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Estimates of the spectrum and frequency of pathogenic variants in Parkinsons disease (PD) in different populations are currently limited and biased. Furthermore, although therapeutic modification of several genetic targets has reached the clinical trial stage, a major obstacle in conducting these trials is that PD patients are largely unaware of their genetic status and, therefore, cannot be recruited. Expanding the number of investigated PD-related genes and including genes related to disorders with overlapping clinical features in large, well-phenotyped PD patient groups is a prerequisite for capturing the full variant spectrum underlying PD and for stratifying and prioritizing patients for gene-targeted clinical trials. The Rostock Parkinsons disease (ROPAD) study is an observational clinical study aiming to determine the frequency and spectrum of genetic variants contributing to PD in a large international cohort. We investigated variants in 50 genes with either an established relevance for PD or possible phenotypic overlap in a group of 12 580 PD patients from 16 countries [62.3% male; 92.0% White; 27.0% positive family history (FH+), median age at onset (AAO) 59 years] using a next-generation sequencing panel. Altogether, in 1864 (14.8%) ROPAD participants (58.1% male; 91.0% White, 35.5% FH+, median AAO 55 years), a PD-relevant genetic test (PDGT) was positive based on GBA1 risk variants (10.4%) or pathogenic/likely pathogenic variants in LRRK2 (2.9%), PRKN (0.9%), SNCA (0.2%) or PINK1 (0.1%) or a combination of two genetic findings in two genes (∼0.2%). Of note, the adjusted positive PDGT fraction, i.e. the fraction of positive PDGTs per country weighted by the fraction of the population of the world that they represent, was 14.5%. Positive PDGTs were identified in 19.9% of patients with an AAO ≤ 50 years, in 19.5% of patients with FH+ and in 26.9% with an AAO ≤ 50 years and FH+. In comparison to the idiopathic PD group (6846 patients with benign variants), the positive PDGT group had a significantly lower AAO (4 years, P = 9 × 10-34). The probability of a positive PDGT decreased by 3% with every additional AAO year (P = 1 × 10-35). Female patients were 22% more likely to have a positive PDGT (P = 3 × 10-4), and for individuals with FH+ this likelihood was 55% higher (P = 1 × 10-14). About 0.8% of the ROPAD participants had positive genetic testing findings in parkinsonism-, dystonia/dyskinesia- or dementia-related genes. In the emerging era of gene-targeted PD clinical trials, our finding that ∼15% of patients harbour potentially actionable genetic variants offers an important prospect to affected individuals and their families and underlines the need for genetic testing in PD patients. Thus, the insights from the ROPAD study allow for data-driven, differential genetic counselling across the spectrum of different AAOs and family histories and promote a possible policy change in the application of genetic testing as a routine part of patient evaluation and care in PD.

Myelodysplastic neoplasm (MDS) is a heterogeneous group of clonal hematological disorders that originate from the hematopoietic and progenitor cells and present with cytopenias and morphologic dysplasia with a propensity to progress to bone marrow failure or acute myeloid leukemia (AML). Genetic evolution plays a critical role in the pathogenesis, progression, and clinical outcomes of MDS. This process involves the acquisition of genetic mutations in stem cells that confer a selective growth advantage, leading to clonal expansion and the eventual development of MDS. With the advent of next-generation sequencing (NGS) assays, an increasing number of molecular aberrations have been discovered in recent years. The knowledge of molecular events in MDS has led to an improved understanding of the disease process, including the evolution of the disease and prognosis, and has paved the way for targeted therapy. The 2022 World Health Organization (WHO) Classification and the International Consensus Classification (ICC) have incorporated the molecular signature into the classification system for MDS. In addition, specific germline mutations are associated with MDS development, especially in pediatrics and young adults. This article reviews the genetic abnormalities of MDS in adults with a brief review of germline predisposition syndromes.

CONTEXT: Next-generation sequencing (NGS) analysis of sporadic medullary thyroid carcinoma (sMTC) has led to increased detection of somatic mutations, including RET M918T, which has been considered a negative prognostic indicator. OBJECTIVE: This study aimed to determine the association between clinicopathologic behavior and somatic mutation identified on clinically motivated NGS. METHODS: In this retrospective cohort study, patients with sMTC who underwent NGS to identify somatic mutations for treatment planning were identified. Clinicopathologic factors, time to distant metastatic disease (DMD), disease-specific survival (DSS), and overall survival (OS) were compared between somatic mutations. RESULTS: Somatic mutations were identified in 191 sMTC tumors, including RET M918T (53.4%), other RET codons (10.5%), RAS (18.3%), somatic RET indels (8.9%), and RET/RAS wild-type (WT) status (8.9%). The median age at diagnosis was 50 years (range, 11-83); 46.1% were female. When comparing patients with RET M918T, RET-Other, and RET WT (which included RAS and RET/RAS WT), there were no differences in sex, TNM category, systemic therapy use, time to DMD, DSS, or OS. On multivariate analysis, older age at diagnosis (HR 1.05, P < .001; HR 1.06, P< .001) and M1 stage at diagnosis (HR 3.17, P = .001; HR 2.98, P = .001) were associated with decreased DSS and OS, respectively, but mutation cohort was not. When comparing RET M918T to RET indels there was no significant difference in time to DMD, DSS, or OS between the groups. CONCLUSION: Somatic RET mutations do not portend compromised DSS or OS in a cohort of sMTC patients who underwent clinically motivated NGS.

