High-throughput Sequencing Approach Research Articles

First Polycipivirus and Unmapped RNA Virus Diversity in the Yellow Crazy Ant, Anoplolepis gracilipes.

The yellow crazy ant, Anoplolepis gracilipes is a widespread invasive ant that poses significant threats to local biodiversity. Yet, compared to other global invasive ant species such as the red imported fire ant (Solenopsis invicta) or the Argentine ant (Linepithema humile), little is known about the diversity of RNA viruses in the yellow crazy ant. In the current study, we generated a transcriptomic database for A. gracilipes using a high throughput sequencing approach to identify new RNA viruses and characterize their genomes. Four virus species assigned to Dicistroviridae, two to Iflaviridae, one to Polycipiviridae, and two unclassified Riboviria viruses were identified. Detailed genomic characterization was carried out on the polycipivirus and revealed that this virus comprises 11,644 nucleotides with six open reading frames. Phylogenetic analysis and pairwise amino acid identity comparison classified this virus into the genus Sopolycivirus under Polycipiviridae, which is tentatively named “Anoplolepis gracilipes virus 3 (AgrV-3)”. Evolutionary analysis showed that AgrV-3 possesses a high level of genetic diversity and elevated mutation rate, combined with the common presence of multiple viral strains within single worker individuals, suggesting AgrV-3 likely evolves following the quasispecies model. A subsequent field survey placed the viral pathogen “hotspot” of A. gracilipes in the Southeast Asian region, a pattern consistent with the region being recognized as part of the ant’s native range. Lastly, infection of multiple virus species seems prevalent across field colonies and may have been linked to the ant’s social organization.

Identification of Circular RNAs in the Ovaries of Hu Sheep and Local Fat-tailed Sheep during the Luteal Phase

Background: Circular RNAs (circRNAs), a novel class of endogenous non-coding RNAs with covalently closed loop structures, play important role in mammalian growth and development by regulating gene expression and participating in various biological processes. The aim of this study was to identify the expression profiles of circRNAs in the ovaries of Hu sheep and local Fat-tailed sheep during the luteal phase to determine the molecular mechanisms underlying the effects of circRNAs on reproduction and follicular development in sheep. Methods: To identify the differentially expressed circRNAs expressed in luteal phase conditions in Hu sheep and fat-tailed sheep, ovarian samples collected during the luteal phase were analyzed using high-throughput sequencing and subsequent transcriptomic approaches and bioinformatics analysis. Result: A total of 2,875 differentially expressed circRNAs were identified by high-through sequencing. Gene Ontology terminology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the host genes were mainly involved in the secretion and metabolism of the various pathway. Quantitative real-time PCR was performed to validate the results of expression profiling. These results contributed to an improved understanding of circRNA and the biological mechanisms of ovarian reproduction in sheep.

Collective and harmonized high throughput barcoding of insular arthropod biodiversity: Toward a Genomic Observatories Network for islands.

Current understanding of ecological and evolutionary processes underlying island biodiversity is heavily shaped by empirical data from plants and birds, although arthropods comprise the overwhelming majority of known animal species, and as such can provide key insights into processes governing biodiversity. Novel high throughput sequencing (HTS) approaches are now emerging as powerful tools to overcome limitations in the availability of arthropod biodiversity data, and hence provide insights into these processes. Here, we explored how these tools might be most effectively exploited for comprehensive and comparable inventory and monitoring of insular arthropod biodiversity. We first reviewed the strengths, limitations and potential synergies among existing approaches of high throughput barcode sequencing. We considered how this could be complemented with deep learning approaches applied to image analysis to study arthropod biodiversity. We then explored how these approaches could be implemented within the framework of an island Genomic Observatories Network (iGON) for the advancement of fundamental and applied understanding of island biodiversity. To this end, we identified seven island biology themes at the interface of ecology, evolution and conservation biology, within which collective and harmonized efforts in HTS arthropod inventory could yield significant advances in island biodiversity research.

Microbiota Succession of Whole and Filleted European Sea Bass (Dicentrarchus labrax) during Storage under Aerobic and MAP Conditions via 16S rRNA Gene High-Throughput Sequencing Approach.

