Generation Sequencing Techniques Research Articles

Gene sequencing applications to combat oral-cavity related disorders: a systematic review with meta-analysis

Gene sequencing (GS) has numerous applications in combatting oral-cavity related disorders, including identifying genetic risk factors for diseases, developing targeted therapies, and improving diagnostic methods. It can help identify specific genetic mutations or variations that increase the risk of developing oral-cavity related disorders, such as oral cancer, periodontal disease, and cleft lip and palate. By the means of the following investigation, our primary objective was to assess the impact of GS technique in diagnosing and potentially treating diseases of the oral cavity by the means of a systematic review and meta-analysis. We commenced by defining the terms "gene sequencing," "oral cavity," and "disorders" as the important elements in our investigation's subject. Next, relevant databases like PubMed, Scopus, Embase, Web of Science, and Google Scholar were searched using keywords and synonyms for each concept, such as "genomic sequencing," "DNA sequencing," "oral health," "oral diseases," "dental caries," "periodontal disease," "oral cancer," and "salivary gland disorders." We combined several search terms, such as "gene sequencing AND oral disorders AND periodontal disease" or "oral cancer OR genomic sequencing," to further hone your search results using Boolean operators like "AND" and "OR." The oral cavity analysis obtained by CS in the selected articles revealed that most of the disorders were, in fact, a direct causal event influenced by the oral microbiome. Moreover, each sampled oral cavity evidenced a different microbial community, which predicted the precipitation of benign as well as malignant conditions, though not on a definitive basis. In the last ten years, genomic sequencing had advanced remarkably as majority of our selected studies observed, making it possible to diagnose and treat a variety of oral and maxillofacial disorders, including cancer. It was also used to ascertain a person's genetic make-up as well as to spot numerous genetic abnormalities that can predispose individuals to diseases. Understanding the different sequencing techniques and the resulting genetic anomalies may help with their clinical application and lead to an improvement in illness diagnosis and prognosis as a whole in the field of dentistry.

Review of Six Different Next Generation Sequencing (NGS) Techniques for Forensic Science, including Advantages and Disadvantages

Next-generation sequencing methods have advanced greatly since Sanger sequencing, allowing for increased data yield, productivity, and utility. Read time can be used to categorize the upcoming technological generations. This article summarizes the differences between the two technological paradigms, the second-generation (short-read) kind, and the third-generation (long-read) variety. Popular technologies such as Ion Torrent and Illumina stand in for short-read sequencing methods, whereas Oxford Nanopore and Pacific Biosciences are used to represent long-read sequencing approaches. The introduction of the first next-generation sequencing (NGS) technology about ten years ago completely transformed the study of genetics. Whole genomes are now mapped and published practically weekly as a result of speed and cost advances. The number of scholarly papers and conference presentations highlighting the forensic uses of NGS in multiple forensic genetic laboratories has somewhat increased from the previous year. These results show that NGS provides new opportunities for forensic genomic investigation. To gather more information from multiple specimens in a single experiment, combinations of different markers, such as Short Tandem Repeats (STRs), Single Nucleotide Polymorphisms (SNPs), insertion/deletions, and mRNA, can be used instead of the usual Polymerase Chain Reactions- CE techniques. The most significant forensic STR loci's true spectrum of variation and hitherto unknown STR alleles have been discovered. We will address the possible use of singlemolecule sequencing and NGS in forensic science.

Multi-modal imaging genetics data fusion by deep auto-encoder and self-representation network for Alzheimer's disease diagnosis and biomarkers extraction