BACKGROUND: Hmong-Mien (HM) speakers are linguistically related and live primarily in China, but little is known about their ancestral origins or the evolutionary mechanism shaping their genomic diversity. In particular, the lack of whole-genome sequencing data on the Yao population has prevented a full investigation of the origins and evolutionary history of HM speakers. As such, their origins are debatable. RESULTS: Here, we made a deep sequencing effort of 80 Yao genomes, and our analysis together with 28 East Asian populations and 968 ancient Asian genomes suggested that there is a strong genetic basis for the formation of the HM language family. We estimated that the most recent common ancestor dates to 5800 years ago, while the genetic divergence between the HM and Tai-Kadai speakers was estimated to be 8200 years ago. We proposed that HM speakers originated from the Yangtze River Basin and spread with agricultural civilization. We identified highly differentiated variants between HM and Han Chinese, in particular, a deafness-related missense variant (rs72474224) in the GJB2 gene is in a higher frequency in HM speakers than in others. CONCLUSIONS: Our results indicated complex gene flow and medically relevant variants involved in the HM speakers evolution history.

Protein display systems are powerful techniques used to identify protein molecules that bind with high affinity to target proteins of interest. The initial challenge in implementing a display system is the construction of a high-diversity naïve library. Here, we describe the methods to generate a designed ankyrin repeat protein (DARPin) display library using degenerate oligonucleotides. Specifically described is the construction of a single DARPin repeat module by overlap extension PCR, concatenation of the module by restriction enzyme digestion and ligation, and incorporation of the concatenated modules into a full-length DARPin sequence in a bacterial cloning or display vector containing the hydrophilic N- and C-terminal capping domains. Protocols for PCR amplification of DARPin sequences to estimate diversity of naïve and enriched libraries via next-generation sequencing are included, as is a simple Linux-based program for analysis of naïve and enriched sequences. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of a single DARPin repeat by overlap extension PCR Basic Protocol 2: Concatenation of DARPin repeats Basic Protocol 3: Ligation of internal repeats into cloning/display vector containing N- and C-terminal capping repeats Basic Protocol 4: Estimation of library size and diversity by next-generation sequencing (NGS) Basic Protocol 5: NGS analysis of naïve and enriched libraries.

Single nucleotide polymorphisms (SNPs) in homologous regions play a critical role in the field of genetics. However, genotyping these SNPs is challenging due to the presence of repetitive sequences within genome, which demand specific method. We introduce a new, mid-throughput method that simplifies SNP genotyping in homologous DNA sequences by utilizing a combination of multiplex kb level PCR (PCR size 2.5k-3.5 kb) for capturing targeted regions and multiplex nested PCR library construction for next-generation sequencing (Multi-kb level capture-seq). First of all, we randomly selected 7 SNPs in homologous regions and successfully captured 6-plex kb level amplicons (one of segments contains 2 SNPs, while the remaining segments each have only one SNP) in a single tube. And then, the amplification products were subjected to multiplex nested PCR for library construction and sequenced on Illumina platform. We tested this strategy using 600 amplicons from 100 samples and accurately genotyped 96.8% of target SNPs with a coverage depth of ≥ 15×. For the uniformity within the samples, over 66.7% (4/6) of the amplicons had a coverage depth above 0.2-fold of average sequencing depth. To validate the accuracy of this approach, we performed Ligase detection reaction PCR for genotyping the 100 samples, and found that the genotyping data was 97.71% consistent with our NGS results. In conclusion, we have developed a highly efficient and accurate method for SNP genotyping in homologous regions, which offers researchers a new strategy to explore the complex regions of genome. [ABSTRACT FROM AUTHOR]

Summary Background Methods Results Conclusion The diagnosis of monogenic obesity is burdened by frequent variants of uncertain significance (VUS). We describe our real‐life approach of variant reassessment over time and we assess whether inconclusive variants are decreasing in monogenic obesity.We tested for monogenic obesity (genes: LEPR, POMC, ADCY3, PCSK1, CARTPT, SIM1, MRAP2, LEP, NTRK2, BDNF, KSR2, MAGEL2, SH2B1, MC4R, MC3R) in 101 children/adolescents (11.7 [7.3–13.7] years, 3.6 [3.3–4.0] z‐BMI) in Verona and 183 (11.3 [8.4–12.2] years, 3.2 [2.7–3.9] z‐BMI) in Naples from January 2020 to February 2023. In March–July 2024 we reassessed the baseline variants by updated software interpretation and literature renavigation.We initially found 20 VUS, 4 Likely Pathogenic (LP), 5 Likely Benign (LB) and 1 benign variant in 33 individuals. At follow‐up, 6 VUS were reclassified as benign/LB, one LP as pathogenic and 3 LB as benign. Overall, 10/30 variants (6/18 in Verona, 3/11 in Naples and a variant found in both centres) were reclassified, leading to a less uncertain report for 13 of 33 variant‐carrying patients. Monogenic obesity was diagnosed in 3 probands in Verona and 4 in Naples, carrying variants at MC4R or NTRK2.Our variant reassessment was effective to improve classification certainty for the 39% of patients and suggested that the molecular diagnosis of monogenic obesity is becoming more accurate over time. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndromes/neoplasms (MDS) and acute myeloid leukaemia (AML) are neoplastic haematopoietic cell proliferations that are diagnosed and classified based on a combination of morphological, clinical and genetic features. Specifically, the percentage of myeloblasts in the blood and bone marrow is a key feature that has historically separated MDS from AML and, together with several other morphological parameters, defines distinct disease entities within MDS. Both MDS and AML have recurrent genetic abnormalities that are increasingly influencing their definitions and subclassification. For example, in 2022, two new MDS entities were recognised based on the presence of SF3B1 mutation or bi‐allelic TP53 abnormalities. Genomic information is more objective and reproducible than morphological analyses, which are subject to interobserver variability and arbitrary numeric cut‐offs. Nevertheless, the integration of genomic data with traditional morphological features in myeloid neoplasm classification has proved challenging by virtue of its sheer complexity; gene expression and methylation profiling also can provide information regarding disease pathogenesis, adding to the complexity. New machine‐learning technologies have the potential to effectively integrate multiple diagnostic modalities and improve on historical classification systems. Going forward, the application of machine learning and advanced statistical methods to large patient cohorts can refine future classifications by advancing unbiased and robust previously unrecognised disease subgroups. Future classifications will probably incorporate these newer technologies and higher‐level analyses that emphasise genomic disease entities over traditional morphologically defined entities, thus promoting more accurate diagnosis and patient risk stratification. [ABSTRACT FROM AUTHOR]