In the present work, the profiles of bacterial communities of whole and filleted European sea bass (Dicentrarchus labrax), during several storage temperatures (0, 4, 8 and 12 °C) under aerobic and Modified Atmosphere Packaging (MAP) conditions, were examined via the 16S rRNA High-Throughput Sequencing (HTS) approach. Sensorial attributes were also assessed to determine products’ shelf-life. Results indicated that shelf-life was strongly dependent on handling, as well as on temperature and atmosphere conditions. HTS revealed the undisputed dominance of Pseudomonas from the very beginning and throughout storage period in the majority of treatments. However, a slightly different microbiota profile was recorded in MAP-stored fillets at the middle stages of storage, which mainly referred to the sporadic appearance of some bacteria (e.g., Carnobacterium, Shewanella, etc.) that followed the dominance of Pseudomonas. It is noticeable that a major difference was observed at the end of shelf-life of MAP-stored fillets at 12 °C, where the dominant microbiota was constituted by the genus Serratia, while the relative abundance of Pseudomonas and Brochothrix was more limited. Furthermore, at the same temperature under aerobic storage of both whole and filleted fish, Pseudomonas almost co-existed with Acinetobacter, while the presence of both Erwinia and Serratia in whole fish was noteworthy. Overall, the present study provides useful information regarding the storage fate and spoilage status of whole and filleted European sea bass, suggesting that different handling and storage conditions influence the shelf-life of sea bass by favoring or delaying the dominance of Specific Spoilage Organisms (SSOs), affecting in parallel to some extent the formation of their consortium that is responsible for products’ sensorial deterioration. Such findings enrich the current knowledge and should be used as a benchmark to develop specific strategies aiming to delay spoilage and thus increase the products’ added value.

The O-glycosylating enzyme GALNT2 acts as an oncogenic driver in non-small cell lung cancer

BackgroundN-Acetylgalactosaminyltransferases (GALNTs), the enzymes that initiate mucin-type O-glycosylation, are closely associated with tumor occurrence and progression. However, a comprehensive analysis of GALNTs in non-small cell lung cancer (NSCLC) is lacking.MethodsThe expression profiles and prognostic values of the GALNT family members in NSCLC were analyzed using publicly available databases. Gain- and loss-of-function experiments were applied to assess the biological function of GALNT2 in NSCLC. High-throughput sequencing and bioinformatics approaches were employed to uncover the regulatory mechanism of GALNT2.ResultsAmong the family members of GALNTs, only GALNT2 was frequently overexpressed in NSCLC tissues and was positively correlated with poor prognosis. In vitro assays showed that GALNT2 knockdown repressed NSCLC cell proliferation, migration, and invasion, but induced apoptosis and cell cycle arrest. Correspondently, GALNT2 overexpression exerted the opposite effects. In vivo experiments demonstrated that knockdown of GALNT2 restrained tumor formation in nude mice. Mechanistic investigations revealed that GALNT2 modified the O-glycosylation of ITGA5 and affected the activation of the PI3K/Akt and MAPK/ERK pathways. Further studies showed that miR-30d was a negative regulator of GALNT2.ConclusionsThese findings suggest that GALNT2 is an oncogene in NSCLC and has the potential as a target for NSCLC therapy.

A high scale SARS-CoV-2 profiling by its whole-genome sequencing using Oxford Nanopore Technology in Kazakhstan.

