Next-generation Sequencing Techniques Research Articles

Structural diversity of bacterial communities in two divergent sunflower rhizosphere soils

PurposeFarming practices on farmlands aim to improve nutrients in the fields or crops, soil quality and functions, as well as boost and sustain crop yield; however, the effect of loss of ecological diversity and degradation have impacted ecosystem functions. The beneficial rhizosphere-microorganism network and crop rotation may enhance a stable ecosystem. The use of next-generation sequencing technique will help characterize the entire bacterial species in the sunflower rhizosphere compared with the nearby bulk soils. We investigated the potential of the bacterial community structure of sunflower rhizosphere and bulk soils cultivated under different agricultural practices at two geographical locations in the North West Province of South Africa.MethodsDNA was extracted from rhizosphere and bulk soils associated with sunflower plants from the crop rotation (rhizosphere soils from Lichtenburg (LTR) and bulk soils from Lichtenburg (LTB) and mono-cropping (rhizosphere soils from Krayburg (KRPR) and bulk soils from Krayburg (KRPB) sites, and sequenced employing 16S amplicon sequencing. Bioinformatics tools were used to analyse the sequenced dataset.ResultsProteobacteria and Planctomycetes dominated the rhizosphere, while Firmicutes and Actinobacteria were predominant in bulk soils. Significant differences in bacterial structure at phyla and family levels and predicted functional categories between soils (P < 0.05) across the sites were revealed. The effect of physicochemical parameters was observed to influence bacterial dispersal across the sites.ConclusionThis study provides information on the predominant bacterial community structure in sunflower soils and their predictive functional attributes at the growing stage, which suggests their future study for imminent crop production and management for enhanced agricultural yields.

Next-Generation Sequencing of Cerebrospinal Fluid for the Diagnosis of VZV-Associated Rhombencephalitis

Rhombencephalitis (RE) is a general term for a group of inflammatory diseases of the rhombencephalon caused by different etiologies. Patients of RE caused by the varicella-zoster virus (VZV) are sporadic in medical practice. The VZV-RE is easily misdiagnosed and causes a poor prognosis for patients. In this study, we analyzed the clinical symptoms and imaging features of five patients with VZV-RE diagnosed by the next-generation sequencing (NGS) technique of cerebrospinal fluid. Magnetic resonance imaging (MRI) examination was used to characterize the imaging of the patients. The McNemar test was used to analyze the cerebrospinal fluid testing (CSF) values and MRI test of the 5 patients. We finally used NGS technology to confirm the diagnosis in 5 patients with VZV-RE. MRI revealed T2/FLAIR high signal lesions in the patients' medulla oblongata, pons, and cerebellum. All patients had early signs of cranial nerve palsy; some had herpes or pain in the corresponding cranial nerve distribution areas. The patients develop headaches, fever, nausea, vomiting, and other signs and symptoms of brainstem cerebellar involvement. McNemar's test showed no statistical difference between multi-mode MRI and CSF values for diagnosing VZV-RE (p = 0.513). This study showed that patients with herpes in the skin and mucous membranes at the distribution area of the cranial nerves and with the underlying disease were prone to RE. We suggest that the NGS analysis should be considered and selected based on the level of parameters, such as MRI lesion characteristics.