Alzheimer's disease (AD) is an incurable neurodegenerative disease, so it is important to intervene in the early stage of the disease. Brain imaging genetics is an effective technique to identify AD-related biomarkers, which can early diagnosis of AD patients once they are clinically verified. With the development of medical imaging and gene sequencing techniques, the association analysis between multi-modal imaging data and genetic data has garnered increasing attention. However, current imaging genetics studies have problem with non-intuitive data fusion. Meanwhile, the characteristics of multi-modal imaging genetics data are high-dimensional, non-linearity, and fewer subjects, so it is necessary to select effective features. In this paper, a multi-modal data fusion framework by deep auto-encoder and self-representation (MFASN) was proposed for early diagnosis of AD. First, a multi-modality brain network was constructed by combining information from the resting-state functional magnetic resonance imaging (fMRI) data and structural magnetic resonance imaging (sMRI) data. Then, we utilized the deep auto-encoder to achieve non-linear transformations and select the informative features. A sparse self-representation module was employed to capture the multi-subspaces structure of the latent representation. At last, a multi-task structured sparse association model was developed to fully mine correlations between the genetic data and multi-modal brain network features. Experiments on AD neuroimaging initiative datasets proved the superiority of the proposed method, while discovering discriminative biomarkers were strongly associated with AD.

Detection of Metabolic Gene of Interest (MGOI) in WGS of Microbacterium barkeri (LMA4)

This communication reports about detection of metabolically active genes of interest cell free extract of actinomycetal isolates from Pond soil. The strain LMA4 previously identified as Microbacterium barkeri, based on biotyping as well as 16 s r RNA sequencing, was observed with moderate bioactivity against test strains of Staphylococcus aureus and Escherichia coli. The Actinomycetal strain was Gram positive, aerial profuse branching bearing rounded spores. The whole genome sequencing was carried using Next Generation Sequencing tools and techniques. The FASTA sequence received from sequencing farm had been subjected to BLASTn analysis and aligned with sequences of databse. The sequence similarity was checked considering percentage of identity, as inferred from online portal. The whole genome sequence of LMA4 had inferred about elution of antibiotic producing genes namely [i] Thiazole synthase protein, [ii] Indole-3-glycerol phosphate synthase Protein, [iii] Antibiotic synthesis MbtH protein, [iv] Antibiotic biosynthesis monooxygenase protein, [v] class II terpene cyclase protein and [vi] YcaO-like family protein in the sequence profile. The genes were identified, ORFs were predicted and more over conserved domain was spotted. The genes were structurally represented using modelling approaches and more so ligand based (target oriented) molecular simulation study had predicted affirmative interactions between receptor and ligand molecule. The receptors were WGS detected gene products, and the ligands were Sortase of Staphylococcus aureus and D-ala-ala ligase of Escherichia coli. The study may pave a way towards extracting the metabolites of interest and implementing as natural cost effective drugs.

Gut microbiota diversity in a dung beetle (Catharsius molossus) across geographical variations and brood ball-mediated microbial transmission.

The dung beetle primarily feeds on the feces of herbivorous animals and play a crucial role in ecological processes like material cycles and soil improvement. This study aims to explore the diversity and composition of the gut microbiota of Catharsius molossus (a renowned dung beetle originating from China and introduced to multiple countries for its ecological value) and exploring whether these gut microbes are transmitted vertically across generations. Using 16S rRNA and ITS rRNA gene sequencing techniques, we described the diversity and composition of gut microbes in C. molossus from different localities and different developmental stages (Egg, young larvae and old larvae). We discovered that the diversity of gut microbiota of dung beetles varied obviously among different geographical localities and different developmental stages, and we also discussed the potential influencing factors. Interestingly, the microbial community structure within the brood balls is more similar to male dung beetle than to that of females, which is consistent with the observation that the brood ball is constructed by the male dung beetle, with the female laying egg in it at the final step. This unique breeding method facilitates offspring in inheriting microbial communities from both the mother and the father. Initially, the larvae's gut microbiota closely mirrors that of the parental gift in these brood balls. As larvae grow, significant changes occur in their gut microbiota, including an increase in symbiotic bacteria like Lactococcus and Enterococcus. Analysis of the gut bacteria of adult dung beetles across various localities and different developmental stages identified nine core genera in adults, contributing to 67.80% of the total microbial abundance, and 11 core genera in beetles at different developmental stages, accounting for 49.13% of the total. Notably, seven genera were common between these two core groups. Our results suggest that Parental gifts can play a role in the vertical transmission of microbes, and the abundance of probiotics increases with larval development, supporting the hypothesis that "larval feeding behavior occurs in two stages: larvae first feed on parental gifts to acquire necessary microbes, then enrich symbiotic microbiota through consuming their own feces."