Severe acute respiratory syndrome (SARS-CoV-2) is responsible for the worldwide pandemic, COVID-19. The original viral whole-genome was sequenced by a high-throughput sequencing approach from the samples obtained from Wuhan, China. Real-time gene sequencing is the main parameter to manage viral outbreaks because it expands our understanding of virus proliferation, spread, and evolution. Whole-genome sequencing is critical for SARS-CoV-2 variant surveillance, the development of new vaccines and boosters, and the representation of epidemiological situations in the country. A significant increase in the number of COVID-19 cases confirmed in August 2021 in Kazakhstan facilitated a need to establish an effective and proficient system for further study of SARS-CoV-2 genetic variants and the development of future Kazakhstan’s genomic surveillance program. The SARS-CoV-2 whole-genome was sequenced according to SARS-CoV-2 ARTIC protocol (EXP-MRT001) by Oxford Nanopore Technologies at the National Laboratory Astana, Kazakhstan to track viral variants circulating in the country. The 500 samples kindly provided by the Republican Diagnostic Center (UMC-NU) and private laboratory KDL “Olymp” were collected from individuals in Nur-Sultan city diagnosed with COVID-19 from August 2021 to May 2022 using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). All samples had a cycle threshold (Ct) value below 20 with an average Ct value of 17.03. The overall average value of sequencing depth coverage for samples is 244X. 341 whole-genome sequences that passed quality control were deposited in the Global initiative on sharing all influenza data (GISAID). The BA.1.1 (n = 189), BA.1 (n = 15), BA.2 (n = 3), BA.1.15 (n = 1), BA.1.17.2 (n = 1) omicron lineages, AY.122 (n = 119), B.1.617.2 (n = 8), AY.111 (n = 2), AY.126 (n = 1), AY.4 (n = 1) delta lineages, one sample B.1.1.7 (n = 1) belongs to alpha lineage, and one sample B.1.637 (n = 1) belongs to small sublineage were detected in this study. This is the first study of SARS-CoV-2 whole-genome sequencing by the ONT approach in Kazakhstan, which can be expanded for the investigation of other emerging viral or bacterial infections on the country level.

Epidermal Barrier Integrity is Associated with Both Skin Microbiome Diversity and Composition in Patients with Acne Vulgaris.

BackgroundAlthough the changed lipid environment of the pilosebaceous unit and the growth of lipophilic Cutibacterium acnes (C. acnes) during puberty has long been considered as the trigger of acne vulgaris, the involvement of the interaction between the epidermal barrier integrity and the skin microbiome in this disease has not been fully elucidated.ObjectiveThe aim of this study was to analyze the differences in the epidermal barrier and skin microbiota in patients with acne vulgaris and their correlation.MethodsThe skin microbial samples and epidermal barrier data from 74 acne patients and 19 healthy subjects were collected in this cross-sectional study. The microbial diversity was analyzed based on a high-throughput sequencing approach that targets the V3-V4 region of the bacteria 16S ribosomal RNA genes.ResultsCompared with healthy controls, acne patients had significantly increased transepidermal water loss (TEWL), pH levels, sebum, porphyrins, and red areas, and reduced skin microbiome diversity according to the goods coverage diversity index (p = 0.021), Shannon diversity index (p = 0.037), and Simpson diversity index (p = 0.023). Moreover, the diversity gradually decreased with the increase in acne grading. Based on the principal coordinate analysis (PCoA) analysis plot, the skin microbiota of acne patients and healthy controls could be divided into two different sets, which could not be used to separate acne patients with different disease severity. Finally, this study found that both TEWL and sebum were negatively associated with the Shannon and Simpson diversity index. Meanwhile, the taxa Enhydrobacter and Stenotrophomonas were positively associated with TEWL, stratum corneum hydration, respectively.ConclusionThis study demonstrated that acne vulgaris exists in patients with both damaged epithelial barriers and associated microbiota dysbiosis; the findings will help improve the understanding of the disease and may contribute to the development of better treatment options.

Biodiversity and Oenological Property Analysis of Non-Saccharomyces Yeasts Isolated from Korla Fragrant Pears (Pyrus sinkiangensis Yu)

Oenological yeasts play a critical role in the winemaking process. In this study, the biodiversity of the non-Saccharomyces yeast was analyzed and monitored using high-throughput sequencing and culture-dependent approaches. Oenological and fermentation characteristics of these native yeasts were further investigated. A total of 241 fungus species and 5 species of culturable non-Saccharomyces yeasts were detected using high-throughput sequencing and culture-dependent approaches, respectively. Five strains of aroma-producing yeasts (K4, K14, K19, K21, and K26) were isolated, and their growth characteristics, carbon source utilization, hydrogen sulfide production performance, and β-glucosidase activity were different. The oenological condition tolerances of most strains were lower than that of commercial S. cerevisiae X16. The co-inoculum of these strains and S. cerevisiae X16 regulated the volatile aroma characteristics of the fermented Korla fragrant pear (KFP) fruit wine, enriching and complicating the aroma flavor. Thus, the combined inoculation of these indigenous wine yeasts and S. cerevisiae has some application potential in the production of KFP wine.

