Next-generation Sequencing Tools Research Articles

Revealing the Satellite DNA Content in Ancistrus sp. (Siluriformes: Loricariidae) by Genomic and Bioinformatic Analysis.

Eukaryotic genomes are composed of simple, repetitive sequences, including satellite DNAs (satDNA), which are noncoding sequences arranged in tandem arrays. These sequences play a crucial role in genomic functions and innovations, influencing processes such as the maintenance of nuclear material, the formation of heterochromatin and the differentiation of sex chromosomes. In this genomic era, advances in next-generation sequencing and bioinformatics tools have facilitated the exhaustive cataloging of repetitive elements in genomes, particularly in non-model species. This study focuses on the satDNA content of Ancistrus sp., a diverse species of fish from the Loricariidae family. The genus Ancistrus shows significant karyotypic evolution, with extensive variability from the ancestral diploid number. By means of bioinformatic approaches, 40 satDNA families in Ancistrus sp., constituting 5.19% of the genome were identified. Analysis of the abundance and divergence landscape revealed diverse profiles, indicating recent amplification and homogenization of these satDNA sequences. The most abundant satellite, AnSat1-142, constitutes 2.1% of the genome, while the least abundant, AnSat40-52, represents 0.0034%. The length of the monomer repeat varies from 16 to 142 base pairs, with an average length of 61 bp. These results contribute to understanding the genomic dynamics and evolution of satDNAs in Ancistrus sp. The study underscores the variability of satDNAs between fish species and provides valuable information on chromosome organization and the evolution of repetitive elements in non-model organisms.

Comprehensive analysis of the CRISPR-Cas systems in Streptococcus thermophilus strains isolated from traditional yogurts.

Phage resistance is crucial for lactic acid bacteria in the dairy industry. However, identifying all phages affecting these bacteria is challenging. CRISPR-Cas systems offer a resistance mechanism developed by bacteria and archaea against phages and plasmids. In this study, 11 S. thermophilus strains from traditional yogurts underwent analysis using next-generation sequencing (NGS) and bioinformatics tools. Initial characterization involved molecular ribotyping. Bioinformatics analysis of the NGS raw data revealed that all 11 strains possessed at least one CRISPR type. A total of 21 CRISPR loci were identified, belonging to CRISPR types II-A, II-C, and III-A, including 13 Type II-A, 1 Type III-C, and 7 Type III-A CRISPR types. By analyzing spacer sequences in S. thermophilus bacterial genomes and matching them with phage/plasmid genomes, notable strains emerged. SY9 showed prominence with 132 phage matches and 30 plasmid matches, followed by SY12 with 35 phage matches and 25 plasmid matches, and SY18 with 49 phage matches and 13 plasmid matches. These findings indicate the potential of S. thermophilus strains in phage/plasmid resistance for selecting starter cultures, ultimately improving the quality and quantity of dairy products. Nevertheless, further research is required to validate these results and explore the practical applications of this approach.

A Food Poisoning Caused by Salmonella Enterica (S. Enteritidis) ST11 Carrying Multi-Antimicrobial Resistance Genes in 2019, China.

