High-throughput Sequencing Approach Research Articles

DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide.

Studies of genome regulation routinely use high-throughput DNA sequencing approaches to determine where specific proteins interact with DNA, and they rely on DNA amplification and short-read sequencing, limiting their quantitative application in complex genomic regions. To address these limitations, we developed Directed Methylation with Long-read sequencing (DiMeLo-seq), which uses antibody-tethered enzymes to methylate DNA near a target protein’s binding sites in situ. These exogenous methylation marks are then detected simultaneously with endogenous CpG methylation on unamplified DNA using long-read, single-molecule sequencing technologies. We optimized and benchmarked DiMeLo-seq by mapping chromatin-binding proteins and histone modifications across the human genome. Furthermore, we identified where centromere protein A (CENP-A) localizes within highly repetitive regions that we re unmappable with short sequencing reads, and we estimated the density of CENP-A molecules along single chromatin fibers. DiMeLo-seq is a versatile method that provides multimodal, genome-wide information for investigating protein-DNA interactions.

Temporal dynamics of Campylobacter and Arcobacter in a freshwater lake that receives fecal inputs from migratory geese

Migratory geese could influence the microbiological water quality; however, their impacts on pathogen dynamics remain largely unknown. In this study, we analyzed the population dynamics of Campylobacter and Arcobacter group bacteria (AGB) in a freshwater lake in Japan over two years. The bacteria were quantified by using both culture-dependent and -independent methods. The potential sources of these bacteria were examined by a high-throughput flaA sequencing approach. Campylobacter was abundantly detected both by culture-dependent and -independent methods in the lake, especially when migratory geese were present in the lake. High-throughput flaA sequencing suggests that geese were the likely source of Campylobacter in the lake. The viable population of Campylobacter exceeds the concentrations that can potentially cause 10−4 infections per person per year when water is used to grow fresh vegetables. The occurrence of AGB, on the other hand, was not directly related to the population of migratory geese. AGB were not detected in geese fecal samples. Diverse AGB flaA genotypes occurred in the lake over multiple seasons. Our results suggest that AGB likely comprise a part of the indigenous microbial population of the lake and grow in response to high nutrient, warm temperature, and low dissolved oxygen concentrations in the lake. Geese therefore can indirectly impact the AGB population by providing nutrients to cause eutrophication and lower the dissolved oxygen concentration. Since geese travel long-distance and disperse their fecal microbiota and nutrients to wide areas, they may have significant impacts on water quality and public health.

Characteristics of intestinal microbiota in male morphotypes of the giant freshwater prawn Macrobrachium rosenbergii

There is a close correlation between intestinal microbiota and development. However, our understanding of the relationship between the intestinal microbial community and phenotypic traits in giant freshwater prawn (GFP), Macrobrachium rosenbergii, remains limited. Here, we explored this association between the gut bacterial community composition and phenotypic traits of male morphotypes of the GFP by using culture-dependent and high-throughput sequencing approaches. The results showed that Lactococcus garvieae was the dominant bacterial species, and accounted for 83.3%, 82.7%, 65.5%, and 40.8% of the total bacteria in the orange claw (OC), small males (SM), blue claw (BC), and old blue claw (OBC) groups, respectively. The relative abundance of the intestinal bacterial communities in the OBC and SM groups differed significantly from that in the OC and BC groups. We also observed statistically significant associations between gut microbiota and phenotypic traits. Moreover, the results obtained by using the conventional culture method further confirmed that L. garvieae was significantly predominant in all groups and more abundant in the OC and BC groups, suggesting an important role of L. garvieae in GFP development. This finding strongly suggests that the intestinal microbiota of GFP is tightly correlated with the morphotypic differentiation likely owing to its effects on nutrient metabolism in the gut.

Effects of Three Pesticides on the Earthworm Lumbricus terrestris Gut Microbiota.