A new flavi-like virus identified in populations of wild carrots

We report the discovery of a new flavi-like virus identified in wild carrots (Daucus carota subsp. carota), using a double-stranded (ds)RNA high-throughput sequencing (HTS) approach. The new virus, tentatively named "carrot flavi-like virus 1" (CtFLV-1), has a large genome of 21.8kb that harbours a single open reading frame encoding a 7,078-aa polyprotein with conserved RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains. The new virus is phylogenetically related to recently described flavi-like viruses from arthropods, but its closest relative is a plant-associated virus, gentian Kobu-sho-associated virus (GKSaV). A pairwise comparison showed that these two viruses share 38.4% amino acid (aa) sequence identity in their polyproteins and 73% and 47.8% aa sequence identity in their conserved RdRp and Hel domains, respectively. Based on their similar genome organization and phylogenetic relationship, GKSaV and CtFLV-1 could form the basis for a new genus of plant-associated viruses, possibly within the family Flaviviridae, for which the name "Koshovirus" is proposed.

FreshOmics: A manually curated and standardized -omics database for investigating freshwater microbiomes.

Freshwater is a critical resource for human survival but severely threatened by anthropogenic activities and climate change. These changes strongly impact the abundance and diversity of the microbial communities which are key players in the functioning of these aquatic ecosystems. Although widely documented since the emergence of high-throughput sequencing approaches, the information on these natural microbial communities is scattered among thousands of publications and it is therefore difficult to investigate the temporal dynamics and the spatial distribution of microbial taxa within or across ecosystems. To fill this gap and in the FAIR principles context we built a manually curated and standardized microbial freshwater -omics database (FreshOmics). Based on recognized ontologies (ENVO, MIMICS, GO, ISO), FreshOmics describes 29 different types of freshwater ecosystems and uses standardized attributes to depict biological samples, sequencing protocols and article attributes for more than 2487 geographical locations across 71 countries around the world. The database contains 24,808 sequence identifiers (i.e., Run_Id / Exp_ID, mainly from SRA/DDBJ SRA/ENA, GSA and MG-RAST repositories) covering all sequence-based -omics approaches used to investigate bacteria, archaea, microbial eukaryotes, and viruses. Therefore, FreshOmics allows accurate and comprehensive analyses of microbial communities to answer questions related to their roles in freshwater ecosystems functioning and resilience, especially through meta-analysis studies. This collection also highlights different sort of errors in published works (e.g., wrong coordinates, sample type, material, spelling).

SpBLRSR: Schatten p-norm Constrained Bounded Low-rank Subspace Recovery for Predicting N7-methylguanosine (m7G)-disease Associations

Background: As an essential positively charged RNA modification, N7-methylguanosine (m7G) has been reported to be associated with multiple diseases including cancers. While transcriptome-wide m7G sites have been identified by high-throughput sequencing approaches, the disease-associated m7G sites are still largely unknown. Therefore, computational methods are urgently needed to predict potential m7G-disease associations, which is crucial for understanding the biosynthetic pathways of tumorigenesis at the epi-transcriptome layer. Objective: We hope to develop an effective computational method that can accurately predict the associations between m7G sites and diseases, and then to prioritizing candidate m7G sites for novel diseases. Method: In this article, we proposed a Schatten p-norm constrained bounded low-rank subspace recovery (SpBLRSR) method for m7G-disease association prediction. An m7G-disease block matrix was built to alleviate the sparseness during the association pattern discovery process. By incorporating the low-rank representation (LRR) model and sparse subspace clustering (SSC) model, SpBLRSR was designed to capture both the global and local structures of the association pattern. Results: Compared with the benchmark methods, SpBLRSR achieved the best performance in predicting associations between m7G sites and disease, and in prioritizing m7G sites for novel diseases. Then the robustness of Schatten p-norm in our method was further validated via a noise contamination experiment. Finally, case study of breast cancer was performed to elucidate the biological meaning of our method. Conclusion: SpBLRSR exploits the disease pathogenesis at the epitranscriptome layer by predicting potential m7A sites for disease.

