequencing, proteomics, metabolomics and DNA-methylation profiling for diagnostics of rare genetic diseases in general and inborn errors of metabolism in particular.

Hemocyte Nuclei Isolation from Adult Drosophila melanogaster for snRNA-seq.

In adult Drosophila, most of the hemocytes are macrophage-like cells (so called plasmatocytes), which serve various functions in organ homeostasis and immune defense. Ontogeny and functions are largely conserved between vertebrate and invertebrate macrophages. Hence, Drosophila offers a powerful genetic toolbox to study macrophage function and genetically modulate these cells. Technological advances in high-throughput sequencing approaches allowed to give an in-depth characterization of vertebrate macrophage populations and their heterogenous composition within different organs as well as changes in disease. Embryonic and larval hemocytes in Drosophila have been recently analyzed in single-cell RNA-sequencing (scRNA-seq) approaches during infection and steady state. These analyses revealed anatomical and functional Drosophila hemocyte subtypes dedicated to specific tasks. Only recently, the Fly Cell Atlas provided a whole transcriptomic single-cell atlas via single-nuclei RNA-sequencing (snRNA-seq) of adult Drosophila including many different tissues and cell types where hemocytes were also included. Yet, a specific protocol to isolate nuclei from adult hemocytes for snRNA-seq and study these cells in different experimental conditions was not available. In this chapter, we give a detailed protocol to purify hemocyte nuclei from adult Drosophila, which can be used in subsequent analyses such as snRNA-seq.

Carrot populations in France and Spain host a complex virome rich in previously uncharacterized viruses.

High-throughput sequencing (HTS) has proven a powerful tool to uncover the virome of cultivated and wild plants and offers the opportunity to study virus movements across the agroecological interface. The carrot model consisting of cultivated (Daucus carota ssp. sativus) and wild carrot (Daucus carota ssp. carota) populations, is particularly interesting with respect to comparisons of virus communities due to the low genetic barrier to virus flow since both population types belong to the same plant species. Using a highly purified double-stranded RNA-based HTS approach, we analyzed on a large scale the virome of 45 carrot populations including cultivated, wild and off-type carrots (carrots growing within the field and likely representing hybrids between cultivated and wild carrots) in France and six additional carrot populations from central Spain. Globally, we identified a very rich virome comprising 45 viruses of which 25 are novel or tentatively novel. Most of the identified novel viruses showed preferential associations with wild carrots, either occurring exclusively in wild populations or infecting only a small proportion of cultivated populations, indicating the role of wild carrots as reservoir of viral diversity. The carrot virome proved particularly rich in viruses involved in complex mutual interdependencies for aphid transmission such as poleroviruses, umbraviruses and associated satellites, which can be the basis for further investigations of synergistic or antagonistic virus-vector-host relationships.

A novel heterozygous ZBTB18 missense mutation in a family with non-syndromic intellectual disability.

Intellectual disability (ID) is a common neurodevelopmental disorder characterized by significantly impaired adaptive behavior and cognitive capacity. High throughput sequencing approaches have revealed the genetic etiologies for 25-50% of ID patients, while inherited genetic mutations were detected in <5% cases. Here, we investigated the genetic cause for non-syndromic ID in a Han Chinese family. Whole genome sequencing was performed on identical twin sisters diagnosed with ID, their respective children, and their asymptomatic parents. Data was filtered for rare variants, and in silico prediction tools were used to establish pathogenic alleles. Candidate mutations were validated by Sanger sequencing. In silico modeling was used to evaluate the mutation's effects on the protein encoded by a candidate coding gene. A novel heterozygous variant in the ZBTB18 gene c.1323C>G (p.His441Gln) was identified. This variant co-segregated with affected individuals in an autosomal dominant pattern and was not detected in asymptomatic family members. Molecular studies reveal that a p.His441Gln substitution disrupts zinc binding within the second zinc finger and disrupts the capacity for ZBTB18 to bind DNA. This is the first report of an inherited ZBTB18 mutation for ID. This study further validates WGS for the accurate molecular diagnosis of ID.

Changes in Bulk and Rhizosphere Soil Microbial Diversity Communities of Native Quinoa Due to the Monocropping in the Peruvian Central Andes.