Purpose: MYB has been shown to play a central role in oncogenesis in a majority of adenoid cystic carcinomas (ACC). Testing for MYB expression via immunohistochemistry (IHC) or testing for the MYB gene fusion by next-generation sequencing (NGS) have become useful tools for the diagnosis of ACC. In addition, detection of MYB expression may have implications for patient management. Methods: A cohort of 35 ACC cases was identified from the archival pathology files of the Massachusetts General Hospital. Cases were tested for MYB expression using a panel of 4 different commercially available MYB antibodies and scored using a modified Allred system. RNA-based NGS for MYB gene fusion detection was also performed. Results: Among 4 different MYB antibodies, the sensitivity for MYB detection ranged from 26 to 97%. When a 30% threshold for determination of MYB immunohistochemical positivity was used, the AB_10900735 IHC clone showed the maximum sensitivity (97%). RNA sequencing revealed 19 (54%) cases positive for MYB fusions, and expression analysis derived from the sequencing data confirmed a significant association between MYB expression and fusion status (p = 0.036). Although less sensitive, the AB_778878 MYB clone showed a significant positive association between IHC staining and MYB RNA expression (R2 = 0.15, p = 0.023). Conclusion: The detection of MYB expression using immunohistochemistry varies significantly depending on the antibody used. Comparison with MYB fusion and transcription levels, as determined by NGS, reveals that MYB has a complex relationship between genetic alterations, transcript levels, and protein abundance. [ABSTRACT FROM AUTHOR]

Dysregulation of DNA methylation is associated with human disease, particularly cancer, and the assessment of aberrant methylation patterns holds great promise for clinical diagnostics. However, DNA polymerases do not effectively discriminate between processing 5‐methylcytosine (5 mC) and unmethylated cytosine, resulting in the silencing of methylation information during amplification or sequencing. As a result, current detection methods require multi‐step DNA conversion treatments or careful analysis of sequencing data to decipher individual 5 mC bases. To overcome these challenges, we propose a novel DNA polymerase‐mediated 5 mC detection approach. Here, we describe the engineering of a thermostable DNA polymerase variant derived from Thermus aquaticus with altered fidelity towards 5 mC. Using a screening‐based evolutionary approach, we have identified a DNA polymerase that exhibits increased misincorporation towards 5 mC during DNA synthesis. This DNA polymerase generates mutation signatures at methylated CpG sites, allowing direct detection of 5 mC by reading an increased error rate after sequencing without prior treatment of the sample DNA. [ABSTRACT FROM AUTHOR]

Background: Brain metastasis (BM) is a rare but severe complication of head and neck squamous cell carcinoma (HNSCC), with limited knowledge of molecular characteristics and immunogenicity. Methods: We analyzed 61 cases of HNSCC-BM from three academic institutions (n = 24) and Foundation Medicine Inc (FMI, n = 37). A subset of cases underwent next-generation sequencing, multiple immunofluorescence, and proximity ligation sequencing. Gene enrichment analysis compared alterations in FMI BM samples (n = 37) with local samples (n = 4082). Results: Demographics included: median age of 59 years, 75% male, 55% current/former smokers, 75% oropharyngeal primary, and 67% human papillomavirus (HPV) +. ATM (54%), KMT2A (54%), PTEN (46%), RB1 (46%), and TP53 (46%) were frequently altered in BM samples from academic centers (62% HPV/p16+). Structural rearrangements ranged from 9 to 90 variants by proximity ligation sequencing. BMs had low densities of CD8+, PD-1+, PD-L1+, and FOXP3 + cells, and 92% had PD-L1 combined positive scores < 1%. CDKN2A (40.5%), TP53 (37.8%), and PIK3CA (27.0%) alterations were common in the FMI BMs (51% HPV+). MAP2K2 alterations and HPV + signature were enriched in FMI BMs compared to local tumors (11.8% vs. 6.4%, P = 0.005 and 51.25% vs. 26.11%, P = 0.001 respectively), and pathogenic TSC1 inactivating mutations were enriched in local tumors (67.3% vs. 37.8%, P = 0.008). Median overall survival from BM diagnosis was 9 months (range 0–27). Conclusions: HNSCC patients with BM frequently have oropharyngeal primary sites and are HPV+. Common molecular alterations in BM samples, including targetable PIK3CA and ATM, were identified. MAP2K2 alterations were enriched and densities of immune cells were low, highlighting potential targets for further research and immunotherapy considerations. [ABSTRACT FROM AUTHOR]

Introduction: Faced to the growing development of collecting systematic molecular analyses in relapsed pediatric cancers to transform their targeted matched therapies, this study aimed to assess the clinical and therapeutic indications of systematic diagnostic genomic explorations performed in pediatric solid cancers to determine which type of screening and if it afford at relapse time an accurate targeted strategy. Methods: A total of 280 patients less than 22 years, referred at the University Hospitals of Strasbourg for a newly diagnosed solid tumor from January 2015 to December 2021, were prospectively genomically investigated since diagnosis. Using 7 different molecular tests going from single-gene methods (IHC, FISH, RT-PCR, Sanger sequencing, droplet digital PCR) to largescale analyses (Next-Generation sequencing, RNAsequencing and FoundationOne®CDx), we explored retrospectively the molecular findings in those pediatric solid tumors (except hematolymphoid cancers) to improve diagnosis, prognosis assessment and relapse therapeutics. Results: One hundred and ninety-eight patients (71%) underwent molecular biology (MB) at diagnosis. Thirty-eight different histologies were grouped into cerebral tumors (30%), sarcomas (26%, bone and soft tissues), various blastomas (27%), and other entities (17%). Over a median 40-month follow-up, the overall survival rate of patients was 85% and the relapse rate 28%. Of the 326 analyses carried out, 245 abnormalities (single nucleotide variations: 50%, fusions: 25%, copy number alteration: 20%) concerning 70 oncogenes were highlighted. The overall clinical impact rate was 84%. Broad-spectrum analyses had a higher therapeutic impact (57%) than the targeted analyses (28%). 75% of broad-spectrum tests found an actionable variant conducting 23% of patients to receive rapidly a matched targeted therapy since first relapse. Conclusion: Our experience highlighted the clinical utility of molecular profiling of solid tumors as soon as at diagnosis in children to expect improving access to innovative agents at relapse. [ABSTRACT FROM AUTHOR]