This study was to identify and analyze the pathogen responsible for food poisoning in a tourist group traveling from Macao to Zhuhai. Samples were obtained from 27 patients of 96 cases, as well as samples of contaminated food in Macau. The collected samples were subjected to serological identification, drug sensitivity analysis, drug resistance gene identification, virulence factor analysis, and tracing. Twenty-six isolates and the salad isolate were S. enteritidis ST11. Isolates from patients were exhibited significant resistance to Penicillin AMP (Ampicillin) and quinolones NAL (Nalidixic acid). Among these isolates, 21 strains were resistant to two or more antibiotics, indicating the multi-drug resistance (MDR). Genomic characteristics and phylogenetic analysis were performed on 9 of the isolates using whole genome sequencing (WGS). The analysis revealed that the resistance to AMP and NAL was primarily caused by a gryA mutation D87Y (9/9, 100%), and the presence of beta-lactam resistance genes blaOXA-1 (1/9, 11.11%), blaTEM-141 (1/9, 11.11%), and blaTEM-1B (8/9, 88.89%). It was also found a strains isolated from patients had two resistance genes to quinolones or beta-lactam drugs (1/8, 12.5%), respectively. The strains were found to possess 165 virulence genes, one adherence class virulence factor, one invasion class virulence factor and various pathogenicity islands, including SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-10, SPI-13, SPI-14, SPI-15, SGI 1, CS54_island, and C63PI-1. Additionally, the virulence plasmids were detected, including IncFIB(s)-IncFII(s)-IncX1 (55.56%), IncFIB(s)-IncFII(s) (33.33%), and IncFIB(s)-IncFII(s)-IncHI2-IncHI2A (11.11%). PFGE (Pulsed Field Gel Electrophoresis) and phylogenetic tree analysis revealed a high degree of similarity between Salmonella isolates from patients and food samples from Macao. This study identified Salmonella enterica ST11 as the cause of the food poisoning outbreak. The findings highlight the importance of phenotypic characterization and next-generation sequencing (NGS) tools in epidemiological studies and emphasize the potential risk of a new emerging multi-antibiotic ST11 clone for S. enteritidis.

Two-Time Multiplexed Targeted Next-Generation Sequencing Might Help the Implementation of Germline Screening Tools for Myelodysplastic Syndromes/Hematologic Neoplasms.

Next-generation sequencing (NGS) tools have importantly helped the classification of myelodysplastic syndromes (MDS), guiding the management of patients. However, new concerns are under debate regarding their implementation in routine clinical practice for the identification of germline predisposition. Cost-effective targeted NGS tools would improve the current standardized studies and genetic counseling. Here, we present our experience in a preliminary study detecting variants using a two-time multiplexed library strategy. Samples from different MDS patients were first mixed before library preparation and later multiplexed for a sequencing run. Two different mixes including a pool of three (3×) and four (4×) samples were evaluated. The filtered variants found in the individually sequenced samples were compared with the variants found in the two-time multiplexed studies to determine the detection efficiency scores. The same candidate variants were found in the two-time multiplexed studies in comparison with the individual tNGS. The variant allele frequency (VAF) values of the candidate variants were also compared. No significant differences were found between the expected and observed VAF percentages in both the 3× (p-value 0.74) and 4× (p-value 0.34) multiplexed studies. Our preliminary results suggest that the two-time multiplexing strategy might have the potential to help reduce the cost of evaluating germline predisposition.

MRD Monitoring By Euroclonality IGH Based NGS Approach Predicts Outcome in Follicular Lymphoma Patients Lacking a Conventional BCL2::IGH Marker: A Substudy from the Fondazione Italiana Linfomi (FIL) FOLL12 Trial