Brugada Syndrome: From Molecular Mechanisms and Genetics to Risk Stratification

Brugada syndrome (BrS) is a rare hereditary arrhythmia disorder, with a distinctive ECG pattern, correlated with an increased risk of ventricular arrhythmias and sudden cardiac death (SCD) in young adults. BrS is a complex entity in terms of mechanisms, genetics, diagnosis, arrhythmia risk stratification, and management. The main electrophysiological mechanism of BrS requires further research, with prevailing theories centered on aberrant repolarization, depolarization, and current-load match. Computational modelling, pre-clinical, and clinical research show that BrS molecular anomalies result in excitation wavelength (k) modifications, which eventually increase the risk of arrhythmia. Although a mutation in the SCN5A (Sodium Voltage-Gated Channel Alpha Subunit 5) gene was first reported almost two decades ago, BrS is still currently regarded as a Mendelian condition inherited in an autosomal dominant manner with incomplete penetrance, despite the recent developments in the field of genetics and the latest hypothesis of additional inheritance pathways proposing a more complex mode of inheritance. In spite of the extensive use of the next-generation sequencing (NGS) technique with high coverage, genetics remains unexplained in a number of clinically confirmed cases. Except for the SCN5A which encodes the cardiac sodium channel NaV1.5, susceptibility genes remain mostly unidentified. The predominance of cardiac transcription factor loci suggests that transcriptional regulation is essential to the Brugada syndrome's pathogenesis. It appears that BrS is a multifactorial disease, which is influenced by several loci, each of which is affected by the environment. The primary challenge in individuals with a BrS type 1 ECG is to identify those who are at risk for sudden death, researchers propose the use of a multiparametric clinical and instrumental strategy for risk stratification. The aim of this review is to summarize the latest findings addressing the genetic architecture of BrS and to provide novel perspectives into its molecular underpinnings and novel models of risk stratification.

Molecular classification and therapeutics in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) encompasses a wide variety of disease states that have to date been subgrouped and characterized based on immunohistochemical methods, which provide limited prognostic value to clinicians and no alteration in treatment regimen. The addition of rituximab to CHOP therapy was the last leap forward in terms of treatment, but regimens currently follow a standardized course when disease becomes refractory with no individualization based on genotype. Research groups are tentatively proposing new strategies for categorizing DLBCL based on genetic abnormalities that are frequently found together to better predict disease course following dysregulation of specific pathways and to deliver targeted treatment. Novel algorithms in combination with next-generation sequencing techniques have identified between 4 and 7 subgroups of DLBCL, depending on the research team, with potentially significant and actionable genetic alterations. Various drugs aimed at pathways including BCR signaling, NF-κB dysfunction, and epigenetic regulation have shown promise in their respective groups and may show initial utility as second or third line therapies to patients with recurrent DLBCL. Implementation of subgroups will allow collection of necessary data to determine which groups are significant, which treatments may be indicated, and will provide better insight to clinicians and patients on specific disease course.

Targeted Sequencing Approach and Its Clinical Applications for the Molecular Diagnosis of Human Diseases.

The outbreak of COVID-19 has positively impacted the NGS market recently. Targeted sequencing (TS) has become an important routine technique in both clinical and research settings, with advantages including high confidence and accuracy, a reasonable turnaround time, relatively low cost, and fewer data burdens with the level of bioinformatics or computational demand. Since there are no clear consensus guidelines on the wide range of next-generation sequencing (NGS) platforms and techniques, there is a vital need for researchers and clinicians to develop efficient approaches, especially for the molecular diagnosis of diseases in the emergency of the disease and the global pandemic outbreak of COVID-19. In this review, we aim to summarize different methods of TS, demonstrate parameters for TS assay designs, illustrate different TS panels, discuss their limitations, and present the challenges of TS concerning their clinical application for the molecular diagnosis of human diseases.

Using Nanocompore to Identify RNA Modifications from Direct RNA Nanopore Sequencing Data

RNA modifications can alter the behavior of RNA molecules depending on where they are located on the strands. Traditionally, RNA modifications have been detected and characterized by biophysical assays, mass spectrometry, or specific next-generation sequencing techniques, but are limited to specific modifications or are low throughput. Nanopore is a platform capable of sequencing RNA strands directly, which permits transcriptome-wide detection of RNA modifications. RNA modifications alter the nanopore raw signal relative to the canonical form of the nucleotide, and several software tools detect these signal alterations. One such tool is Nanocompore, which compares the ionic current features between two different experimental conditions (i.e., with and without RNA modifications) to detect RNA modifications. Nanocompore is not limited to a single type of RNA modification, has a high specificity for detecting RNA modifications, and does not require model training. To use Nanocompore, the following steps are needed: (i) the data must be basecalled and aligned to the reference transcriptome, then the raw ionic current signals are aligned to the sequences and transformed into a Nanocompore-compatible format; (ii) finally, the statistical testing is conducted on the transformed data and produces a table of p-value predictions for the positions of the RNA modifications. These steps can be executed with several different methods, and thus we have also included two alternative protocols for running Nanocompore. Once the positions of RNA modifications are determined by Nanocompore, users can investigate their function in various metabolic pathways. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: RNA modification detection by Nanocompore Alternate Protocol 1: RNA modification detection by Nanocompore with f5c Alternate Protocol 2: RNA modification detection by Nanocompore using Nextflow.