Addressing nontarget amplification in DNA metabarcoding studies of arthropod-feeding rodents.

High-throughput sequencing approaches have revolutionized how we study animal diets by enabling the detection of dietary components from the metabarcoding of DNA in excrement. Mitochondrial cytochrome oxidase C subunit I (mtCOI) DNA metabarcoding is commonly used to study the diets of arthropod-feeding animals; however, this approach is susceptible to nontarget amplification of the consumer species mtCOI locus. Nontarget amplification is often an unforeseen complication that can drastically reduce the quality and utility of the results generated by high-throughput amplicon sequencing. By interrogating the diets of new world rodents in the genus Neotoma (woodrats) in both natural and captive settings, we demonstrate that nontarget amplification can drastically reduce the total read abundance of detected arthropod taxa in fecal samples and inhibit downstream analyses of dietary diversity and composition metrics. Using the results from these investigations, we offer a guide on how to identify concerns for nontarget amplification when selecting degenerate primers for DNA metabarcoding studies and recommend several approaches that can reduce or eliminate nontarget amplification. Lastly, for the community interested in investigating the diets of arthropod-feeding rodents, we generated a database containing the degree of mismatch between publicly available Rodentia mtCOI sequences and four common universal mtCOI primer sets to be used as a resource for inferring the relative risk of nontarget amplification when designing arthropod metabarcoding studies in rodent systems. This guide will be especially useful for researchers working with consumer species that have not previously been studied.

The Landscape of Liver Chromatin Accessibility and Conserved Non-coding Elements in Larimichthys crocea, Nibea albiflora, and Lateolabrax maculatus.

Large yellow croaker (Larimichthys crocea), yellow drum (Nibea albiflora), and Chinese seabass (Lateolabrax maculatus) are important economic marine fishes in China. The conserved non-coding elements (CNEs) in the liver tissues of the three kinds of fish are directly or indirectly involved in the regulation of gene expression and affect liver functions. However, the fishes' CNEs and even chromatin accessibility landscape have not been effectively investigated. Hence, this study established the landscapes of the fishes' genome-wide chromatin accessibility and CNEs by detecting regions of the open chromatin in their livers using an assay for transposase-accessible chromatin by high-throughput sequencing (ATAC-seq) and comparative genomics approach. The results showed that Smad1, Sp1, and Foxl1 transcription factor binding motifs were considerably enrichedin thechromatin accessibility landscape in theliver of the three species, and the three transcription factors (TFs) had a wide range of common targets. The hypothetical gene set was targeted by one, two, or all three TFs, which was much higher than would be expected for an accidental outcome. The gene sets near the CNEs were mainly enriched through processes such as a macromolecule metabolic process and ribonucleoprotein complex biogenesis. The active CNEs were found in the promoter regions of genes such as ap1g1, hax1, and ndufs2. And 5 CNEs were predicted to be highly conserved active enhancers. These results demonstrated that Smad1, Sp1, and Foxl1 might be related to the liver function in the three fishes. In addition, we found a series of ATAC-seq-labeled CNEs located in the gene promoter regions, and highly conserved H3k27ac + -labeled CNEs located in the liver function genes. The highly conserved nature of these regulatory elements suggests that they play important roles in the liver in fish. This study minedthelandscape of chromatin accessibility and CNEs of three important economic fishes to fill the knowledge gaps in this field. Moreover, the work provides useful data for the industrial application and theoretical research of these three fish species.

Application of CRISPR/Cas9 Genome Editing System to Reduce the Pre- and Post-Harvest Yield Losses in Cereals

Cereals are an important source of food for millions of people across low-middle-income countries. Cereals are considered a staple food for poor people. The majority of the people are depending upon agricultural occupation. Agriculture provides a primary source of income for many farmers in low-middle-income countries. The pre- and post-harvest loss of crop yield affects farmers and is a major problem in achieving food security. Biotic and abiotic factors cause pre- and post-harvest loss of crop yield worldwide. It significantly affects the economic status of farmers as well as low-middle-income countries. Many advanced technologies are available for resolving the pre- and post-harvest loss of crop yield. The past few decades have seen remarkable progress in crop improvement. Especially high-throughput genome sequencing approaches contributed to advancement in the crop improvement. Genome-editing has also been considered a key tool for crop improvement. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become a potent genome editing system for modifying key traits in cereal crops. CRISPR/Cas9 system offers new opportunities for addressing pre-and post-harvest constraints affecting cereal grain production and storage. In this review, we discuss the application of the CRISPR/Cas9 genome editing system to reduce pre-and post-harvest yield loss in cereal crops. It may promote the economic status of farmers and reduce food demand in the coming decades.

Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates Conserved Protospacer Selection Mechanisms Between Diverse CRISPR-Cas Types.

Adaptation of clustered regularly interspaced short palindromic repeats (CRISPR) arrays is a crucial process responsible for the unique, adaptive nature of CRISPR-Cas immune systems. The acquisition of new CRISPR spacers from mobile genetic elements has previously been studied for several types of CRISPR-Cas systems. In this study, we used a high-throughput sequencing approach to characterize CRISPR adaptation of the type V-A system from Francisella novicida and the type V-B system from Alicyclobacillus acidoterrestris. In contrast to other class 2 CRISPR-Cas systems, we found that for the type V-A and V-B systems, the Cas12 nucleases are dispensable for spacer acquisition, with only Cas1 and Cas2 (type V-A) or Cas4/1 and Cas2 (type V-B) being necessary and sufficient. Whereas the catalytic activity of Cas4 is not essential for adaptation, Cas4 activity is required for correct protospacer adjacent motif selection in both systems and for prespacer trimming in type V-A. In addition, we provide evidence for acquisition of RecBCD-produced DNA fragments by both systems, but with spacers derived from foreign DNA being incorporated preferentially over those derived from the host chromosome. Our work shows that several spacer acquisition mechanisms are conserved between diverse CRISPR-Cas systems, but also highlights unexpected nuances between similar systems that generally contribute to a bias of gaining immunity against invading genetic elements.

Bacterial Microbiome in the Phyllo-Endosphere of Highly Specialized Rock Spleenwort.

Bacteria communities associated with plants have been given increasing consideration because they are arguably beneficial to their host plants. To understand the ecological and evolutionary impact of these mutualistic associations, it is important to explore the vast unknown territory of bacterial genomic diversity and their functional contributions associated with the major branches of the tree-of-life. Arguably, this aim can be achieved by profiling bacterial communities by applying high throughput sequencing approaches, besides establishing model plant organisms to test key predictions. This study utilized the Illumina Miseq reads of bacterial 16S rRNA sequences to determine the bacterial diversity associated with the endosphere of the leaves of the highly specialized rock spleenwort Asplenium delavayi (Aspleniaceae). By documenting the bacterial communities associated with ferns collected in natural occurrence and cultivation, this study discovered the most species-rich bacterial communities associated with terrestrial ferns reported until now. Despite the substantial variations of species diversity and composition among accessions, a set of 28 bacterial OTUs was found to be shared among all accessions. Functional analyses recovered evidence to support the predictions that changes in bacterial community compositions correspond to functional differentiation. Given the ease of cultivating this species, Asplenium delavayi is introduced here as a model organism to explore the ecological and evolutionary benefits created by mutualistic associations between bacteria and ferns.

Genome-wide genetic diversity may help identify fine-scale genetic structure among lake whitefish spawning groups in Lake Erie

In Lake Erie, lake whitefish Coregonus clupeaformis supported lucrative fisheries before populations were decimated by overfishing and water quality degradation. In recent years, there has been a renewed interest in lake whitefish and management of the fishery they support. Lake whitefish spawn on several reefs throughout Lake Erie, but the relative recruitment dynamics and contributions of spawning groups to the fishery are not well understood. Modern high-throughput sequencing approaches offer new opportunities to census population diversity and to identify subtle differences among closely related populations. We used high-throughput sequencing data to evaluate the genetic structure and diversity of lake whitefish collected opportunistically across broad spatial scales in Lake Erie. Using RAD-capture (Rapture), we sequenced and genotyped individuals (N = 88) from the west, central, and east basin of Lake Erie at 120,268 single nucleotide polymorphisms (SNPs). Lake whitefish from Niagara and Crib Reefs (west basin) diverged from the three collections. Interestingly, these were the only lake whitefish collected during the act of spawning (late November), and all other fish were collected pre-spawn (August-early November). These results suggest that some lake whitefish spawning reefs may be reproductively isolated, though definition of these groups into stocks will require more intentional sampling during the act of spawning.