Background. Despite durable clinical responses achieved by chemo-immunotherapy in follicular lymphoma (FL) patients, most of them eventually relapse. Actually, minimal residual disease (MRD) analysis based on the detection of BCL2::IGH rearrangement by PCR is the most widely used and standardized approach, able to early identify patients at high risk of disease reappearance. Nevertheless, this tool fails the molecular marker definition in up to 45% of cases, hampering the disease monitoring in a large group of patients. In the last decade, the continuous improvement of next-generation sequencing (NGS) techniques allowed to introduce these novel tools for MRD monitoring in some hematologic diseases, as acute lymphoblastic leukemia and multiple myeloma, thus reducing PCR limitations and increasing the number of patients eligible for MRD. Nonetheless, applications of NGS tools for MRD detection in FL are still scant. Aims. Therefore, we tested the feasibility of the EuroClonality (EC) IGH-based NGS approach [Brüggeman, Leukemia 2019] to identify novel molecular markers suitable for MRD monitoring in the context of the large prospective trial “FOLL12” (EudraCT: 2012-003170-60) by the Fondazione Italiana Linfomi (FIL), where 57% of patients were already studied for MRD by classic PCR methods targeting the BCL2::IGH rearrangement.[Luminari, JCO2022] Methods. Advanced FL patients received upfront treatment with either R-CHOP or BR, followed by a combined PET/MRD response-based maintenance with rituximab. Bone marrow (BM) samples were centralized to the FIL MRD Network for standardized MRD analysis. Baseline gDNA of “no marker” patients was screened by IGH-based NGS using EC-IGH two-steps PCR approach in the EuroMRD certified lab of Torino university; then MRD was monitored in those patients harboring a IGH clonal marker at baseline by one step EC-IGH method. The identification of baseline clonotypes and MRD clones was performed by ARRest/Interrogate tool. Results. 343/786 enrolled patients (43%) lacked a conventional BCL2::IGH marker and 161/343 (47%) showed morphologic BM lymphoma infiltration: of these, 124 (77%) had an available BM baseline sample for IGH screening and were thus included in the analysis. Overall, IGH clonotypes suitable as novel molecular markers were identified in 75/124 patients (60%), of which 73 monoclonal and 2 biclonal. Of this series, 47 patients had centralized a BM sample for MRD analysis at end of induction. Median age of this subset was 59 years (33-81), 53% were female, all were Ann Arbor stage IV, 34% were FLIPI intermediate and 43% high risk, respectively. Moreover, 56% received R-CHOP and 44% BR as induction treatment, resulting in a 79% CR rate and 89% MRD negativity rate (in line with the results of the general trial population). PFS at 3 years for this series was 69%: however, the 5 patients who scored MRD positive (MRD+) showed statistically significant worse PFS at 3 years than MRD negative cases (22% vs 73%, respectively, p=0.0069, Figure 1). Notably, 4 out of 5 MRD+ patients experienced POD24. Focusing on biclonal cases, one presented superimposable MRD trends between clones (both achieving MRD negativity), while in the other both clones remained MRD positive but showing one Log difference between them. Interestingly, only one of the two clones showed a MRD increase in the following timepoint (6 months after end of induction), heralding the clinical relapse subsequently occurred at month 12 (Figure 2). On the other hand, no IGH clonotype was identified in the first 17 patients who were screened for a novel MRD marker despite the absence of BM infiltration. Conclusions. The first data on the large-scale application of NGS-based marker screening tools in FL patients lacking a conventional BCL2::IGH marker for MRD enrolled in a prospective clinical trial showed that the EC IGH-based NGS approach was able to provide a new molecular marker in 60% of patients with evidence of BM infiltration (that means about the 28% of “no marker” FL patients); importantly, this approach was predictive of worse PFS for MRD+ cases. Overall, these data are promising for the wide employment of novel, effective MRD monitoring tools, able to recover more than one fourth of FL patients lacking a conventional molecular marker and so, by combining conventional and NGS-based techniques, potentially rising the percentage of MRD evaluable cases up to at least 70% cases.

Genome mining reveals novel biosynthetic gene clusters in entomopathogenic bacteria

The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study, we conducted genome mining to explore the biosynthetic potential of entomopathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus. By utilizing next-generation sequencing and bioinformatics tools, we identified novel biosynthetic gene clusters (BGCs) in the genomes of the bacteria, specifically plu00736 and plu00747. These clusters were identified as unidentified non-ribosomal peptide synthetase (NRPS) and unidentified type I polyketide synthase (T1PKS) clusters. These BGCs exhibited unique genetic architecture and encoded several putative enzymes and regulatory elements, suggesting its involvement in the synthesis of bioactive secondary metabolites. Furthermore, comparative genome analysis revealed that these BGCs were distinct from previously characterized gene clusters, indicating the potential for the production of novel compounds. Our findings highlighted the importance of genome mining as a powerful approach for the discovery of biosynthetic gene clusters and the identification of novel bioactive compounds. Further investigations involving expression studies and functional characterization of the identified BGCs will provide valuable insights into the biosynthesis and potential applications of these bioactive compounds.