A Perspective Review on Understanding Drought Stress Tolerance in Wild Banana Genetic Resources of Northeast India.

The enormous perennial monocotyledonous herb banana (Musa spp.), which includes dessert and cooking varieties, is found in more than 120 countries and is a member of the order Zingiberales and family Musaceae. The production of bananas requires a certain amount of precipitation throughout the year, and its scarcity reduces productivity in rain-fed banana-growing areas due to drought stress. To increase the tolerance of banana crops to drought stress, it is necessary to explore crop wild relatives (CWRs) of banana. Although molecular genetic pathways involved in drought stress tolerance of cultivated banana have been uncovered and understood with the introduction of high-throughput DNA sequencing technology, next-generation sequencing (NGS) techniques, and numerous "omics" tools, unfortunately, such approaches have not been thoroughly implemented to utilize the huge potential of wild genetic resources of banana. In India, the northeastern region has been reported to have the highest diversity and distribution of Musaceae, with more than 30 taxa, 19 of which are unique to the area, accounting for around 81% of all wild species. As a result, the area is regarded as one of the main locations of origin for the Musaceae family. The understanding of the response of the banana genotypes of northeastern India belonging to different genome groups to water deficit stress at the molecular level will be useful for developing and improving drought tolerance in commercial banana cultivars not only in India but also worldwide. Hence, in the present review, we discuss the studies conducted to observe the effect of drought stress on different banana species. Moreover, the article highlights the tools and techniques that have been used or that can be used for exploring and understanding the molecular basis of differentially regulated genes and their networks in different drought stress-tolerant banana genotypes of northeast India, especially wild types, for unraveling their potential novel traits and genes.

Plant species shape the bacterial communities on the phyllosphere in a hyper-arid desert

Microorganisms are an important component of global biodiversity. However, they are vulnerable to hyper-arid climates in desert regions. Xerophytes are desert vegetation with unique biodiversity. However, little is known about the identities and communities of phyllosphere epiphytic microorganisms inhabiting the xerophyte leaf surface in the hot and dry environment. The diversity and community composition of phyllosphere epiphytes on different desert plants in Gansu, China, was investigated using the next-generation sequencing technique, revealing the diversity and community composition of the phyllosphere epiphytic bacteria associated with desert xerophytes. In addition, the ecological functions of the bacterial communities were investigated by combining the sequence classification information and prokaryotic taxonomic function annotation (FAPROTAX). This study determined the phyllosphere bacterial community composition, microbial interactions, and their functions. Despite harsh environments in the arid desert, we found that there are still diverse epiphytic bacteria on the leaves of desert plants. The bacterial communities mainly included Actinobacteria (52.79%), Firmicutes (31.62%), and Proteobacteria (12.20%). Further comparisons revealed different microbial communities, including Firmicutes at the phylum and Paenibacillaceae at the family level, in the phyllosphere among different plants, suggesting that the host plants had strong filter effects on bacteria. Co-occurrence network analysis revealed positive relationships were dominant among different bacterial taxa. The abundance of Actinobacteria and Proteobacteria was positively correlated, demonstrating their mutual relationship. On the other hand, the abundance of Firmicutes was negatively correlated, which suggested that they inhibit the growth of other bacterial taxa. FAPROTAX prediction revealed that chemoheterotrophy (accounting for 39.02% of the community) and aerobic chemoheterotrophy (37.01%) were the main functions of the leaf epiphytic bacteria on desert plants. This study improves our understanding of the community composition and ecological functions of plant-associated microbial communities inhabiting scattered niches in the desert ecosystem. In addition, the study provides insight into the biodiversity assessment in the desert region.