Genetic Profile of Patients with Limb-Girdle Muscle Weakness in the Chilean Population.

Hereditary myopathies are a group of genetically determined muscle disorders comprising more than 300 entities. In Chile, there are no specific registries of the distinct forms of these myopathies. We now report the genetic findings of a series of Chilean patients presenting with limb-girdle muscle weakness of unknown etiology. Eighty-two patients were explored using high-throughput sequencing approaches with neuromuscular gene panels, establishing a definite genetic diagnosis in 49 patients (59.8%) and a highly probable genetic diagnosis in eight additional cases (9.8%). The most frequent causative genes identified were DYSF and CAPN3, accounting for 22% and 8.5% of the cases, respectively, followed by DMD (4.9%) and RYR1 (4.9%). The remaining 17 causative genes were present in one or two cases only. Twelve novel variants were identified. Five patients (6.1%) carried a variant of uncertain significance in genes partially matching the clinical phenotype. Twenty patients (24.4%) did not carry a pathogenic or likely pathogenic variant in the phenotypically related genes, including five patients (6.1%) presenting an autoimmune neuromuscular disorder. The relative frequency of the different forms of myopathy in Chile is like that of other series reported from different regions of the world with perhaps a relatively higher incidence of dysferlinopathy.

Multiplexed Assembly and Annotation of Synthetic Biology Constructs Using Long-Read Nanopore Sequencing.

Recombinant DNA is a fundamental tool in biotechnology and medicine. These DNA sequences are often built, replicated, and delivered in the form of plasmids. Validation of these plasmid sequences is a critical and time-consuming step, which has been dominated for the last 35 years by Sanger sequencing. As plasmid sequences grow more complex with new DNA synthesis and cloning techniques, we need new approaches that address the corresponding validation challenges at scale. Here we prototype a high-throughput plasmid sequencing approach using DNA transposition and Oxford Nanopore sequencing. Our method, Circuit-seq, creates robust, full-length, and accurate plasmid assemblies without prior knowledge of the underlying sequence. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating full-length, contiguous plasmid maps. We then leverage our long-read data to characterize epigenetic marks and estimate plasmid contamination levels. Circuit-seq scales to large numbers of samples at a lower per-sample cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, while low equipment costs make it practical for individual laboratories.

Physicochemical Variables Better Explain Changes in Microbial Community Structure and Abundance under Alternate Wetting and Drying Events

Soil microbial communities play an important role in nutrient cycling; however, their response under repeated long-term fertilization has attracted little attention and needs further appraisal. A 14-day incubation study compared the relative abundance, diversity, and composition of bacterial and fungal microbial communities in soils treated with long-term applications of chemical fertilizer (CF), pig manure plus chemical fertilizer (PMCF), and rice straw plus chemical fertilizer (SRCF) in a paddy field. A high-throughput sequencing approach was applied to assess the diversity and composition of microbial community. Results revealed the Shannon index of the bacterial community decreased with fertilizer addition but increased in case of fungal community. The abundance of the Actinobacteria was higher in the PMCF, while Proteobacteria were higher in the CF and SRCF treatments than those in the unamended control under alternate wetting and drying (AWD) and permanent flooding (PF). In addition, chemical fertilizer history increased the abundance of Firmicutes under AWD. Initially, Nitrospira were found higher in the unamended control than in the amended treatments, but an increase was observed with time in fertilized treatments. Among all genera, Proteobacteria were the most abundant bacterial genus. The main properties that markedly affected the bacterial communities were SOC (R2 = 0.4037, p < 0.02), available P (R2 = 0.3273, p < 0.05), and NO3− (R2 = 0.3096, p < 0.08). Soil physicochemical factors and biogenic factors explained a variation of 46.27% and 29.35%, respectively. At the same time, 4.59% was the combined effect of physicochemical and biogenic factors. Our results suggested that the physicochemical properties had a more significant impact on bacterial activities than water regime by increasing N and organic matter concentrations in the soils.