Earthworms play a vital role in the terrestrial ecosystem functioning and maintenance of soil fertility. However, many pesticides, for example, imidacloprid, benomyl, and metribuzin that are world-widely used in agriculture, may be potentially dangerous to earthworms. At the same time, standard tests for pesticides acute and chronic toxicity do not reflect all aspects of their negative impact and might not be enough sensitive for effective assessment. In this paper, we studied the effects of non-lethal concentrations of imidacloprid, benomyl, and metribuzin on the gut bacterial community of Lumbricus terrestris using high-throughput sequencing approach. We found that pesticides reduced the total bacterial diversity in the earthworm’s gut even at the recommended application rate. Under the applied pesticides, the structure of the gut prokaryotic community underwent changes in the relative abundance of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Planctomyces, Verrucomicrobia, and Cyanobacteria, as well as the genera Haliangium, Gaiella, Paenisporosarcina, Oryzihumus, Candidatus Udaeobacter, and Aquisphaera. Moreover, the pesticides affected the abundance of Verminephrobacter—the earthworms’ nephridia specific symbionts. In general, the negative impact of pesticides on bacterial biodiversity was significant even under pesticides content, which was much lower than their acute and chronic toxicity values for the earthworms. These results highlighted the fact that the earthworm’s gut microbial community is highly sensitive to soil contamination with pesticides. Therefore, such examination should be considered in the pesticide risk assessment protocols.

Archaea is more important than bacteria in driving soil stoichiometry in phosphorus deficient habitats

Phosphorus deficiency is a critical limit on the cycling of carbon (C), nitrogen (N) and phosphorus (P) in forest ecosystems. Despite the pivotal roles of microbes in driving the biogeochemical cycling of C/N/P, our knowledge on the relationships of soil bacteria and archaea to P deficiency in forest ecosystems remains scarce. Here, we studied 110 acidic soils (average pH 4.5) collected across 700-km subtropical forests with a gradient of available phosphorus (AP) ranging from 0.21 to 17.6 mg/kg. We analyzed the soil C/N/P stoichiometry and studied soil bacterial and archaeal diversity/abundance via high throughput sequencing and qPCR approaches. Our results show that soil P decoupled with N or C when below 3 mg/kg but coupled with C and N when above 3 mg/kg. Archaeal diversity and abundance were significantly higher in low AP (< 3 mg/kg) soils than in high AP (>3 mg/kg) soils, while bacterial were less changed. Compared with bacteria, archaea are more strongly related with soil stoichiometry (C:N, C:P, N:P), especially when AP was less than 3 mg/kg. Taxonomic and functional composition analysis further confirmed that archaeal rather than bacterial taxonomic composition was significantly related with functional composition of microbial communities. Taken together, our results show that archaea are more important than bacteria in driving soil stoichiometry in phosphorus deficient habitats and suggest a niche differentiation of soil bacteria and archaea in regulating the soil C/N/P cycling in subtropical forests.

DNA Metabarcoding Enables High-Throughput Detection of Spotted Wing Drosophila (Drosophila suzukii) Within Unsorted Trap Catches

The spotted wing drosophila (Drosophila suzukii, Matsumara) is a rapidly spreading global pest of soft and stone fruit production. Due to the similarity of many of its life stages to other cosmopolitan drosophilids, surveillance for this pest is currently bottlenecked by the laborious sorting and morphological identification of large mixed trap catches. DNA metabarcoding presents an alternative high-throughput sequencing (HTS) approach for multi-species identification, which may lend itself ideally to rapid and scalable diagnostics of D. suzukii within unsorted trap samples. In this study, we compared the qualitative (identification accuracy) and quantitative (bias toward each species) performance of four metabarcoding primer pairs on D. suzukii and its close relatives. We then determined the sensitivity of a non-destructive metabarcoding assay (i.e., which retains intact specimens) by spiking whole specimens of target species into mock communities of increasing specimen number, as well as 29 field-sampled communities from a cherry and a stone fruit orchard. Metabarcoding successfully detected D. suzukii and its close relatives Drosophila subpulchrella and Drosophila biarmipes in the spiked communities with an accuracy of 96, 100, and 100% respectively, and identified a further 57 non-target arthropods collected as bycatch by D. suzukii surveillance methods in a field scenario. While the non-destructive DNA extraction retained intact voucher specimens, dropouts of single species and entire technical replicates suggests that these protocols behave more similarly to environmental DNA than homogenized tissue metabarcoding and may require increased technical replication to reliably detect low-abundance taxa. Adoption of high-throughput metabarcoding assays for screening bulk trap samples could enable a substantial increase in the geographic scale and intensity of D. suzukii surveillance, and thus likelihood of detecting a new introduction. Trap designs and surveillance protocols will, however, need to be optimized to adequately preserve specimen DNA for molecular identification.