Quinoa (Chenopodium quinoa) is a highly nutritious crop that is resistant to adverse conditions. Due to the considerable increase in its commercial production in Andean soils, the plant is suffering the negative effects of monocropping, which reduces its yield. We used for the first time a high-throughput Illumina MiSeq sequencing approach to explore the composition, diversity, and functions of fungal and bacterial communities of the bulk and rhizosphere in soils of native C. quinoa affected by monocropping in the central Andes of Peru. The results showed that the bacterial and fungal community structure among the treatments was significantly changed by the monocropping and the types of soil (rhizosphere and bulk). Also, in soils subjected to monocropping, there was an increase in Actinobacteria and a decrease in Proteobacteria, and the reduction in the presence of Ascomycota and the increase in Basidiomycota. By alpha-diversity indices, lower values of bacteria and fungi were observed in the monoculture option compared to the soil not affected by monocropping, and sometimes significant differences were found between both. We detected differentially abundant phytopathogenic fungi and bacteria with growth-stimulating effects on plants. Also, we denoted a decrease in the abundance of the functional predictions in bacteria in the monocropped soils. This research will serve as a starting point to explore the importance and effects of microorganisms in degraded soils and their impact on the growth and quality of quinoa crops.

Mitochondrial DNA is a target of HBV integration

Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular carcinoma (HCC) development remains unclear. In this study, we apply a high-throughput HBV integration sequencing approach that allows sensitive identification of HBV integration sites and enumeration of integration clones. We identify 3339 HBV integration sites in paired tumour and non-tumour tissue samples from 7 patients with HCC. We detect 2107 clonally expanded integrations (1817 in tumour and 290 in non-tumour tissues), and a significant enrichment of clonal HBV integrations in mitochondrial DNA (mtDNA) preferentially occurring in the oxidative phosphorylation genes (OXPHOS) and D-loop region. We also find that HBV RNA sequences are imported into the mitochondria of hepatoma cells with the involvement of polynucleotide phosphorylase (PNPASE), and that HBV RNA might have a role in the process of HBV integration into mtDNA. Our results suggest a potential mechanism by which HBV integration may contribute to HCC development.

The utility of transcriptomics in the conservation of sensitive and economically important species

The utility of transcriptomics in the conservation of sensitive and economically important species

Composition and Distribution Characteristics of Rhizosphere Bacterial Community of Ammodendron bifolium Growing in Takeermohuer Desert Are Different from Those in Non-rhizosphere.

Soil microorganisms play important roles in vegetation establishment and soil biogeochemical cycling. Ammodendron bifolium is a dominant sand-fixing and endangered plant in Takeermohuer Desert, and bacterial community associated with this plant rhizosphere is still unclear. In this study, we studied the composition and diversity of bacterial community from A. bifolium rhizosphere and bulk soil at different soil depths (i.e., 0-40cm, 40-80cm, 80-120cm) using traditional bacterial isolation and high-throughput sequencing approaches, and preliminarily analyzed the edaphic factors influencing the structure of bacterial communities. Results showed that Takeermohuer Desert with high salinity has been an oligotrophic environment, while the rhizosphere exhibited eutrophication resulting from high content SOM (soil organic matter) and SAN (soil alkaline nitrogen) compared with bulk soil. The dominant bacterial groups in the desert were Actinobacteria (39.8%), Proteobacteria (17.4%), Acidobacteria (10.2%), Bacteroidetes (6.3%), Firmicutes (6.3%), Chloroflexi (5.6%), and Planctomycetes (5.0%) at the phyla level. However, the relative abundances of Proteobacteria (20.2%) and Planctomycetes (6.1%) were higher in eutrophic rhizosphere, and Firmicutes (9.8%) and Chloroflexi (6.9%) relatively higher in barren bulk soil. A large number of Actinobacteria were detected in all soil samples, of which the most abundant genus was Streptomyces (5.4%) and Actinomadura (8.2%) in the bulk soil and rhizosphere, respectively. The Chao1 and PD indexes in rhizosphere were significantly higher than those in bulk soil at the same soil depth, and tended to decrease with increasing soil depth. Co-occurrence network analyses showed that the keystone species in Takeermohuer Desert were Actinobacteria, Acidobacteria, Proteobacteria, and Chlorofexi. Furthermore, the major environmental factors affecting rhizosphere bacterial community were EC (electrical conductivity), SOM, STN (soil total nitrogen), SAN, and SAK (soil available potassium), while bulk soil were distance and C/N (STC/STN). We concluded that A. bifolium rhizosphere bacterial community is different from non-rhizosphere in composition, distribution, and environmental influencing factors, which will have important significances for understanding their ecological functions and maintaining biodiversity.