Investigation of the Genetic Etiology in Idiopathic Generalized Epileptic Disorders by Targeted Next-generation Sequencing Technique

Idiopathic generalized epilepsy is the most common group of epilepsy disorders in children and adolescents. Various types of genetic abnormality were identified among the hereditary factors that explain epilepsy. To determine the variations in the etiopathogenesis, treatment protocol planning, and prognosis of idiopathic generalized epilepsy using the next-generation sequencing method. A cross-sectional study. This study included 32 patients with idiopathic generalized epilepsy. Genomic DNA was obtained from peripheral venous blood samples taken from the patients. A total of 18 genes encoding ion channel subunits that are involved in monogenic disorders and are associated with idiopathic generalized epilepsy were included. The targeted custom next-generation sequencing panel was designed to cover all coding exons and all exon/intron splice site regions of 18 genes. We detected 9 (28%) variations, including 1 likely pathogenic (a variant in the SCN1A gene) and 8 of unknown clinical significance (2 in the CLCN2 genes, GABBR2, SCN1B, SLC2A1, SLC4A10 genes, and 2 in the TBC1D24 gene). Study results should be supported by functional advanced studies, with increased existing knowledge in the relevant variations.

Update of pediatric bone tumors-other mesenchymal tumors of bone, hematopoietic neoplasms of bone, and WHO classification of undifferentiated small round cell sarcomas of bone.

There are numerous bone tumors in the pediatric population, with imaging playing an essential role in diagnosis and management. Our understanding of certain bone tumors has rapidly evolved over the past decade with advancements in next-generation genetic sequencing techniques. This increased level of understanding has altered the nomenclature, management approach, and prognosis of certain lesions. We provide a detailed update of bone tumors that occur in the pediatric population with emphasis on the recently released nomenclature provided in the 5th edition of the World Health Organization Classification of Soft Tissue and Bone Tumours. We discuss other mesenchymal tumors of bone, hematopoietic neoplasms of bone, and WHO classification of undifferentiated small round cell sarcomas of bone. We have detailed osteogenic tumors and osteoclastic giant cell-rich tumors, as well as notochordal tumors, chondrogenic tumors, and vascular tumors of the bone in separate manuscripts.

Targeting lipid metabolism as a new therapeutic strategy for inherited cardiomyopathies.

Inherited cardiomyopathies caused by pathological genetic variants include multiple subtypes of heart disease. Advances in next-generation sequencing (NGS) techniques have allowed for the identification of numerous genetic variants as pathological variants. However, the disease penetrance varies among mutated genes. Some can be associated with more than one disease subtype, leading to a complex genotype-phenotype relationship in inherited cardiomyopathies. Previous studies have demonstrated disrupted metabolism in inherited cardiomyopathies and the importance of metabolic adaptations in disease onset and progression. In addition, genotype- and phenotype-specific metabolic alterations, especially in lipid metabolism, have been revealed. In this mini-review, we describe the metabolic changes that are associated with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which account for the largest proportion of inherited cardiomyopathies. We also summarize the affected expression of genes involved in fatty acid oxidation (FAO) in DCM and HCM, highlighting the potential of PPARA-targeting drugs as FAO modulators in treating patients with inherited cardiomyopathies.

Evaluating the role of next-generation sequencing and radiological techniques in rare disease diagnosis

Aim: This article evaluates the utility of next-generation sequencing (NGS) and radiological techniques in the diagnosis of rare diseases, emphasizing the challenges and opportunities presented by these technologies. Methods: A comprehensive review of existing literature on NGS technologies, including first, second, and third-generation sequencing methods, as well as their applications in genomics, transcriptomics, and epigenomics, was conducted alongside radiological imaging techniques such as MRI and CT scans. Results: NGS has revolutionized rare disease diagnosis by enabling high-throughput, cost-effective sequencing, facilitating the identification of pathogenic mutations, and advancing personalized medicine. Radiological techniques provide complementary insights into anatomical abnormalities and disease progression. Despite significant advantages, challenges such as data interpretation, cost, and ethical considerations persist. Conclusion: NGS and radiological imaging offer transformative potential in rare disease diagnosis, enhancing our understanding of genetic and anatomical aspects of disorders and enabling targeted therapeutic approaches. Continued technological advancements and integrative analyses with other omics data and imaging findings will further enhance their diagnostic utility.