Structural shifts in sea ice prokaryotic communities across a salinity gradient in the subarctic

Current knowledge of the processes that shape prokaryotic community assembly in sea ice across polar ecosystems is scarce. Here, we coupled culture-dependent (bacterial isolation on R2A medium) and culture-independent (high-throughput 16S rRNA gene sequencing) approaches to provide the first comprehensive assessment of prokaryotic communities in the late winter ice and its underlying water along a natural salinity gradient in coastal Hudson Bay, an iconic cryo-environment that marks the ecological transition between Canadian Subarctic and Arctic biomes. We found that prokaryotic community assembly processes in the ice were less selective at low salinity since typical freshwater taxa such as Frankiales, Burkholderiales, and Chitinophagales dominated both the ice and its underlying water. In contrast, there were sharp shifts in community structure between the ice and underlying water samples at sites with higher salinity, with the orders Alteromonadales and Flavobacteriales dominating the ice, while the abovementioned freshwater taxa dominated the underlying water communities. Moreover, primary producers including Cyanobium (Cyanobacteria, Synechococcales) may play a role in shaping the ice communities and were accompanied by known Planctomycetes and Verrucomicrobiae taxa. Culture-dependent analyses showed that the ice contained pigment-producing psychrotolerant or psychrophilic bacteria from the phyla Proteobacteria, Actinobacteriota, and Bacteroidota, likely favored by the combination of low temperatures and the seasonal increase in sunlight. Our findings suggest that salinity, photosynthesis and dissolved organic matter are the main drivers of prokaryotic community structure in the late winter ice of coastal Hudson Bay, the ecosystem with the fastest sea ice loss rate in the Canadian North.

A new high-throughput HBV integration sequencing approach shows that mitochondrial DNA is frequently targeted by virus integration in liver cells with active HBV replication

Background: Although research on HBV DNA integration has made relevant progress, important aspects remain unclear. Development of new integration detection methods may help in improving knowledge in the field and in HBV-related tumorigenesis.

Microbial communities of ascocarps and soils in a natural habitat of Tuber indicum.

Truffles are the fruiting bodies of hypogeous fungi in the genus Tuber. Some truffle species usually grow in an area devoid of vegetation, called brûlé, but limited knowledge is available on the microbial composition and structure of them. Here, we investigated the bacterial and fungal communities of Tuber indicum ascocarps and soils inside and outside a characteristic brûlé from a poplar plantation with no truffle production history in northeastern China using a high-throughput sequencing approach. A predominance of members of the bacterial phylum Proteobacteria was observed in all samples. Members of Bacillus were the main genera in the ascocarps, while members of Lysobacter and unidentified Acidobacteria were more abundant in the soil. In addition, members of Gibberella, Fusarium, and Absidia were the dominant fungi in the ascocarps, while members of Tuber were enriched in the ascocarps and soils inside the brûlé. Some mycorrhization helper bacteria (Rhizobium) and ectomycorrhiza-associated bacteria (Lysobacter) were detected, indicating their potential roles in the complex development of underground fruiting bodies and brûlé formation. These findings may contribute to the protection and cultivation of truffles.

Downregulation of Three Novel miRNAs in the Lymph Nodes of Sheep Immunized With the Brucella suis Strain 2 Vaccine.