AbstractBackgroundAims/ObjectivesMaterial and MethodsResultsConclusions and SignificanceWhile cochlear implantation (CI) and electric acoustic stimulation (EAS) have a positive outcome in most cases, their effectiveness varies depending on the etiology of the hearing loss. Among the various etiologies, genetic factors are the leading cause of hearing loss and may impact CI and EAS outcomes.To reveal the genetic background of the hearing loss in CI/EAS patients in each ethnic population, we undertook a multi-center study involving the genetic testing of hearing loss in CI/EAS patients from 10 centers.Saliva samples and clinical information for the patients and their family members were obtained and next-generation sequencing analysis using a panel carrying 63 deafness genes was then performed.Genetic testing successfully identified the causative gene variants in 54.5% (48/88) of patients with pre-lingual onset hearing loss (onset under 6 years) and in 12% (12/95) of those with late-onset hearing loss (onset at 6 years or more).We clearly indicated that genetic factors are the most common cause of hearing loss regardless of ethnic background. Saliva-based genetic testing is a useful tool for multi-center studies seeking to clarify the genetic causes of hearing loss in CI or EAS patients between countries separated by distance. [ABSTRACT FROM AUTHOR]

ABSTRACT PTPRQ plays an important role in the development of inner ear hair cell stereocilia. While many autosomal recessive variants in PTPRQ have been identified as the pathogenic cause for nonsyndromic hearing loss (DFNB84A), so far only one autosomal dominant PTPRQ variant, c.6881G>A (p.Trp2294*), has been reported for late‐onset, mild‐to‐severe hearing loss (DFNA73). By using targeted next‐generation sequencing, this study identified a novel PTPRQ truncating pathogenic variant, c.3697del (p.Leu1233Phefs*11), from a Chinese Han family that co‐segregated with autosomal dominant, postlingual, progressive hearing loss. A Ptprq‐3700del knock‐in mouse model was generated by CRISPR‐Cas9 and characterized for its hearing function and inner ear morphology. While the homozygous knock‐in mice exhibit profound hearing loss at all frequencies at the age of 3 weeks, the heterozygous mutant mice resemble the human patients in mild, progressive hearing loss from age 3 to 12 weeks, primarily affecting high frequencies. At this stage, the homozygous knock‐in mice have a normal hair cell count but disorganized stereocilia. Cochlear proteosome analysis of the homozygous mutant mice revealed differentially expressed genes and pathways involved in oxidative phosphorylation, regulation of angiogenesis and synaptic vesicle cycling. Our study provides a valuable animal model for further functional studies of the pathogenic mechanisms underlying DFNA73. [ABSTRACT FROM AUTHOR]

Background: Preconception expanded carrier screening (ECS) is a genetic test that enables the identification of at-risk carriers of recessive disorders by screening for up to hundreds of genes. Next-generation sequencing (NGS) development has paved the way for its integration into ECS. This study aims to identify the carrier genetic status of couples experiencing or anticipating conception challenges through NGS-based ECS and to gain an overview of the rare genetic disorders in a population with increased consanguinity. Methods: Thirty couples who presented to the Genetic Disease Clinic between 2015 and 2024 with failed reproductive outcomes or with a positive personal or family history of genetic disorders and underwent ECS were included and retrospectively analyzed. Results: Fifty-four individuals (90.00%) were found to carry at least one variant of 95 identified genes, totaling 174 variants. Six individuals (10.00%) tested negative for any variant. Seven individuals had one variant (11.67%), 13 had two variants (21.67%), and 34 had 3 or more variants (56.67%). The most common variants identified were of HBA, HBB, CYP21A2, and G6PD genes. Most of the detected variants were unknown or unexpected (n = 143, 82.18%). Eight couples carried two or more variants in common. Consanguinity was reported in 14 couples (46.67%). Conclusions: Preconception ECS is crucial for reproductive planning, permitting couples to evaluate their combined genetic risks and make informed decisions, reducing the chance of having children with genetic disorders. [ABSTRACT FROM AUTHOR]

Tay-Sachs disease (TSD) is a rare genetic disorder with diverse clinical manifestations, often leading to underdiagnosis due to symptom similarities with other neurological conditions. In this study, we aimed to identify the genetic mutations underlying late-onset TSD in a 27-year-old patient with progressive neurological symptoms. Whole-exome sequencing revealed two hexA gene mutations associated with TSD: a previously known variant, c.805G > A (p.Gly269Ser), and a novel splice-site mutation, c.346 + 2dupT. Through clinical assessments, genetic analysis, and functional investigations--including RNA sequencing and enzymatic activity assays--we confirmed the pathogenicity of the novel mutation. Our findings highlight the efficacy of advanced genomic technologies in diagnosing intricate genetic disorders and emphasize the significance of functional validation to confirm the effects of mutations. Identifying compound heterozygous mutations in the hexA gene also provides insight into Mendelian inheritance patterns. This case highlights the diagnostic challenges posed by overlapping clinical phenotypes and emphasizes the need for increased genetic awareness among clinicians. Accurate diagnosis of TSD has significant implications for patients and their families, allowing for informed genetic counseling and guiding clinical management decisions. While current treatment options are limited, timely and accurate diagnosis holds promise for future research and therapeutic interventions. This study highlights the value of a multidisciplinary approach in exploring the molecular basis of complex genetic diseases and informing clinical decisions. [ABSTRACT FROM AUTHOR]