Prevalence and diversity of ascarid and strongylid nematodes in Australian Thoroughbred horses using next-generation sequencing and bioinformatic tools

The study presents the results of a cross-sectional survey to describe the epidemiology of ascarid and strongylid nematodes in horses, the impact of diverse climatic conditions on parasite diversity and the levels of faecal egg shedding in different age groups of managed Thoroughbred horses. Individual faecal samples (n = 1377) collected from 62 Thoroughbred farms across four climatic zones in Australia were analysed using the modified McMaster technique for faecal egg counts (FECs) and strongylid nematodes were identified utilising PCR-directed next-generation sequencing (NGS) of the second internal transcribed spacer of the nuclear ribosomal DNA (ITS-2). Across all age groups, the prevalence of ascarid and strongylid nematodes was 12% (95% confidence interval 10–14%) and 72% (70–74%), respectively. Based on strongylid FECs, yearlings had the highest prevalence (89%) followed by weanlings (83%), foals (79%), wet mares (61%), dry mares (59%) and stallions (54%). However, for Parascaris spp., foals had the highest prevalence (46%) followed by weanlings (32%) and yearlings (13%). The highest mean FECs for Parascaris spp. were observed in foals (418 eggs per gram [EPG] of faeces) while those for strongylids were in yearlings (1002 EPG). Of the adult horses (mares and stallions), 67% (489 of 729) and 11% (77 of 729) were low (i.e., ≤250 EPG) and moderate (i.e., 251–500 EPG) strongylid egg-shedders, respectively. Strongylid egg shedding varied across climatic zones, with the highest mean FECs in the summer rainfall (723 EPG) followed by non-seasonal rainfall (629 EPG), winter rainfall (613 EPG), and Mediterranean (606 EPG) rainfall zones. Twenty-three nematode species were detected using NGS, with Cylicostephanus longibursatus (28%), Cylicocyclus nassatus (23%) and Coronocyclus coronatus (23%), being the most abundant species. Three species of Strongylus (i.e., S. vulgaris, S. equinus and S. edentatus) were also detected. The nemabiome composition, species richness and relative abundance varied within horse age and between climatic zones. These empirical findings provide a comprehensive understanding of the prevalence of parasites within horse populations and the multifaceted factors that influence their occurrence, thereby allowing for the formulation of tailored strategies aimed at parasite control in domestic horses.

Implementation of Exome Sequencing to Identify Rare Genetic Diseases.

Modern high-throughput genomic testing using next-generation sequencing (NGS) has led to a significant increase in the successful diagnosis of rare genetic disorders. Recent advances in NGS tools and techniques have led to accurate and timely diagnosis of a large proportion of genetic diseases by finding sequence variations in clinical samples. One of the NGS techniques, exome sequencing (ES), is considered as a powerful and easily approachable method for genetic disorders in terms of rapid and cost-effective diagnostic yields. In this chapter, we describe an overview of whole exome sequencing (ES) in the context of experimental and analytical methodologies. Approaches to ES include sequencing capture technique, quality control processes at various stages of sequencing analysis, exome data filtering strategy that incorporates both primary and secondary filtering, and prioritization of candidate variants in diagnosing genetic diseases.

Cilia Disorders in the Genomics Era: Historical Overview and Commentary on Ciliopathy Diagnostics

Introduction: Motile and sensory (primary) cilia are organelles that are found on the surface of almost all cells. Defects in cilia cause a number of multi-organ diseases known as ciliopathies, which have clinically heterogeneous symptoms. This heterogeneity makes diagnosing cilia disorders challenging and clinicians often rely on genetic sequencing to delineate ciliopathies from other diseases. However, there is not a consensus on which sequencing tools are most optimal for ciliopathy diagnosis and research. Methods: Here I review the implications of next-generation sequencing tools for ciliopathy diagnostics. I describe landmark studies that showed ciliopathies as genetic conditions and transition to the advantages and challenges of using next-generation sequencing techniques. In particular, I compare studies that utilized targeted sequencing with those that used whole-exome and/or whole-genome sequencing. Discussion: High throughput screens can identify novel cilia genes and show promise as a robust diagnostic tool in clinical settings. Moreover, I compare the effectiveness of whole-exome and whole-genome sequencing both for basic science research and clinical applications, arguing that whole-exome sequencing is a sufficient first pass in clinical settings. I also acknowledge that ciliopathies are associated with many, both significant and insignificant, genetic variants making interpreting next-generation sequencing data an ongoing challenge for scientists and clinicians. Conclusion: This review demonstrates the increasing body of knowledge on cilia genomics and highlights that next-generation sequencing will be integral towards optimizing diagnostics for these heterogeneous and debilitating group of disorders.