Ovine and caprine brucellosis, both caused by Brucella melitensis, lead to substantial economic losses in the animal industry and health problems in human populations. Brucella suis strain 2 (B.suis S2), as a live attenuated vaccine, is used extensively in China to prevent brucellosis. It has been proven that microRNA (miRNAs) are involved in the immunopathogenesis of brucellosis; however, the miRNA-driven mechanism of immune response to B.suis S2 in vivo remains unknown. To determine which new miRNAs are involved in the host immune response to B.suis S2 and elucidate the function of these miRNAs, we performed a comprehensive analysis of miRNA expression profiles in sheep immunized with B.suis S2 using the high-throughput sequencing approach. The submandibular lymphatic nodes from sheep seropositive for Brucella were collected at 7, 14, 21, 30, 60 and 90 days post-immunization. MiRNA sequencing analysis revealed that 282 differentially expressed miRNAs (|log2 fold-change |>0.5 and p < 0.05) were significantly enriched in the immune pathways, including the NF-kappa B signaling pathway, B cell receptor signaling pathway, p53 signaling pathway and complement and coagulation cascades. Increasing the threshold to |log2 fold change|>1 and p < 0.01 revealed 48 differentially expressed miRNAs, 31 of which were novel miRNAs. Thirteen of these novel miRNAs, which were differentially expressed for at least two time points, were detected via RT-qPCR assays. The novel_229, novel_609, novel_973 and oar-miR-181a assessed by RT-qPCR were detectable and consistent with the expression patterns obtained by miRNA sequencing. Functional analyses of these miRNAs demonstrated that their target genes participated in the immune response pathways, including the innate and adaptive immunity pathways. The immune-related target genes of novel_229 included ENSOARG00000000649 and TMED1, as well as LCN2, PDPK1 and LPO were novel_609 target genes. The immune-related target genes of novel_973 included C6orf58, SPPL3, BPIFB1, ENSOARG00000021083, MPTX1, CCL28, FGB, IDO1, OLR1 and ENSOARG00000020393. The immune-related target genes of oar-miR-181a included ENSOARG00000002722, ARHGEF2, MFAP4 and DOK2. These results will deepen our understanding of the host miRNA-driven defense mechanism in sheep immunized with B.suis S2 vaccine, and provide the valuable information for optimizing vaccines and developing molecular diagnostic targets.

Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding

Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepare a mock eukaryotic community from the marine environment, equal amounts of plasmid DNA from 40 microalgal species were mixed and used for amplicon-sequencing by a high-throughput sequencing approach. To compare the differences in PCR kits used for this experiment, we focused on the following seven parameters: 1) Quality, 2) Chimera, 3) Blast top hit accuracy, 4) Deletion, 5) Insertion, 6) Base substitution and 7) Amplification bias amongst species. The results showed statistically significant differences (p &amp;lt; 0.05) for all of the seven parameters depending on the PCR kits used. These differences may result from the different DNA polymerases included in each kit, although the result can also be influenced by PCR reaction conditions. Simultaneous analysis of several parameters suggested that kits containing KOD plus Neo (TOYOBO) and HotStart Taq DNA polymerase (BiONEER, CA, US) at the annealing temperature of 65 °C displayed better results in terms of parameters associated with chimeras, top hit similarity and deletions.

The swine enteric virome in a commercial production system and its association with neonatal diarrhea

Swine are an important food source worldwide and producers may not always be aware of the variety of the pathogens infecting their herds, particularly viruses. In this study, 12 enteric viruses were monitored in a total of 181 diarrheic and healthy piglets; namely porcine astrovirus groups 1–5 (poAstV1−5), rotavirus A and C (RVA-RVC), caliciviruses (CaVs), porcine kobuvirus (PoK), hepatitis E virus (HEV), and torque teno sus virus 1 and k2 (TTsuV1-k2). All animals were sampled before 3 weeks of age, and then at 5, 12 and 20 weeks of age. In addition to the 12 targeted viruses, the virome of 12 piglets at 4 different life stages was characterized using a high-throughput sequencing approach. The presence of CaV (sapovirus), poAstV-3 or poAstV-5 was found to be a risk factor for neonatal diarrhea. Co-infections with RVC and poAstV-2, poAstV-3, and poAstV-4 and CaV co-infected with PoK or poAstV-4 were also found to be risk factors for diarrhea in piglets. RVC, PoK, poAstV-3 and poAstV-4 were the most prevalent viruses in piglets below 3 weeks of age. PoAstV-2, poAstV-4, TTsuV1 and TTsuVk2 were found to be the most prevalent viruses infecting piglets of 20 weeks of age. The enteric virome composition varied between healthy and diarrheic piglets. The alpha and beta diversity of the enteric viromes varied from under 3 weeks of age to 20 weeks and was mainly supported by phages. Overall, this study sheds new light on enteric virome dynamics and the virome’s relationship with neonatal diarrhea.