Next Generation Sequencing for the Analysis of Parvovirus B19 Genomic Diversity.

Parvovirus B19 (B19V) is a ssDNA human virus, responsible for an ample range of clinical manifestations. Sequencing of B19V DNA from clinical samples is frequently reported in the literature to assign genotype (genotypes 1-3) and for finer molecular epidemiological tracing. The increasing availability of Next Generation Sequencing (NGS) with its depth of coverage potentially yields information on intrinsic sequence heterogeneity; however, integration of this information in analysis of sequence variation is not routinely obtained. The present work investigated genomic sequence heterogeneity within and between B19V isolates by application of NGS techniques, and by the development of a novel dedicated bioinformatic tool and analysis pipeline, yielding information on two newly defined parameters. The first, α-diversity, is a measure of the amount and distribution of position-specific, normalised Shannon Entropy, as a measure of intra-sample sequence heterogeneity. The second, σ-diversity, is a measure of the amount of inter-sample sequence heterogeneity, also incorporating information on α-diversity. Based on these indexes, further cluster analysis can be performed. A set of 24 high-titre viraemic samples was investigated. Of these, 23 samples were genotype 1 and one sample was genotype 2. Genotype 1 isolates showed low α-diversity values, with only a few samples showing distinct position-specific polymorphisms; a few genetically related clusters emerged when analysing inter-sample distances, correlated to the year of isolation; the single genotype 2 isolate showed the highest α-diversity, even if not presenting polymorphisms, and was an evident outlier when analysing inter-sample distance. In conclusion, NGS analysis and the bioinformatic tool and pipeline developed and used in the present work can be considered effective tools for investigating sequence diversity, an observable parameter that can be incorporated into the quasispecies theory framework to yield a better insight into viral evolution dynamics.

Investigation of BRCA2 Gene K3326X Variant in Patients with Breast and Ovarian Cancer by Next-Generation Sequencing Technique

Investigation of BRCA2 Gene K3326X Variant in Patients with Breast and Ovarian Cancer by Next-Generation Sequencing Technique

Molecular characterization & recombination analysis of complete enterovirus-88 isolated from acute flaccid paralysis cases in India.

Focus on non-polio enteroviruses (NPEVs) causing acute flaccid paralysis (AFP) due to myelitis has increased with the containment of the poliovirus. Enterovirus-B88 (EV-B88) has been associated with the AFP cases in Bangladesh, Ghana, South Africa, Thailand and India. In India, EV-B88 infection was linked to AFP a decade ago; however, to date, no complete genome has been made available. In this study, the complete genome sequence of EV-B88 was identified and reported from two different States (Bihar and Uttar Pradesh) in India using the next-generation sequencing technique. Virus isolation was performed on the three AFP suspected cases as per the WHO-recommended protocol. Samples showing cytopathic effects in the human Rhabdocarcinoma were labelled as NPEVs. Next-generation sequencing was performed on these NPEVs to identify the aetiological agent. The contiguous sequences (contigs) generated were identified, and reference-based mapping was performed. EV-B88 sequences retrieved in our study were found to be 83 per cent similar to the EV-B88 isolate from Bangladesh in 2001 (strain: BAN01-10398; Accession number: AY843306.1). Recombination analyses of these samples demonstrate recombination events with sequences from echovirus-18 and echovirus-30. Recombination events in the EV-B serotypes are known, and this work reconfirms the same for EV-B88 isolates also. This study is a step in increasing the awareness about EV-B88 in India and emphasizes future studies to be conducted in the identification of other types of EV present in India.

Characterization of the complete mitochondrial genome and phylogenetic analysis of Epiverta chelonia (Coleoptera: Coccinellidae)

Epiverta chelonia (Mader 1933; Coleoptera: Coccinellidae) is an important economically and scientifically valuable insect. In this study, the first complete mitochondrial genome of E. chelonia was sequenced and characterized using next-generation sequencing techniques. The circular mitogenome of E. chelonia consists of 17,347 bp including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (D-loop). The base composition was AT-biased (75.77%). Bayesian Inference and Maximum likelihood phylogenetic trees strongly supported the monophyly of Coccinellinae. Also, E. chelonia was supported as the sister group of Subcoccinella vigintiquatuorpunctata, within Epilachninae. Thus, the E. chelonia mitochondrial genome will be a fundamental resource for understanding the molecular phylogenetic relationships of the species-rich family Coccinellidae of Coleoptera.