The efficacy and cost-effectiveness of Multigene Panel Next-Generation Sequencing (NGS) in directing patients towards genomically matched therapies remain uncertain. This study investigated metastatic colorectal cancer (mCRC) patients who underwent NGS analysis on formalin-fixed paraffin-embedded tumor samples. Data from 179 patients were analyzed, revealing no mutations in 39 patients (21.8%), one mutation in 83 patients (46.4%), and two or more mutations in 57 patients (31.8%). KRAS mutations were found in 87 patients (48.6%), including KRAS G12C mutations in 5 patients (2.8%), PIK3CA mutations in 40 patients (22.4%), and BRAF mutations in 26 patients (14.5%). Less common mutations were identified: ERBB2 in five patients (2.8%) and SMO in four patients (2.2%). Additionally, MAP2K1, CTNNB1, and MYC were mutated in three patients (2.4%). Two mutations (1.1%) were observed in ERBB3, RAF1, MTOR, JAK1, and FGFR2. No significant survival differences were observed based on number of mutations. In total, 40% of patients had druggable molecular alterations, but only 1.1% received genomically guided treatment, suggesting limited application in standard practice. Despite this, expanded gene panel testing can identify actionable mutations, aiding personalized treatment strategies in metastatic CRC, although current eligibility for biomarker-guided trials remains limited. [ABSTRACT FROM AUTHOR]

Trimming adapters and low-quality bases from next-generation sequencing (NGS) data is crucial for optimal analysis. We evaluated six trimming programs, implementing five different algorithms, for their effectiveness in trimming adapters and improving quality, contig assembly, and single-nucleotide polymorphism (SNP) quality and concordance for poliovirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and norovirus paired data sequenced on Illumina iSeq and MiSeq platforms. Trimmomatic and BBDuk effectively removed adapters from all datasets, unlike FastP, AdapterRemoval, SeqPurge, and Skewer. All trimmers improved read quality (Q ≥ 30, 87.8 − 96.1%) compared to raw reads (83.6 − 93.2%). Trimmers implementing traditional sequence-matching (Trimmomatic and AdapterRemoval) and overlapping algorithm (FastP) retained the highest-quality reads. While all trimmers improved the maximum contig length and genome coverage for iSeq and MiSeq viral assemblies, BBDuk-trimmed reads assembled the shortest contigs. SNP concordance was consistently high (> 97.7 − 100%) across trimmers. However, BBDuk-trimmed reads had the lowest quality SNPs. Overall, the two adapter trimmers that utilized the traditional sequence-matching algorithm performed consistently across the viral datasets analyzed. Our findings guide software selection and inform future versatile trimmer development for viral genome analysis. [ABSTRACT FROM AUTHOR]

Background: Cardiovascular diseases are prevalent worldwide with any age, and it is characterized by sudden blockage of blood flow to heart and permanent damage to the heart muscle, whose cause and underlying molecular mechanisms are not fully understood. This investigation aimed to explore and identify essential genes and signaling pathways that contribute to the progression of MI. Methods: The aim of this investigation was to use bioinformatics and next-generation sequencing (NGS) data analysis to identify differentially expressed genes (DEGs) with diagnostic and therapeutic potential in MI. NGS dataset (GSE132143) was downloaded from the Gene Expression Omnibus (GEO) database. DEGs between MI and normal control samples were identified using the DESeq2 R bioconductor tool. The gene ontology (GO) and REACTOME pathway enrichment analyses of the DEGs were performed using g:Profiler. Next, four kinds of algorithms in the protein–protein interaction (PPI) were performed to identify potential novel biomarkers. Next, miRNA-hub gene regulatory network analysis and TF-hub gene regulatory network were constructed by miRNet and NetworkAnalyst database, and Cytoscape software. Finally, the diagnostic effectiveness of hub genes was predicted by receiver operator characteristic curve (ROC) analysis and AUC more than 0.800 was considered as having the capability to diagnose MI with excellent specificity and sensitivity. Results: A total of 958 DEGs were identified, consisting of 480 up-regulated genes and 478 down-regulated genes. The enriched GO terms and pathways of the DEGs include immune system, neuronal system, response to stimulus and multicellular organismal process. Ten hub genes (namely cftr, cdk1, rps13, rps15a, rps27, notch1, mrpl12, nos2, ccdc85b and atn1) were obtained via protein–protein interaction analysis results. MiRNA-hub gene regulatory network and TF-hub gene regulatory network showed that hsa-mir-409-3p, hsa-mir-3200-3p, creb1 and tp63 might play an important role in the MI. Conclusions: Analysis of next-generation sequencing dataset combined with global network information and validation presents a successful approach to uncover the risk hub genes and prognostic markers of MI. Our investigation identified four risk- and prognostic-related gene signatures, including cftr, cdk1, rps13, rps15a, rps27, notch1, mrpl12, nos2, ccdc85b and atn1. This gene sets contribute a new perspective to improve the diagnostic, prognostic, and therapeutic outcomes of MI. [ABSTRACT FROM AUTHOR]