A novel victorivirus from the phytopathogenic fungus Neofusicoccum parvum.

Neofusicoccum parvum is an important plant-pathogenic ascomycetous fungus that causes trunk diseases in a variety of plants. A limited number of reports on mycoviruses from this fungus are available. Here, we report the characterization of a novel victorivirus, Neofusicoccum parvum victorivirus 3 (NpVV3). An agarose gel dsRNA profile of a Pakistani strain of N. parvum, NFN, showed a band of ~5 kbp that was not detectable in Japanese strains of N. parvum. Taking a high-throughput and Sanger sequencing approach, the complete genome sequence of NpVV3 was determined to be 5226 bp in length with two open reading frames (ORF1 and ORF2) that encode a capsid protein (CP) and an RNA-dependent RNA polymerase (RdRP). The RdRP appears to be translated by a stop/restart mechanism facilitated by the junction sequence AUGucUGA, as is found in some other victoriviruses. BLASTp searches showed that NpVV3 CP and RdRP share the highest amino acid sequence identity (80.5% and 72.4%, respectively) with the corresponding proteins of NpVV1 isolated from a French strain of N. parvum. However, NpVV3 was found to be different from NpVV1 in its terminal sequences and the stop/restart facilitator sequence. NpVV3 particles ~35 nm in diameter were partially purified and used to infect an antiviral-RNA-silencing-deficient strain (∆dcl2) of an experimental ascomycetous fungal host, Cryphonectria parasitica. NpVV3 showed symptomless infection in the new host strain.

Distinct Phyllosphere Microbiome of Wild Tomato Species in Central Peru upon Dysbiosis

Plants are colonized by myriads of microbes across kingdoms, which affect host development, fitness, and reproduction. Hence, plant microbiomes have been explored across a broad range of host species, including model organisms, crops, and trees under controlled and natural conditions. Tomato is one of the world’s most important vegetable crops; however, little is known about the microbiota of wild tomato species. To obtain insights into the tomato microbiota occurring in natural environments, we sampled epiphytic microbes from leaves of four tomato species, Solanum habrochaites, S. corneliomulleri, S. peruvianum, and S. pimpinellifolium, from two geographical locations within the Lima region of Peru over 2 consecutive years. Here, a high-throughput sequencing approach was applied to investigate microbial compositions including bacteria, fungi, and eukaryotes across tomato species and geographical locations. The phyllosphere microbiome composition varies between hosts and location. Yet, we identified persistent microbes across tomato species that form the tomato microbial core community. In addition, we phenotypically defined healthy and dysbiotic samples and performed a downstream analysis to reveal the impact on microbial community structures. To do so, we compared microbial diversities, unique OTUs, relative abundances of core taxa, and microbial hub taxa, as well as co-occurrence network characteristics in healthy and dysbiotic tomato leaves and found that dysbiosis affects the phyllosphere microbial composition in a host species-dependent manner. Yet, overall, the present data suggests an enrichment of plant-promoting microbial taxa in healthy leaves, whereas numerous microbial taxa containing plant pathogens occurred in dysbiotic leaves.Concluding, we identify the core phyllosphere microbiome of wild tomato species, and show that the overall phyllosphere microbiome can be impacted by sampling time point, geographical location, host genotype, and plant health. Future studies in these components will help understand the microbial contribution to plant health in natural systems and can be of use in cultivated tomatoes.

The influence of benzoyl peroxide on skin microbiota and the epidermal barrier for acne vulgaris.