Genomic epidemiological investigation of an outbreak of Serratia marcescens neurosurgical site infections associated with contaminated haircutting toolkits in a hospital barber shop

Nine surgical site infections caused by Serratia marcescens were diagnosed in neurosurgical patients in a 3500-bed hospital between 2nd February and 6th April 2022. To trace the source of infections caused by S.marcescens to expedite termination of the outbreak and prevent future epidemics. A review of all surgical procedures and cultures yielding S.marcescens since February 2022 was conducted. Samples were collected from patients and environmental sources. S.marcescens isolates were characterized by antibiotic susceptibility testing. Whole-genome sequencing (WGS) was used to investigate genetic relationships. Resistance genes, virulence genes and plasmid replicons were identified. S.marcescens was isolated from patients' puncture fluid, cerebrospinal fluid and other secretions, and was also cultured from the barbers' haircutting tools, including leather knives, slicker scrapers and razors. In total, 15 isolates were obtained from patients and eight isolates were obtained from haircutting tools. All isolates exhibited identical antibiotic resistance patterns. WGS revealed close clustering among the 23 isolates which differed significantly from previous strains. Three resistance genes and nine virulence-associated genes were detected in all isolates, and 19 of 23 isolates harboured an MOBP-type plasmid. The results confirmed an outbreak of S.marcescens, which was traced to contaminated haircutting tools in the hospital barber shop. The outbreak ended after extensive reinforcement of infection control procedures and re-education of the barbers. These results highlight the risk of postoperative infections related to pre-operative skin preparation, and demonstrate the value of next-generation sequencing tools to expedite outbreak investigations.

Comparative Genetic Characterization of CTX-M-Producing Escherichia coli Isolated from Humans and Pigs with Diarrhea in Korea Using Next-Generation Sequencing.

Pathogenic E. coli causes intra- and extraintestinal diseases in humans and pigs and third-generation cephalosporins are the primary option for the treatment of these diseases. The objective of this study was to investigate the characteristics and correlation between CTX-M-producing E. coli from humans and pigs regarding CTX-M-producing E. coli using next-generation sequencing and bioinformatic tools. Among the 24 CTX-M-producing E. coli, three types of CTX-M genes (CTX-M-12, CTX-M-14, and CTX-M-15) were detected in humans and four types of CTX-M genes (CTX-M-14, CTX-M-15, CTX-M-55, and CTX-M-101) were detected in pigs. A total of 24 CTX-M-producing E. coli isolates also showed the following antimicrobial resistance genes: other B-Lactam resistance gene (75.0%); aminoglycoside resistance genes (75.0%); phenicol resistance genes (70.8%); tetracycline resistance genes (70.8%); sulfonamide resistance genes (66.7%); quinolone resistance genes (62.5%); trimethoprim resistance genes (54.2%); and fosfomycin resistance genes (8.3%). FII (92.3%) and FIB (90.9%) were the most common plasmid replicon in humans and pigs, respectively. A total of thirty-eight different genes associated with virulence 24 CTX-M-producing E. coli and all isolates contained at least more than one virulence gene. A total of 24 CTX-M-producing E. coli isolates showed 15 diverse sequence types (STs): thirteen isolates from human belonged to 6 different STs, and 11 isolates from pig belonged to 9 different STs. The presence of virulence genes in E. coli together with antimicrobial resistance genes (including CTX-M genes) emphasizes the necessity of comprehensive surveillance and persistent monitoring of the food chain to avoid all types of bacterial contamination, regardless of human or pig origin.

The interplay between vector microbial community and pathogen transmission on the invasive Asian tiger mosquito, Aedes albopictus: current knowledge and future directions.