Background: Endometriosis is a common cause of endometrial-type mucosa outside the uterine cavity with symptoms such as painful periods, chronic pelvic pain, pain with intercourse and infertility. However, the early diagnosis of endometriosis is still restricted. The purpose of this investigation is to identify and validate the key biomarkers of endometriosis. Methods: Next-generation sequencing dataset GSE243039 was obtained from the Gene Expression Omnibus database, and differentially expressed genes (DEGs) between endometriosis and normal control samples were identified. After screening of DEGs, gene ontology (GO) and REACTOME pathway enrichment analyses were performed. Furthermore, a protein–protein interaction (PPI) network was constructed and modules were analyzed using the Human Integrated Protein–Protein Interaction rEference database and Cytoscape software, and hub genes were identified. Subsequently, a network between miRNAs and hub genes, and network between TFs and hub genes were constructed using the miRNet and NetworkAnalyst tool, and possible key miRNAs and TFs were predicted. Finally, receiver operating characteristic curve analysis was used to validate the hub genes. Results: A total of 958 DEGs, including 479 upregulated genes and 479 downregulated genes, were screened between endometriosis and normal control samples. GO and REACTOME pathway enrichment analyses of the 958 DEGs showed that they were mainly involved in multicellular organismal process, developmental process, signaling by GPCR and muscle contraction. Further analysis of the PPI network and modules identified 10 hub genes, including vcam1, snca, prkcb, adrb2, foxq1, mdfi, actbl2, prkd1, dapk1 and actc1. Possible target miRNAs, including hsa-mir-3143 and hsa-mir-2110, and target TFs, including tcf3 (transcription factor 3) and clock (clock circadian regulator), were predicted by constructing a miRNA-hub gene regulatory network and TF-hub gene regulatory network. Conclusions: This investigation used bioinformatics techniques to explore the potential and novel biomarkers. These biomarkers might provide new ideas and methods for the early diagnosis, treatment and monitoring of endometriosis. [ABSTRACT FROM AUTHOR]

Background/Objectives: the vaginal microbiota is known to confer protection in the genital ecosystem, due to the predominance of different Lactobacillus species, playing a crucial role in women's health; alterations in the composition of the microbial communities in the vagina can be associated with the development of bacterial vaginosis (BV). Current therapy for BV involves oral or intravaginal administration of metronidazole or clindamycin, albeit the high recurrence rates suggest a need for alternative therapeutic tools, such as probiotics. Herein, the diversity and composition of vaginal microbiota in women with asymptomatic BV was investigated before and after the oral administration of a multi-strain probiotic formulation. Methods: a prospective observational pilot study with pre–post design was carried out from 1 June 2022, to 31 December 2022, on reproductive-age women with asymptomatic BV, as diagnosed via Nugent score, and matched healthy controls. The probiotic was administered to all study participants as acid-resistant oral capsules (twice daily), and a vaginal swab was collected at baseline and after 2 months of treatment, for the metagenomic analysis of 16s rDNA. Results: the diversity and richness of the vaginal microbiota in women with BV were significantly reduced after 2 months of supplementation with the oral probiotic, as evidenced by measures of α-diversity. Interestingly, some bacterial genera typically associated with dysbiosis, such as Megasphaera spp., were significantly decreased; whereas, at the same time, Lactobacillus spp. Doubled. Conclusions: our preliminary results suggest that the multi-strain oral probiotic is a beneficial treatment specifically targeting the dysbiotic vaginal microenvironment. [ABSTRACT FROM AUTHOR]

Celiac disease is an autoimmune enteropathy caused by the ingestion of minute amounts of gluten in a subset of genetically predisposed individuals. Its onset occurs at different ages and with variable symptoms. The gut microbiome may contribute to this variability. This review aims to provide an overview of the available research on celiac disease gut microbiome and identify the knowledge gap that could guide future studies. Following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analysis extension for Scoping Reviews (PRISMA-ScR), four electronic databases were searched for literature from January 2000 to July 2023 addressing celiac disease gut microbiome characterization using next-generation sequencing (NGS) approaches. From the 489 publications retrieved, 48 publications were selected and analyzed, focusing on sample characterization (patients, controls, and tissues) and methodologies used for NGS microbiome analysis and characterization. The majority of the selected publications regarded children and adults, and four were randomized clinical trials. The number of participants per study greatly varied and was typically low. Feces were the most frequently tested sample matrix, and duodenal samples were analyzed in one-third of the studies. Incomplete and diverse information on the methodological approaches and gut microbiome results was broadly observed. While similar trends regarding the relative abundance of some phyla, such as Pseudomonadota (former Proteobacteria), were detected in some studies, others contradicted those results. The observed high variability of technical approaches and possibly low power and sample sizes may prevent reaching a consensus on celiac disease gut microbiome composition. Standardization of research protocols to allow reproducibility and comparability is required, as interdisciplinary collaborations to further data analysis, interpretation, and, more importantly, health outcome prediction or improvement. [ABSTRACT FROM AUTHOR]

Detecting pathogens in endangered animal populations is vital for understanding and mitigating threats to their survival. The critically endangered western ground parrot (Pezoporus flaviventris, WGP), with a population as low as 150 individuals in Australia, faces an imminent risk of extinction. Despite this urgency, research on viral pathogens in this species remains limited. This study aimed to identify and characterise viruses present in faecal samples from seven individual WGP using a viral metagenomic approach. Analysis of the sequenced datasets revealed the presence of a novel virus belonging to the Parvoviridae family, named psittaciform chaphamaparvovirus 7 (PsChPV‐7). The genome of PsChPV‐7 contains typical structural and functional gene sequences found in Parvoviridae but is highly divergent, indicating its classification as a distinct species. Phylogenetic analysis placed PsChPV‐7 within a unique sub‐clade of the Chaphamaparvovirus genus, suggesting its evolutionary significance as an ancient lineage within this group. These findings may contribute to the development of strategic management and biosecurity plans aimed at conserving this endangered WGP. [ABSTRACT FROM AUTHOR]