Identification of a large intra-exonic deletion in BRCA2 exon 18 in a pancreatic ductal adenocarcinoma

By 2030, pancreatic cancer will become the second leading cause of cancer-related deaths in the United States and in Europe. The management of patients with advanced pancreatic cancer relies on chemotherapy and poly (ADP-ribose) polymerase inhibitors for patients who carry BRCA1/2 inactivating alterations. Some variants, such as large insertion/deletions (Indels), inactivating BRCA1/2 and therefore of clinical relevance can be hard to detect by next-generation sequencing techniques. Here we report a 47-year-old patient presenting with pancreatic cancer whose tumour harbours a large somatic intra-exonic deletion of BRCA2 of 141 bp. This BRCA2 deletion, located in the C-terminal domain, can be considered as pathogenic and consequently affect tumorigenesis because it is involved in the interaction between the DSS1 protein and DNA. Thanks to the optimized bioinformatics algorithm, this intermediate size deletion in BRCA2 was identified, enabling personalized patient management via the inclusion of the patients in a clinical trial.

Role of epigenetics in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, associated with poor survival outcomes. Lack of early diagnosis, resistance to conventional therapeutic treatments (including immunotherapy) and recurrence are some of the major hurdles in PDAC and contribute to its poor survival rate. While the risk of genetic predisposition to cancers is widely acknowledged and understood, recent advances in whole-genome and next-generation sequencing techniques have led to the acknowledgment of the role played by epigenetics, especially in PDAC. Epigenetic changes are heritable genetic modifications that influence gene expression without altering the DNA sequence. Epigenetic mechanisms (e.g., DNA methylation, post-translational modification of histone complexes and ncRNA) that result in reversible changes in gene expression are increasingly understood to be responsible for tumor initiation, development and even escape from immune surveillance. Our review seeks to highlight the various components of the epigenetic machinery that are known to be implicated in PDAC initiation and development and the feasibility of targeting these components to identify novel pharmacological strategies that could potentially lead to breakthroughs in PDAC treatment.

Advances in sequencing-based studies of microDNA and ecDNA: Databases, identification methods, and integration with single-cell analysis

Extrachromosomal circular DNA (eccDNA) is a class of circular DNA molecules that originate from genomic DNA but are separate from chromosomes. They are common in various organisms, with sizes ranging from a few hundred to millions of base pairs. A special type of large extrachromosomal DNA (ecDNA) is prevalent in cancer cells. Research on ecDNA has significantly contributed to our comprehension of cancer development, progression, evolution, and drug resistance. The use of next-generation (NGS) and third-generation sequencing (TGS) techniques to identify eccDNAs throughout the genome has become a trend in current research. Here, we briefly review current advances in the biological mechanisms and applications of two distinct types of eccDNAs: microDNA and ecDNA. In addition to presenting available identification tools based on sequencing data, we summarize the most recent efforts to integrate ecDNA with single-cell analysis and put forth suggestions to promote the process.

Comparison of Two Variant Analysis Programs for Next-Generation Sequencing Data of Whole Mitochondrial Genome.

With advance of next-generation sequencing (NGS) techniques, the need for mitochondrial DNA analysis is increasing not only in the forensic area, but also in medical fields. Two commercial programs, Converge Software (CS) and Torrent Variant Caller for variant calling of NGS data, were compared with a considerable amount of sequence data of 50 samples with a homogeneous ethnicity. About 2,300 variants were identified and the two programs showed about 90% of consistency. CS, a dedicated analysis program for mitochondrial DNA, showed some advantages for forensic use. By additional visual inspection, several causes of discrepancy in variant calling results were identified. Application of different notation rules for mitochondrial sequence and the minor allele frequency close to detection threshold were the two most significant reasons. With prospective improvement of each program, researchers and practitioners should be aware of characteristics of the analysis program they use and prepare their own strategies to determine variants.