The invasive Asian tiger mosquito Aedes albopictus is nowadays broadly distributed with established populations in all continents except Antarctica. In the invaded areas, this species represents an important nuisance for humans and, more relevant, it is involved in the local transmission of pathogens relevant under a public health perspective. Aedes albopictus is a competent vector of parasites such as Dirofilaria and viruses including dengue virus, Zika virus, and chikungunya virus, among others. The mosquito microbiota has been identified as one of the major drivers of vector competence, acting upon relevant vector functions as development or immunity. Here, we review the available literature on the interaction between Ae. albopictus microbiota and pathogen transmission and identify the knowledge gaps on the topic. Most studies are strictly focused on the interplay between pathogens and Wolbachia endosymbiont while studies screening whole microbiota are still scarce but increasing in recent years, supported on Next-generation sequencing tools. Most experimental trials use lab-reared mosquitoes or cell lines, exploring the molecular mechanisms of the microbiota-pathogen interaction. Yet, correlational studies on wild populations are underrepresented. Consequently, we still lack sufficient evidence to reveal whether the microbiota of introduced populations of Ae. albopictus differ from those of native populations, or how microbiota is shaped by different environmental and anthropic factors, but especially, how these changes affect the ability of Ae. albopictus to transmit pathogens and favor the occurrence of outbreaks in the colonized areas. Finally, we propose future research directions on this research topic.

A Prospect for Genome Wide Association Studies in Crops

Genome-wide association studies (GWAS) test hundreds of thousands of genetic variants across many genomes to find those statistically associated with a specific trait or disease. A Comprehensive review of studies on biotic resistance, abiotic tolerance, yield-associated characteristics, and metabolic composition is provided. Genome-wide SNP maps have characterized population structure concerning the geographic origin and morphological type and identified patterns of ancient crop diffusion to diverse agroclimatic regions. To better understand the genomic patterns of crop diversification, nucleotide diversity variation, linkage disequilibrium, and recombination rates across the genome are quantified. Results from GWAS can be used for a range of applications. The GWAS approach has proven highly suitable for identifying common SNP-based variants with moderate to significant effects on phenotype. However, the genetic factors underlying some of these associations have been characterized. The vast majority remain unexplained. The development of next-generation sequencing and bioinformatics tools has dramatically improved and is currently being implemented to decipher the genetic diversity of targeted traits. The significant drawbacks are the need for large population size, the cost of preparing DNA samples, and less knowledge about the risk of the quality. To overcome this drawback, researchers have upgraded the statistical approaches, proper imputation of genotypes, and advanced approaches such as nested association mapping and candidate gene association mapping. The primary benefit is one-time genotyping and repeated phenotyping in different environmental conditions. Int. J. Appl. Sci. Biotechnol. Vol 11(2): 54-59.

Harmonized molecular classification; assessment of a single-test ProMisE NGS tool

ObjectivesDespite recommendations for integrating molecular classification of endometrial cancers (EC) into pathology reporting and clinical management, uptake is inconsistent. To assign ProMisE subtype, all molecular components must be available (POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)) and often these are assessed at different stages of care and/or at different centres resulting in delays in treatment. We assessed a single-test DNA-based targeted next generation sequencing (NGS) molecular classifier (ProMisE NGS), comparing concordance and prognostic value to the original ProMisE classifier. MethodsDNA was extracted from formalin-fixed paraffin embedded (FFPE) ECs that had previously undergone ProMisE molecular classification (POLE sequencing, IHC for p53 and MMR). DNA was sequenced using the clinically validated Imagia Canexia Health Find It™ amplicon-based NGS gene panel assay to assess for pathogenic POLE mutations (unchanged from original ProMisE), TP53 mutations (in lieu of p53 IHC), and microsatellite instability (MSI) (in lieu of MMR IHC),with the same order of segregation as original ProMisE used for subtype assignment. Molecular subtype assignment of both classifiers was compared by concordance metrics and Kaplan-Meier survival statistics. ResultsThe new DNA-based NGS molecular classifier (ProMisE NGS) was used to determine the molecular subtype in 164 ECs previously classified with ProMisE. 159/164 cases were concordant with a kappa statistic of 0.96 and an overall accuracy of 0.97. Prognostic differences in progression-free, disease-specific and overall survival between the four molecular subtypes were observed for the new NGS classifier, recapitulating the survival curves of the original ProMisE classifier. ProMisE NGS was 100% concordant between matched biopsy and hysterectomy samples. ConclusionProMisE NGS is feasible on standard FFPE material, demonstrates high concordance with the original ProMisE classifier and maintains prognostic value in EC. This test has the potential to facilitate implementation of molecular classification of EC at the time of first diagnosis.