The disordered skin microbiome has been reported to contribute to the pathogenesis of acne vulgaris, for which benzoyl peroxide (BPO) has long been recommended as the first-line therapy. However, there are no data regarding the effect of BPO treatment on skin microbiota and the epidermal barrier in young adults with acne vulgaris. Thirty-three patients with acne vulgaris and 19 healthy controls were enrolled in the study. All patients received topical treatment with BPO 5% gel for 12 weeks. The epidermal barrier was analyzed at baseline and after treatment. Microbial diversity was analyzed using a high-throughput sequencing approach targeting the V3-V4 region of 16S rRNA genes. After receiving treatment with BPO, patients had significant improvement in their Global Acne Grading System (GAGS) score, porphyrin, and red areas (p < 0.05), and the presence of sebum, stratum corneum hydration (SCH), and transepidermal water loss (TEWL) increased (p < 0.05). When compared with baseline, microbial diversity was significantly reduced after treatment, as calculated by the goods coverage (p=0.0017), Shannon (p=0.0094), and Simpson (p=0.0017) diversity indices. The prevalence of the genus Cutibacterium (before treatment: 5.64 [3.50, 7.78] vs. after treatment: 2.43 [1.81, 3.05], p=0.011) was significantly reduced after treatment while Staphylococcus (before treatment: 43.80 [36.62, 50.98] vs. after treatment: 53.38 [44.88, 61.87], p=0.075) tended to increase. The abundance of Staphylococcus was negatively associated with SCH (p=0.008, r=-0.286). Despite its contribution to an improved GAGS score, BPO treatment for acne vulgaris may reduce microbial diversity and damage the epidermal barrier.

Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation.

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

A high-throughput amplicon sequencing approach for population-wide species diversity and composition survey.

Management of agricultural pests requires an understanding of pest species diversity, their interactions with beneficial insects and spatial-temporal patterns of pest abundance. Invasive and agriculturally important insect pests can build up very high populations, especially in cropping landscapes. Traditionally, sampling effort for species identification involves small sample sizes and is labour intensive. Here, we describe a multiprimer high throughput sequencing (HTS) metabarcoding method and associated analytical workflow for a rapid, intensive, high-volume survey of pest species compositions. We demonstrate our method using the taxonomically challenging Bemisia pest cryptic species complex as examples. The whiteflies Bemisia including the"tabaci" species are agriculturally important capable of vectoring diverse plant viruses that cause diseases and crop losses. Our multiprimer metabarcoding HTS amplicon approach simultaneously process high volumes of whitefly individuals, with efficiency to detect rare (i.e., 1%) test-species, while our improved whitefly primers for metabarcoding also detected beneficial hymenopteran parasitoid species from whitefly nymphs. Field-testing our redesigned Bemisia metabarcoding primer sets across the Tanzania, Uganda and Malawi cassava cultivation landscapes, we identified the sub-Saharan Africa 1 Bemisia putative species as the dominant pest species, with other cryptic Bemisia species being detected at various abundances. We also provide evidence that Bemisia species compositions can be affected by host crops and sampling techniques that target either nymphs or adults. Our multiprimer HTS metabarcoding method incorporated two overlapping amplicons of 472bp and 518bp that spanned the entire 657bp 3' barcoding region for Bemisia, and is particularly suitable to molecular diagnostic surveys of this highly cryptic insect pest species complex that also typically exhibited high population densities in heavy crop infestation episodes. Our approach can be adopted to understand species biodiversity across landscapes, with broad implications for improving transboundary biosecurity preparedness, thus contributing to molecular ecological knowledge and the development of control strategies for high-density, cryptic, pest-species complexes.

Chimerism-Based Tolerance to Kidney Allografts in Humans: Novel Insights and Future Perspectives