The peripheral TCR repertoire diversity indexes can be used as an independent indicator for long term survival rate of ovarian cancer patients.

e17558 Background: According to GLOBOCAN 2020 data, ovarian cancer (OC) ranks 19th in incidence and 15th in mortality with a rate of 1.6% and 2.1%, respectively, High-grade serous OC is the most common sub-type of malignant ovarian tumors, remains the leading cause of cancer-related death among all gynecological cancers in the developed parts of the world. In the United States in 2016, 5-year survival rate was only 10-25% in stage III or IV OC patients, and there was considerable variability in survival in an advanced stage. At present, there is no reliable molecular marker to identify patients with better or worse prognosis. As is known to all, the adaptive immune system defends the body against different foreign antigens, using two main types of antigen receptors: T-cell receptors (TCRs) and antibodies. Now we use high-throughput sequencing approach to analyze the expressed TCRβ repertoires in the peripheral blood of OC patients and healthy women donors. Methods: The experimental design and participant recruitment were approved by Medical Ethics Committee of West China Second University Hospital (Ethics Approval for Research: 2022). Total RNA was extracted from PBMC using TRIZOL reagent. High through put sequencing of the TCRβ gene, assignment of the Vβ, Dβ, and Jβ germline gene, and TCR diversity indexes analyses were conducted essentially following our previously published protocols. Statistical analysis and graph drawing were performed using R language (R 4.0.3). Results: Comparison of the TCR repertoires between the 232 OC patients (OC group) and the 250 healthy women donors (HD group) revealed significant differences. For examples, the VJ combinations of the OC group were distinct from those of the HD group. In addition, the TCR repertoire diversity indexes of the OC group were significantly lower than those of the HD group ( p &lt; 0.001), including D50s, Simpson indexes, the number of highly expressed clones (HECs), and the frequencies of the largest TCR clones. As reported before, the TCR repertoire diversity indexes decreased gradually with increasing age in the HD group (all p &lt; 0.001). Moreover, the TCR repertoire diversities in the survival group were significantly higher than those in the death group (D50: p = 0.0342; the number of HECs: p = 0.0414). Using as the cut off value, the OC patients with D50 &lt; 0.032 had significantly higher mortality rates compared to the OC patients with D50 &gt; 0.032 ( p = 0.0019). Similarly, using the numbers of HECs or the frequencies of the largest TCR clones can also distinguish OC patients in long term survival. Conclusions: Analyzing the peripheral TCR repertoires in the OC and HD revealed significant differences, and the peripheral TCR repertoire diversity indexes may be used as a potential predictor for long term survival for ovarian cancer.

Single-cell detection of primary transcripts, their genomic loci and nuclear factors by 3D immuno-RNA/DNA FISH in T cells.

Over the past decades, it has become increasingly clear that higher order chromatin folding and organization within the nucleus is involved in the regulation of genome activity and serves as an additional epigenetic mechanism that modulates cellular functions and gene expression programs in diverse biological processes. In particular, dynamic allelic interactions and nuclear locations can be of functional importance during the process of lymphoid differentiation and the regulation of immune responses. Analyses of the proximity between chromatin and/or nuclear regions can be performed on populations of cells with high-throughput sequencing approaches such as chromatin conformation capture ("3C"-based) or DNA adenine methyltransferase identification (DamID) methods, or, in individual cells, by the simultaneous visualization of genomic loci, their primary transcripts and nuclear compartments within the 3-dimensional nuclear space using Fluorescence In Situ Hybridization (FISH) and immunostaining. Here, we present a detailed protocol to simultaneously detect nascent RNA transcripts (3D RNA FISH), their genomic loci (3D DNA FISH) and/or their chromosome territories (CT paint DNA FISH) combined with the antibody-based detection of various nuclear factors (immunofluorescence). We delineate the application and effectiveness of this robust and reproducible protocol in several murine T lymphocyte subtypes (from differentiating thymic T cells, to activated splenic and peripheral T cells) as well as other murine cells, including embryonic stem cells, B cells, megakaryocytes and macrophages.