Molecular study of hpmA and ureR genes among Proteus mirabilis isolates inpatient with urinary tract infection

The most frequently occurring human bacterial infections are urinary tract infections (UTIs). P. mirabilis is one of the common causes of UTIs in humans with complicated UTIs, the formation of stones in the kidney, and long-term indwelling catheters. This study aims to isolate, and identify Proteusmirabilis from patients with UTIs using traditional methods and 16s rRNA gene sequencing techniques, as well as detect some virulence genes (hpm A and ureR) in Proteus mirabilis by using polymerase chain reaction (PCR). A total of five isolates of Proteus mirabilis were isolated from patients with urinary tract infections in June 2022. Identification of isolates depends on microscopic and cultural characteristics and traditional biochemical tests, finally, isolates are identified by target gene 16s rRNA using PCR technique, in addition, this study investigates the genes responsible for virulence factors such as hpmA and hpmR.In this study laboratory diagnosis and molecular analysis (16s rRNA gene) revealed that the five isolates are Proteus mirabilis also this study shows the presence of the hpmAgenes by PCR techniques in the isolates 100%. Our study has shown that 16s rRNA was a strong eclectic power and an excellent method for the identification of bacteria also these study frequency ofureR and hpmA in P.mirabilis isolated from UTIs and these genes have a manifest role in the infection.

Unraveling intragenomic polymorphisms in the high-quality genome of Hypoxylaceae: a comprehensive study of the rDNA cistron

Sequencing ribosomal DNA loci, such as ITS and LSU, are among the first steps to characterize and identify newly isolated fungal strains. Substantial doubt has recently been raised concerning the unequivocal suitability of using this sequence information for inferring inter- and intraspecific relationships, as especially the ITS locus was repeatedly shown to harbor high intragenomic plasticity. The evolutionary mechanisms leading to this phenomenon are poorly understood, but this is about to change, owing to the availability of third generation sequencing techniques. The current study attempts a comprehensive analysis of rDNA cistrons in the genomes of over 40 selected species of Hypoxylaceae (Xylariales), using a combination of 2nd generation (Illumina) and 3rd generation sequencing technologies (Oxford Nanopore), along with extensive usage of bioinformatic tools to obtain high-quality genomes. Our findings revealed high intragenomic polymorphisms in distinct Hypoxylaceae species, indicating the presence of deep rDNA paralogs. Phylogenetic inference of the ITS rDNA showed low support for the primary clades within Hypoxylaceae, while inference using the protein encoding gene RPB2 showed better support. We also found that proteinogenic genes are not prone to intraspecific and intragenomic polymorphisms as much as the rDNA loci. Our study highlights the limitations of solely relying on the rDNA cistron as a barcode for fungi. We propose TUB2 as a new primary barcoding marker for Hypoxylaceae and other Xylariales. Moreover, we demonstrate the feasibility to retrieve some DNA loci that had hitherto not been used for classical phylogenetic studies (such as TEF1) in Xylariales directly from genomes without the necessity to work with primers.

Bird migration research today: some achievements and new challenges

This review deals with the topics of bird migration research that have seen significant progress in the recent years. In these fields, earlier views have been significantly re-considered. They include the manifestation of migration, i.e. length of migratory flights, their speed, duration of stopovers, flight altitude, which taken together form the spatio-temporal realization of migration. New data on this array of research questions make it possible to ask new questions about physiological adaptations that make such flights possible, and factors that govern these processes. Significant progress is also evident in the study of long-distance orientation and navigation of migrating birds. However, it mainly concerns the mechanisms of magnetoreception and use of magnetic cues for orientation. Besides, students of bird migration could not ignore the ‘-omics’ revolution, which, like the studies of the manifestation of migration, was mainly caused by technological advances, mainly by the advent of next generation sequencing techniques. The new data obtained by these methods raised the hope to sort out the genetic basis of endogenous control of bird migration. However, the gap between the expectations and the actual results remains. To close this gap, new research into the molecular and cellular mechanisms that underlie migratory behavior is necessary. The results available now mainly refer to the processes that are relatively well understood, mainly the mechanisms of energy turnover and stress response.