Assessment of the Microbial Communities in Soil Contaminated with Petroleum Using Next-Generation Sequencing Tools

Microbial communities are known to play a principal role in petroleum degradation. This study tries to determine the composition of bacteria in selected crude oil-contaminated soil from Tabasco and Tamaulipas states, Mexico. We determined the microbial populations living under these conditions. We evaluated the structure and diversity of bacterial communities in the contaminated soil samples. The most abundant phylum is proteobacteria. Next Generation Sequencing (NGS) analysis of the sampled soils from both states revealed that this phylum has the most relative abundance among the identified bacteria phyla. The heatmap represented the relative percentage of each genus within each sample and clustered the four samples into two groups. Moreover, this allowed us to identify many genera in alkaline soil from Tamaulipas, such as Skermanella sp., Azospirillum sp. and Unclassified species from the Rhodospirillaceae family in higher abundance. Meanwhile, in acidic soil from Tabasco, we identified Thalassospira, Unclassified members of the Sphingomonadaceae family and Unclassified members of the Alphaproteobacteria class with higher abundance. Alpha diversity analysis showed a low diversity (Shannon and Simpson index); Chao observed species in both Regions. These results suggest that the bacteria identified in these genera may possess the ability to degrade petroleum, and further studies in the future should elucidate their role in petroleum degradation.

Early diagnosis of congenital muscular pathologies using next-generation sequencing: experiences from a tertiary center in Morocco

BackgroundCongenital muscular dystrophies (CMD) and congenital myopathies (CM) are clinically and genetically heterogeneous groups of neuromuscular disorders resulting in prenatal or early-onset hypotonia, muscle weakness, myogenic pattern, and dystrophic or myopathic features on muscle biopsy. In this study, we provide a genetic and molecular characterization of CMD and CM in Moroccan patients.Patients and methodsIn this cohort, we investigated 23 Moroccan patients from 21 families who consented to genetic testing. Firstly, genetic analysis in the probands was conducted by next-generation sequencing (NGS) technology using two approaches: targeted NGS gene panel and clinical exome sequencing to study the mutational spectrum and to achieve an accurate diagnosis of these hereditary myopathies in Morocco.ResultsNGS data analysis revealed 16 pathogenic variants harbored in 17 unrelated patients that were genetically resolved. The phenotypic forms identified were in order: LAMA2-related CMD (52.94%), LMNA-CMD (23.53%), and RYR1-related congenital myopathy (17.65%). The congenital titinopathy group was less frequent (5.88%). Here, we identified two novel recessive variants in LAMA2 gene: c.2164G > A (p.Glu722Lys), and c.(6992 + 1_6993-1)_(7300 + 1_7301-1)del p.(Pro2332Glnfs*10). Additionally, we expanded the phenotypic spectrum of a known heterozygous LMNA c.1718C > T p.(Ser573Leu) variant, and we report it for the first time to a form of CMD.ConclusionsThe introduction of the NGS tool in clinical practice allowed us to improve the diagnosis and the management of these neuromuscular diseases and to highlight the importance of molecular genetic diagnosis of these disorders that are underestimated in the Moroccan population.

The developmental dynamics in cool season legumes with focus on chickpea.