This study examines a case involving a 7-year-old child who developed dengue symptoms following Qdenga vaccination. Despite initial negative diagnostic results, molecular analysis confirmed an infection with DENV4. Next-generation sequencing detected viral RNA from both DENV2 and DENV4 serotypes, which were identified as vaccine-derived strains using specific primers. Phylogenetic analysis further confirmed that these sequences belonged to the Qdenga vaccine rather than circulating wild-type viruses. This case underscores the critical need for precise diagnostic interpretation in vaccinated individuals to avoid misdiagnosis and to strengthen public health surveillance. A comprehensive understanding of vaccine-induced viremia is essential for refining dengue surveillance, improving diagnostic accuracy, and informing public health strategies in endemic regions. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is an acute, highly hemorrhagic viral disease in domestic pigs and wild boars. The disease is caused by African swine fever virus, a double stranded DNA virus of the Asfarviridae family. ASF can be classified into 25 different genotypes, based on a 478 bp fragment corresponding to the C-terminal sequence of the B646L gene, which is highly conserved among strains and encodes the major capsid protein p72. The C-terminal end of p72 has been used as a PCR target for quick diagnosis of ASF, and its characterization remains the first approach for epidemiological tracking and identification of the origin of ASF in outbreak investigations. Recently, a new classification of ASF, based on the complete sequence of p72, reduced the 25 genotypes into only six genotypes; therefore, it is necessary to have the capability to sequence the full-length B646L gene (p72) in a rapid manner for quick genotype characterization. Here, we evaluate the use of an amplicon approach targeting the whole B646L gene, coupled with nanopore sequencing in a multiplex format using Flongle flow cells, as an easy, low cost, and rapid method for the characterization and genotyping of ASF in real-time. [ABSTRACT FROM AUTHOR]

The high genetic diversity of influenza viruses means that traditional serological assays have too low throughput to measure serum antibody neutralization titers against all relevant strains. To overcome this challenge, we developed a sequencing-based neutralization assay that simultaneously measures titers against many viral strains using small serum volumes using a workflow similar to traditional neutralization assays. The key innovation is to incorporate unique nucleotide barcodes into the hemagglutinin (HA) genomic segment, and then pool viruses with numerous different barcoded HA variants and quantify the infectivity of all of them simultaneously using next-generation sequencing. With this approach, a single researcher performed the equivalent of 2,880 traditional neutralization assays (80 serum samples against 36 viral strains) in approximately 1 month. We applied the sequencing-based assay to quantify the impact of influenza vaccination on neutralization titers against recent human H1N1 strains for individuals who had or had not also received a vaccine in the previous year. We found that the viral strain specificities of the neutralizing antibodies elicited by vaccination vary among individuals and that vaccination induced a smaller increase in titers for individuals who had also received a vaccine the previous year--although the titers 6 months after vaccination were similar in individuals with and without the previous-year vaccination. We also identified a subset of individuals with low titers to a subclade of recent H1N1 even after vaccination. We provide an experimental protocol (dx.doi.org/10.17504/protocols.io.kqdg3xdmpg25/v1) and computational pipeline (https://github.com/jbloomlab/seqneut-pipeline) for the sequencing-based neutralization assays to facilitate the use of this method by others. [ABSTRACT FROM AUTHOR]

Background and Aims: Cancer has grabbed the attention of scientists and medical professionals all over the world much more than any other disease. In the past few decades, the medical field has improved quite a lot but progress in the path to find a solution for cancer is very less. As the popularity of invasive technologies is diminishing in cancer treatment, scientists have come up withminimally invasive or non‐invasive alternatives, among which liquid biopsy, by far is the most suitable. Methods: Liquid biopsy is used to analyse nucleic acids, subcellular components and circulating tumour cells in various biological fluids for diagnosis of cancer. It can also be used to know the efficacy of cancer drugs in a patient by analysing multiple samples. Outcomes: Liquid biopsy is becoming standard of care as it allows biopsy of those samples in which solid tumour biopsies are not possible. The diversity of sampling procedures, such as collection of urine for urothelial carcinoma or bladder or prostate cancer and phlebotomy for other types of cancer, make liquid biopsy one of the best methods for diagnosis of cancer. Conclusion: This review aims in discussing the several techniques used for the detection of cancer biomarkers and some clinical manifestations due to the changes in the biomarkers which are analysed by liquid biopsy. [ABSTRACT FROM AUTHOR]

The decline of European ash by dieback caused by Hymenoscyphus fraxineus together with stem collar necroses and rots caused by various fungi has been investigated intensively during the last years. Nevertheless, hitherto nearly nothing is known about the species diversity of the fungal rhizobiome of ash trees. Here we investigated the fine roots of affected ash trees on 15 sampling sites in 6 federal countries of Germany. Fine-root samples have been treated in three different sample regimes each as root-adhering soil, unsterilized fine roots and sterilized fine roots. The samples of trees in sampling sites were pooled to get an overview of the species-richness in the area. The next-generation sequencing platform Oxford Nanopore MinION was used to sequence the entire ITS of pooled probes. Most abundant phyla in all samples were the Basidiomycota and Ascomycota. Species richness in sterilized roots was significantly different from unsterilized roots and root-adhering soil. Surprisingly most abundant genera in sterilized roots were the genera Mycena, Mycenella and Delicatula, all of them agaricoids with saprophytic lifestyle. Eleven genera of Glomeromycota have been detected in various abundances, whereas the detection of H. fraxineus was neglectable. [ABSTRACT FROM AUTHOR]

Background: Circulating tumor DNA (ctDNA) has emerged as a promising biomarker for noninvasive cancer diagnostics, particularly in the context of metastatic non‐small‐cell lung cancer (NSCLC). Detecting targetable variants through ctDNA analysis offers the potential to guide treatment decisions, especially in cases where tissue samples are insufficient or unavailable. Method: In this study, we developed and validated a next‐generation sequencing panel targeting 101 cancer‐related genes (101‐test) to detect somatic variants in ctDNA from a large cohort of Chinese patients with metastatic NSCLC. The performance of the 101‐test was assessed by evaluating its limit of detection (LOD), accuracy, and precision in identifying molecular variants. Additionally, the concordance between ctDNA and tissue samples for detecting targetable variants was analyzed in 904 patients. Results: The 101‐test demonstrated a LOD of 0.38% for single‐nucleotide variants (SNVs), 0.33% for insertions and deletions (InDels), and 0.33% for fusions. Sensitivity was 98.3% for SNVs, 100% for InDels, and 100% for fusions when compared to digital droplet PCR (ddPCR)/breakpoint PCR reference methods. The by‐variant sensitivity for somatic variants was 97.5%, with a specificity of 99.9% between tumor‐only and tumor‐normal analyses. In a real‐world cohort, the concordance between ctDNA and tissue samples for identifying targetable variants was 72.2% (457/633). Notably, the EGFR S768I variant, recently recommended by clinical guidelines, achieved an 80% concordance rate. Furthermore, 4.3% of patients (27/633) with targetable variants were identified exclusively through ctDNA testing. Conclusion: The ctDNA‐based 101‐test is a highly sensitive and specific tool for detecting targetable variants in metastatic NSCLC, particularly in cases with insufficient tissue samples. The findings support the use of ctDNA testing as a reliable and complementary method to traditional tissue‐based molecular analysis, enhancing the precision of treatment strategies for NSCLC patients. [ABSTRACT FROM AUTHOR]