The composition of the blood microbiota and its relationship to osteoporosis-related clinical parameters

Osteoporosis, a systemic bone disease, is characterized by decreased bone mass, deterioration of skeletal structure, and increased bone susceptibility. Age, environment, hormone levels, nutrition, and immunity are all factors that influence bone mass. Currently, intestinal flora has been recently recognized as a key regulator of bone mass. The blood's microbiome role in bone health and in the pathogenesis of osteoporosis remains unknown. In this study, the abundance of various blood's microbial taxa in osteoporosis patients were analyzed. We investigated the associations between prominent bacterial taxa and other clinical indicators (i.e. biochemical, blood cell counts and CT scan). DNA was extracted from the whole blood samples of patients with degenerative bone diseases with or without osteoporosis (i.e. n = 8; ST and n = 12, T group) and healthy controls (n = 4, N group). The 16S rRNA gene sequencing technique was utilized to characterize the blood microbiome taxaThe Shannon–Winner and dilution curves revealed that all the characterized species in the sample and the sequencing data were reliable. The number of bacterial taxa in blood and annotated operational taxonomic units were positively correlated with neutrophils. This support that bacteria exist within or adhere to the neutrophil's membrane. The abundance of Yersinia ruckeri, Rhodanobacter_uncultured bacterium, Enterobacter spp., and Raoultella spp increased in the ST group as compared with the N group. Hence, indicate their potential role in the onset and progression of osteoporosis. These findings provide new insights into the association between blood microbiota and bone health. This study could open a new horizon in exploring the clinical application of blood microbiome to improve bone health.

Comparison of Circulating Variants during the Beginning, Middle and the End of the 4th Wave of COVID-19 in Tehran Province, Iran in 2021.

Whole viral genome sequencing with next generation sequencing (NGS) technique is useful tool for determining the diversity of variants and mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study we have attempted to characterize the mutations and circulating variants of the SARSCoV-2 genome during the 4th wave of COVID-19 pandemic in Tehran, Iran in 2021. We performed complete genome sequencing of 15 SARS-CoV-2 detected from 15 COVID-19 patients during the 4th wave of COVID-19 pandemic with NGS. Three groups of the patients at the beginning, middle and the end of the 4th wave were compared together. We detected alpha and delta variants during the 4th wave of the pandemic. The results illustrated a dominance of amino acid substitution D614G in spike, and the most frequent mutants were N-R203K, G204R, S235F, nsp12-P323L, nsp6-G106del, G107del and F108del. The detection of the virus mutations is a useful procedure for identifying the virus behavior and its genetic evolution in order to improve the efficacy of the monitoring strategies and therapeutic measures.

A Comparative Study of Antibacterial Activity of Leaf and Root Extracts of Sida acuta Burm. F. Against Staphylococcus sciuri Isolated From Acne Using Computer-Aided Technique

This study investigated the significant anti-bacteriological efficacy between leaf and root of Sida acuta Burm. F. extracts against acne inducing bacteria. Twelve samples were collected from twelve persons with facial acne. Nutrient agar was used for the isolation of the bacteria and 16S rRNA gene sequencing technique was used for molecular identification. Bioinformatics analysis was carried out using Basic Local Alignment Search Tools (BLAST) for similarity, Cluster W software for multiple sequence alignment and MEGA 7 software for the phylogenetic analysis. Antibacterial susceptibility test of the bacterial isolates was carried out using the agar well diffusion method. A total number of six bacterial species were identified as Staphylococcus sciuri. The study revealed that control antibiotic- Ciprofloxacin (CFX) inhibited the growth of all the bacterial isolates. Staphylococcus sciuri was susceptible to leaf ethanol extract of Sida acuta at 75 μg/ml and 100 μg/ml and was susceptible to the aqueous root extract at 25 μg/ml and 100 μg/ml while it was susceptible to the ethanol root extract at 2.5 μg/ml. From this study, it was shown that the aqueous root extracts had more zones of inhibition with the maximum inhibition of 45mm at 100 μg/ml. Hence, Sida acuta Burm. F. is a powerful antimicrobial agent.