Chronic rejection and immunosuppression-related toxicity severely affect long-term outcomes of kidney transplantation. The induction of transplantation tolerance – the lack of destructive immune responses to a transplanted organ in the absence of immunosuppression – could potentially overcome these limitations. Immune tolerance to kidney allografts from living donors has been successfully achieved in humans through clinical protocols based on chimerism induction with hematopoietic cell transplantation after non-myeloablative conditioning. Notably, two of these protocols have led to immune tolerance in a significant fraction of HLA-mismatched donor-recipient combinations, which represent the large majority of cases in clinical practice. Studies in mice and large animals have been critical in dissecting tolerance mechanisms and in selecting the most promising approaches for human translation. However, there are several key differences in tolerance induction between these models and humans, including the rate of success and stability of donor chimerism, as well as the relative contribution of different mechanisms in inducing donor-specific unresponsiveness. Kidney allograft tolerance achieved through durable full-donor chimerism may be due to central deletion of graft-reactive donor T cells, even though mechanistic data from patient series are lacking. On the other hand, immune tolerance attained with transient mixed chimerism-based protocols initially relies on Treg-mediated suppression, followed by peripheral deletion of donor-reactive recipient T-cell clones under antigenic pressure from the graft. These conclusions were supported by data deriving from novel high-throughput T-cell receptor sequencing approaches that allowed tracking of alloreactive repertoires over time. In this review, we summarize the most important mechanistic studies on tolerance induction with combined kidney-bone marrow transplantation in humans, discussing open issues that still need to be addressed and focusing on techniques developed in recent years to efficiently monitor the alloresponse in tolerance trials. These cutting-edge methods will be instrumental for the development of immune tolerance protocols with improved efficacy and to identify patients amenable to safe immunosuppression withdrawal.

A multispecies amplicon sequencing approach for genetic diversity assessments in grassland plant species.

Grasslands are widespread and economically relevant ecosystems at the basis of sustainable roughage production. Plant genetic diversity (PGD; i.e., within‐species diversity) is related to many beneficial effects on the ecosystem functioning of grasslands. The monitoring of PGD in temperate grasslands is complicated by the multiplicity of species present and by a shortage of methods for large‐scale assessments. However, the continuous advancement of high‐throughput DNA sequencing approaches has improved the prospects of broad, multispecies PGD monitoring. Among them, amplicon sequencing stands out as a robust and cost‐effective method. Here, we report a set of 12 multispecies primer pairs that can be used for high‐throughput PGD assessments in multiple grassland plant species. The target loci were selected and tested in two phases: a “discovery phase” based on a sequence capture assay (611 nuclear loci assessed in 16 grassland plant species), which resulted in the selection of 11 loci; and a “validation phase”, in which the selected loci were targeted and sequenced using multispecies primers in test populations of Dactylis glomerata L., Lolium perenne L., Festuca pratensis Huds., Trifolium pratense L. and T. repens L. The multispecies amplicons had nucleotide diversities per species from 5.19 × 10−3 to 1.29 × 10−2, which is in the range of flowering‐related genes but slightly lower than pathogen resistance genes. We conclude that the methodology, the DNA sequence resources, and the primer pairs reported in this study provide the basis for large‐scale, multispecies PGD monitoring in grassland plants.

Community-Based 16S rDNA Fingerprinting Analysis of Geographically Distinct Marine Sediments of Unexplored Coastal Regions of Palk Bay and Gulf of Mannar.

The present study aims to carefully delineate the bacterial community composition in marine sediments from different geographical coastal regions of Palk Bay and Gulf of Mannar that are known for human recreational activities. Bacterial richness in different marine sediments was assessed using 16S rRNA gene-based Denaturing Gradient Gel Electrophoresis (DGGE) which is a widely deployed fingerprinting technique. The DGGE profiles revealed that the bacterial community profiles of sediment from different coastal regions were complex and dynamic. The most dominant phylum present in the marine sediment samples were Proteobacteria followed by Cyanobacteria, Bacteriodetes, Firmicutes, Acidobacteria, and Actinobacteria. Cosmopolitan presence of Thioalkalivibrio sp. was observed in all the marine sediments. Sequencing of the abundant band reveals the presence of Vibrio spp. in all the marine sediments. Comparative illumina data analysis revealed the presence of 51 different Vibrio species in which Vibrio alginolyticus holds the highest abundance (67.2%) followed by V. harveyi (13.5%). This is the one of the very few reports that compared the complex microbial community composition of the marine sediments of different geographical regions of unexplored coastal region. Further in-depth analysis needs to be taken to understand the presence of complex microbial compositions and their functions through high-throughput whole metagenome sequencing and metaproteomic approaches.