Chickpea is one of the most widely consumed grain legume world-wide. Advances in next-generation sequencing and genomics tools have led to genetic dissection and identification of potential candidate genes regulating agronomic traits in chickpea. However, the developmental particularities and its potential in reforming the yield and nutritional value remain largely unexplored. Studies in crops such as rice, maize, tomato and pea have highlighted the contribution of key regulator of developmental events in yield related traits. A comprehensive knowledge on the development aspects of a crop can pave way for new vistas to explore. Pea and Medicago are the close relatives of genus Cicer and the basic developmental events in these legumes are similar. However, there are some distinct developmental features in chickpea which hold potential for future crop improvement endeavours. The global chickpea germplasm encompasses wide range of diversities in terms of morphology at both vegetative and reproductive stages. There is an immediate need for understanding the genetic and molecular basis of this diversity and utilizing them for the yield contributing trait improvement. The review discusses some of the key developmental events which have potential in yield enhancement and the lessons which can be learnt from model legumes in this regard.

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

Bicuspid Aortic Valves: an Up-to-Date Review on Genetics, Natural History, and Management.

Bicuspid aortic valve (BAV) is the most common congenital cardiac abnormality. It has a wide spectrum of clinical manifestations including aortic regurgitation (AR), aortic stenosis, and an associated aortopathy with a small but increased risk of aortic dissection. This review describes current knowledge of BAV, from anatomy and genetics to a discussion of multifaceted strategies utilized in the management of this unique patient population. This review will also highlight critical knowledge gaps in areas of basic and clinical research to enhance further understanding of this clinical entity. The current knowledge regarding pathophysiologic mechanisms, screening, and surveillance guidelines for BAV and the associated aortopathy is discussed. We also discuss current management techniques for aortic valve repair versus replacement, indications for aortic surgery (root or ascending aorta), and the emergence of the Ross procedure as a viable management option not only in children, but also in adolescents and adults. The varied clinical phenotype of the BAV, resulting in its specific complex hemodynamic interactions, renders it an entity which is separate and distinct from the tricuspid aortic valve pathologies. While various aortic histopathologic and protein alterations in BAV patients have been described, it remains unclear if these changes are causal or the result of hemodynamic alterations imposed by sheer stress on the intrinsically dysfunctional BAV. Medical management for patients with BAV with AS, AI, or dilated aortic roots/ascending aortas remains challenging and needs further investigation. More than 50% of patients with BAV will undergo AVR during their lifetime, and more than 25% of patients with BAV undergo aortic surgery performed for dilation of the aortic root or ascending aorta, often concurrently with AVR. The search for the ultimate genetic or epigenetic cause of the different bicuspid phenotypes will ultimately be facilitated by the next-generation sequencing tools that allow for study of large populations at low cost. Improvements in diagnostic and stratification criteria to accurately risk assess BAV patients are critical to this process.

Environmental Epigenetics in Soil Ecosystems: Earthworms as Model Organisms.

One of the major emerging concerns within ecotoxicology is the effect of environmental pollutants on epigenetic changes, including DNA methylation, histone modifications, and non-coding RNAs. Epigenetic mechanisms regulate gene expression, meaning that the alterations of epigenetic marks can induce long-term physiological effects that can even be inherited across generations. Many invertebrate species have been used as models in environmental epigenetics, with a special focus on DNA methylation changes caused by environmental perturbations (e.g., pollution). Among soil organisms, earthworms are considered the most relevant sentinel organisms for anthropogenic stress assessment and are widely used as standard models in ecotoxicological testing of soil toxicity. In the last decade, several research groups have focused on assessing the impact of environmental stress on earthworm epigenetic mechanisms and tried to link these mechanisms to the physiological effects. The aim of this review is to give an overview and to critically examine the available literature covering this topic. The high level of earthworm genome methylation for an invertebrate species, responsiveness of epigenome to environmental stimuli, availability of molecular resources, and the possibility to study epigenetic inheritance make earthworms adequate models in environmental epigenomics. However, there are still many knowledge gaps that need to be filled in, before we can fully explore earthworms as models in this field. These include detailed characterization of the methylome using next-generation sequencing tools, exploration of multigenerational and transgenerational effects of pollutants, and information about other epigenetic mechanisms apart from DNA methylation. Moreover, the connection between epigenetic effects and phenotype has to be further explored.