Molecular analysis of adenovirus strains responsible for gastroenteritis in children, under five, in Tunisia

Purpose of workEnteric Adenovirus (EAdV) is recognized as one of the most commonly identified agents responsible for severe acute gastroenteritis (AGEs) in the stools of infants.We sought to determine the rate of human adenovirus (HAdV) infections, and the genotypic characterization of circulating strains of HAdV in children under 5 years of age with AGEs in university and regional hospitals, located in the Center-East of Tunisia, from January 2014 to December 2016. MethodsA classic PCR was performed on 582 stool samples taken within 5 days of the onset of symptoms. Chosen positive samples were sequenced, and some of the results were confirmed by the Next Generation Sequencing technique (NGS). Partial nucleotide sequences of the Hexon gene obtained in this study were compared with the NCBI GenBank database using BLAST. Multiple sequence alignment and phylogenetic analysis were conducted using MEGA6 software. The phylogenetic tree was generated using the maximum-likelihood method and bootstrap analysis was performed with 1000 replications. ResultsOut of 582 samples, 52 (8.93 %) cases were positive for HAdV, with a male predominance (57.4 %). Phylogenetic analyses showed that Tunisian HAdV strains clustered into five HAdV lineages corresponding to serotypes F41 (14/28), C2 (9/28), C5 (3/28), E4 (1/28), and A18 (1/28). HAdV was more frequent in children aged up to 12 months, as compared to the other age groups. The HAdV activity was noted in almost all the months of the year with a peak in autumn, in 2014 and 2015, and in winter in 2016. ConclusionThis study showed that infections with HAdV species were frequent in children suffering from AGE with the predominance of HAdV F41 and C2. This result underlines the importance of regular monitoring of circulating genotypes, and it could be useful for future epidemiological research.

Genetic Mutations in Primary Refractory Diffuse Large B-Cell Lymphoma: Next Generation Sequencing Analysis

Introduction Diffuse large B-cell lymphoma (DLBCL) accounts for 30-40% of newly diagnosed adult non-Hodgkin lymphoma. About 60-70% of DLBCL patients could be cured with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), while the other patients show chemo-refractory or relapse of disease and have poor prognosis. The prognostic difference between DLBCL patients is not fully understood, and many attempts to classify DLBCL have been made. Recently, with the development of the next generation sequencing (NGS) technique, Schmitz et al. identified four prominent genetic subtypes named MCD, BN2, N1, EZB. In this study, we performed targeted deep sequencing in DLBCL patients and analyzed whether there is any genetic difference between the patients who are primary refractory disease and the patients who are not. Methods Totally, 77 patients who was diagnosed with DLBCL and treated with R-CHOP from January 2021 to April 2023 were included in this analysis. Formalin-fixed, paraffin-embedded (FFPE) tumor samples obtained at diagnosis were archived. Genomic DNA was extracted using QIAmp DNA FFPE tissue kit according to the manufacturer's protocol. Genetic profiling included the targeted deep sequencing of 424 genes, which had been selected based on recurrent mutations from previous studies, and sequenced using an Illumina Nextseq. The threshold of mutation positivity as a variant allele frequency ≥2% for SNV/Indel and as a copy number ≥4.0(gain) for CNV. Patients were assigned as germinal center B-cell (GCB) or non-GCB subtype based on the immunohistochemistry (IHC) algorithm. BCL-2 and MYC FISH were conducted in patients with double-expressor DLBCL with non-GCB subtype. Results Median age of the patients were 66 years (24-86 years), and 49 patients (63.6%) were male. Forty-five patients (58.5%) were advanced stage. Twenty-four patients (31.2%) were GCB subtype and 51 patients (66.2%) were non-GCB subtype. Among the GCB type DLBCL patients, 10 patients were double expressor DLBCL, and there was no case of double hit lymphoma. As a result of targeted deep sequencing, 317 mutations were found in at least 1 patient, and the patients had a median number of 16 mutations (4-50). KMT2D was the most commonly detected mutation (22/77, 29.3%), followed by MYD88 (19/77, 25.3%), PCLO (19/77, 25.3%), PIM1 (19/77, 25.3%), TP53 (18/77, 24.0%). According to NGS analysis, 26 patients (33.85%) were assigned to MCD subgroup, 13 patients (16.9%) to BN2, 11 patients (14.3%) to N1, 10 patients (13.0%) to EZB, and 17 patients (17%) were not assigned to any subrgroup. Median follow-up of the patients was 13.8 months (0.4-22.8 months), and 12 patients (15.6%) relapsed and 5 patients (6.5%) died. Median PFS and OS were not reached. Among relapsed patients, 7 patients were primary refractory to R-CHOP. In primary refractory DLBCL patients, TP53 (3/7, 42.9%) and CIITA (3/7, 42.9%) were most commonly detected. CIITA is known to be associated with immune escape and treatment resistance. CIITA, RASSF54, STAT6, SYK significantly more mutated in the primary refractory patients than the other ( CIITA, 42.9% vs. 8.6%, p=0.031; RASSF54, 28.6% vs. 2.9%, p=0.039; STAT6, 28.6% vs. 1.4%, p=0.021; SYK, 28.6% vs. 2.9%, p=0.039) (Table 1). Conclusion CIITA, RASSF54, STAT6, SYK were frequently mutated in primary refractory DLBCL in this study. Because of the small sample size, however, the prognostic significance of these mutations needed to be studied in further researches.

Comprehensive genomic profiling for oncological advancements by precision medicine.

Considerable advancements in next generation sequencing (NGS) techniques have sparked the use of comprehensive genomic profiling (CGP) as a guiding tool for precision-centered oncological treatments. The past two decades have seen the completion of the human genome project, and the consequential invention of NGS. High-throughput sequencing technologies support the discovery and commonplace use of individualized cancer treatments, specifically immune-centered checkpoint inhibitor therapies, and oncogene and tumor suppressor gene targeted therapies. Nevertheless, CGP is not commonly used in all clinical settings. This review investigates the clinically relevant applications of CGP. Studies published between the years 2000-2023 have shown substantial evidence of the benefits of integrating CGP into routine care practice, while also making important comparisons to current-standard oncological treatment strategies. Findings of a comprehensive genomic profile includes predictive, prognostic, and diagnostic biomarkers, together with somatic mutation identification which can indicate the efficacy of immunotherapies and molecularly guided therapies. This review highlights the importance of CGP in identifying driver mutations in tumors that subsequently can be effectively targeted with molecular therapeutics and lead to drug discovery, allowing for increased precision in treating tumors selectively based on their specific genetic mutations, thereby improving patient outcomes.

Sputum microbiota and inflammatory subtypes in asthma, COPD, and its overlap

BackgroundAirway microbiota in asthma-COPD overlap (ACO) remains unknown. ObjectiveThis study with ACO-enriched population aimed to clarify airway microbiota in ACO and in mixed granulocytic inflammation, often detected in ACO and chronic airway diseases. MethodsThis is an observational cross-sectional study. Patients with asthma with airflow limitation, ACO and COPD were enrolled. Blood tests, pulmonary function, exhaled nitric oxide and sputum tests were conducted. Sputum microbiota was evaluated using the 16S rRNA gene sequencing technique. ResultsA total of 112 patients (13 asthma, 67 ACO, 32 COPD) were examined. There were no significant differences in α-diversity among the three diseases. The relative abundances of phylum Bacteroidetes, class Bacteroidia, and genus Porphyromonas were associated with decreased eosinophilic inflammation, and were significantly lower in ACO than in COPD. In a comparison of sputum inflammatory subtypes, the proportion of Haemophilus was numerically highest in the mixed granulocytic subtype, followed by the neutrophilic subtype. Likewise, the proportion of Haemophilus was the highest in the intermediate-high (2%–8%) sputum eosinophil group and lowest in the severe (8%≤) eosinophil group. Clinically, Haemophilus proportion was associated with sputum symptoms. Finally, the proportion of Streptococcus was associated with higher blood eosinophil counts and severest airflow limitation. ConclusionBacteroidia and Porphyromonas abundances in sputum are associated with the eosinophil-low phenotype, and ACO may be characterised by a decrease in these taxa. A mild elevation of sputum eosinophil does not preclude the presence of Haemophilus, which should be noted in the management of obstructive airway diseases.

Non-invasive prenatal testing: a revolutionary journey in prenatal testing.

Non-invasive prenatal testing (NIPT) is a pioneering technique that has consistently advanced the field of prenatal testing to detect genetic abnormalities and conditions with the aim of decreasing the incidence and prevalence of inherited conditions. NIPT remains a method of choice for common autosomal aneuploidies, mostly trisomy 21, and several monogenic disorders. The advancements in gene sequencing techniques have expanded the panel of conditions where NIPT could be offered. However, basic research on the impact of several genetic conditions lags behind the methods of detection of these sequence aberrations, and the impact of the expansion of NIPT should be carefully considered based on its utility. With interest from commercial diagnostics and a lack of regulatory oversight, there remains a need for careful validation of the predictive values of different tests offered. NIPT comes with many challenges, including ethical and economic issues. The scientific evidence, technical feasibility, and clinical benefit of NIPT need to be carefully investigated before new tests and developments are translated into clinical practice. Moreover, the implementation of panel expansion of NIPT should accompany expert genetic counseling pre- and post-testing.

Improving diagnostic precision in primary ovarian insufficiency using comprehensive genetic and autoantibody testing.

Is it possible to find the cause of primary ovarian insufficiency (POI) in more women by extensive screening? Adding next generation sequencing techniques including a POI-associated gene panel, extended whole exome sequencing data, as well as specific autoantibody assays to the recommended diagnostic investigations increased the determination of a potential etiological diagnosis of POI from 11% to 41%. POI affects ∼1% of women. Clinical presentations and pathogenic mechanisms are heterogeneous and include genetic, autoimmune, and environmental factors, but the underlying etiology remains unknown in the majority of cases. Prospective cross-sectional study of 100 women with newly diagnosed POI of unknown cause consecutively referred to Haukeland University Hospital, Bergen, Norway, January 2019 to December 2021. In addition to standard recommended diagnostic investigations including screening for chromosomal anomalies and premutations in the fragile X mental retardation 1 gene (FMR1) we used whole exome sequencing, including targeted analysis of 103 ovarian-related genes, and assays of autoantibodies against steroid cell antigens. We identified chromosomal aberrations in 8%, FMR1 premutations in 3%, genetic variants related to POI in 16%, and autoimmune POI in 3%. Furthermore in 11% we identified POI associated genetic Variants of unknown signifcance (VUS). A homozygous pathogenic variant in the ZSWIM7 gene (NM_001042697.2) was found in two women, corroborating this as a novel cause of monogenic POI. No associations between phenotypes and genotypes were found. Use of candidate genetic and autoimmune markers limit the possibility to discover new markers. To further investigate the genetic variants, family studies would have been useful. We found a relatively high proportion of genetic variants in women from Africa and lack of genetic diversity in the genomic databases can impact diagnostic accuracy. Since no specific clinical or biochemical markers predicted the underlying cause of POI discussion of which tests should be part of diagnostic screening in clinical practice remains open. New technology has altered the availability and effectiveness of genetic testing, and cost-effectiveness analyses are required to aid sustainable diagnostics. The study was supported by grants and fellowships from Stiftelsen Kristian Gerhard Jebsen, the Novonordisk Foundation, the Norwegian Research Council, University of Bergen, and the Regional Health Authorities of Western Norway. The authors declare no conflict of interest. NCT04082169.

Development and validation of a next-generation sequencing panel for clinical pharmacogenetics

The rapid clinical implementation of next generation sequencing techniques is due to its ability to sequence a large number of genetic regions at lower costs than conventional techniques. However, its use in the field of pharmacogenetics is still very limited. ObjectiveDesign, development, implementation and validation of a clinical pharmacogenetics next-generation sequencing panel. MethodWe developed a panel of hybrid capture probes (SureSelect®) for the analysis of the genetic regions of clinical interest collected by literature search and using Illumina HiSeq 1500® sequencing platform. We developed a bioinformatic algorithm for variant annotation, haplotype inference and determination of structural variants in the genes of interest. The results obtained were validated with Coriell® reference material from the pharmacogenetic repositories. ResultsThe developed panel allows the study of a total of 12,794 regions comprised in 389 genes. Validation results showed a sensitivity greater than 99% for single nucleotide variants and small INDELs. Haplotype imputation was consistent with the consensus results in the characterized reference materials. Furthermore, the developed tool was able to correctly identify different types of CYP2D6 copy number variations as well as a wide variety of HLA-B alleles. ConclusionsThis technology represents an appropriate alternative for its clinical use with advantages over conventional techniques in its throughput and complex gene study capabilities (CYP2D6, HLA-B).

Prickly Ash Seeds improve immunity of Hu sheep by changing the diversity and structure of gut microbiota.

Prickly Ash Seeds (PAS), as a traditional Chinese medicinal herb, have pharmacological effects such as anti-asthma, anti-thrombotic, and anti-bacterial, but their impact on gut microbiota is still unclear. This study used a full-length 16 s rRNA gene sequencing technique to determine the effect of adding PAS to the diet on the structure and distribution of gut microbiota in Hu sheep. All lambs were randomly divided into two groups, the CK group was fed with a basal ration, and the LZS group was given a basal diet with 3% of PAS added to the ration. The levels of inflammatory factors (IL-10, IL-1β, and TNF-α) in intestinal tissues were measured by enzyme-linked immunosorbent assay (ELISA) for Hu sheep in the CK and LZS group. The results indicate that PAS can increase the diversity and richness of gut microbiota, and can affect the community composition of gut microbiota. LEfSe analysis revealed that Verrucomicrobiota, Kiritimatiella, WCHB 41, and uncultured_rumen_bacterium were significantly enriched in the LZS group. KEGG pathway analysis found that LZS was significantly higher than the CK group in the Excretory system, Folding, sorting and degradation, and Immune system pathways (p < 0.05). The results of ELISA assay showed that the level of IL-10 was significantly higher in the LZS group than in the CK group (p < 0.05), and the levels of TNF-α and IL-1β were significantly higher in the CK group than in the LZS group (p < 0.05). LEfSe analysis revealed that the dominant flora in the large intestine segment changed from Bacteroidota and Gammaproteobacteria to Akkermansiaceae and Verrucomicrobiae after PAS addition to Hu sheep lambs; the dominant flora in the small intestine segment changed from Lactobacillales and Aeriscardovia to Kiritimatiellae and WCHB1 41. In conclusion, the addition of PAS to sheep diets can increase the number and types of beneficial bacteria in the intestinal tract, improve lamb immunity, and reduce intestinal inflammation. It provides new insights into healthy sheep production.

ESR1 Gene Mutations and Liquid Biopsy in ER-Positive Breast Cancers: A Small Step Forward, a Giant Leap for Personalization of Endocrine Therapy?

The predominant forms of breast cancer (BC) are hormone receptor-positive (HR+) tumors characterized by the expression of estrogen receptors (ERs) and/or progesterone receptors (PRs). Patients with HR+ tumors can benefit from endocrine therapy (ET). Three types of ET are approved for the treatment of HR+ BCs and include selective ER modulators, aromatase inhibitors, and selective ER downregulators. ET is the mainstay of adjuvant treatment in the early setting and the backbone of the first-line treatment in an advanced setting; however, the emergence of acquired resistance can lead to cancer recurrence or progression. The mechanisms of ET resistance are often related to the occurrence of mutations in the ESR1 gene, which encodes the ER-alpha protein. As ESR1 mutations are hardly detectable at diagnosis but are present in 30% to 40% of advanced BC (ABC) after treatment, the timeline of testing is crucial. To manage this resistance, ESR1 testing has recently been recommended; in ER+ HER2- ABC and circulating cell-free DNA, so-called liquid biopsy appears to be the most convenient way to detect the emergence of ESR1 mutations. Technically, several options exist, including Next Generation Sequencing and ultra-sensitive PCR-based techniques. In this context, personalization of ET through the surveillance of ESR1 mutations in the plasma of HR+ BC patients throughout the disease course represents an innovative way to improve the standard of care.