Background: Urothelial carcinoma presents as non-muscle-invasive bladder cancer (NMIBC) in ~75% of primary cases. Addressing the limitations of the TNM and WHO04/16 classification systems, this study investigates genetic alterations, the mitotic activity index (MAI), and immunohistochemistry (IHC) markers CK20, p53, and CD25 as better prognostic biomarkers in NMIBC. Methods: Using the Oncomine™ Focus Assay for targeted next-generation sequencing (NGS), 409 single-nucleotide variations (SNVs) and 193 copy number variations (CNVs) were identified across 287 patients with TaT1 tumors. Results: FGFR3 and PIK3CA alterations were significantly more prevalent in Ta tumors, while T1 tumors had significant ERBB2 alterations. Low-grade (LG) tumors were enriched with FGFR3 alterations, while high-grade (HG) tumors were significantly associated with ERBB2 alterations, as well as FGFR1 and CCND1 amplifications. FGFR3 alterations were linked to shorter recurrence-free survival (RFS; p = 0.033) but improved progression-free survival (PFS; p < 0.001). Conversely, ERBB2 alterations (p < 0.001), ERBB3 mutations (p = 0.044), and both MYC (p < 0.001) and MYCN (p = 0.011) amplifications were associated with shorter PFS. Survival analysis of gene sets revealed inverse associations between PIK3CA and ERBB2 (p = 0.003), as well as PIK3CA and MYC (p = 0.005), with PFS. Conclusions: In multivariate Cox regression, MAI was the strongest predictor for PFS. Integrating genetic alterations and histopathological features may improve risk stratification in NMIBC. [ABSTRACT FROM AUTHOR]

Background: The rearranged during transfection (RET) proto-oncogene fusion is common in papillary thyroid cancer (PTC), varying across ethnic groups. However, comprehensive comparisons of RET fusion types are limited. This study aims to identify predominant RET fusions and analyze their clinicopathological characteristics in a cohort of Chinese thyroid cancer cases. Methods: This single-center retrospective cohort study analyzed thyroid cancer data, utilizing next-generation sequencing on formalin-fixed, paraffin-embedded tissue samples. Detailed clinicopathological data of thyroid cancer cases with RET fusions were collected. Results: Among 2300 thyroid cancer cases, RET fusions were exclusively found in PTC or differentiated high-grade thyroid carcinoma (DHGTC) cases (2234 cases), absent in other types (66 cases). Of the 2234 PTC or DHGTC cases, 113 (5.06%) exhibited RET fusions, including 100 primary cases. Coiled-coil domain containing 6 (CCDC6)-RET fusions predominated (78.0%, 78/100), with nuclear receptor coactivator 4 (NCOA4)-RET fusions representing 22.0% (22/100). NCOA4-RET fusions were more prevalent in patients aged 45 years and older (54.5% vs. 28.2%, p = 0.021) and DHGTC cases (p < 0.05) and associated with higher rates of lymph node metastases (90.9% vs. 67.9%, p = 0.032). CCDC6-RET fusion exhibited a higher prevalence of Hashimoto's thyroiditis (HT) (67.9% vs. 22.7%, p < 0.001) and elevated thyroglobulin antibody levels (14.11 [1.86–174.32] IU/mL vs. 2.01 [1.14–15.41] IU/mL, p = 0.018). Moreover, CCDC6-RET fusion predominantly occurred in classical PTC (56.4%, 44/78) and infiltrative follicular PTC (17.9%, 14/78), whereas NCOA4-RET fusion was more frequent in classical PTC (36.4%, 8/22), solid PTC (27.3%, 6/22), and DHGTC (27.3%, 6/22). RET fusions with compound mutations were associated with older age (≥45 years) and bilateral thyroid involvement. Follow-up data showed a higher recurrence rate in the RET fusion group compared with the BRAFV600E mutation group (5.0% vs. 0.0%, p = 0.018). Although the NCOA4-RET group showed a numerically higher recurrence rate compared with CCDC6-RET (9.1% vs. 3.8%), this difference was not statistically significant (p = 0.559). Conclusions:RET fusions are specific to PTC or DHGTC cases among Chinese thyroid cancer cases. CCDC6-RET and NCOA4-RET fusions exhibited distinct clinicopathological features, with NCOA4-RET being more aggressive. [ABSTRACT FROM AUTHOR]

Objectives: To compare the clinicopathological, molecular, and immune features of conventional and high‐grade transformation (HGT) secretory carcinoma (SC) in salivary glands. Materials and Methods: The clinicopathological data of 88 cases including 74 conventional SCs and 14 SCs with HGT were reviewed. Targeted next‐generation sequencing was performed in 11 SCs with HGT and 7 conventional SCs. The level of PD‐L1 and CD8+ TILs was determined by immunohistochemistry. Results: Compared with the conventional group, the rates of nodal metastasis, local recurrence, distant metastasis and mortality were significantly higher in the HGT cohort. Mutations of ARID1A/B, KMT2A, HOXD13, NRG1 and ETV6 genes were identified in HGT SCs. A recurrent E307G mutation in GATA6 gene was also observed in two cases. Two deceased HGT patients with distant metastasis harboured NOTCH3 mutations. ETV6‐RET translocation was prone to occur in the HGT SCs. Additionally, PD‐L1 expression was low, and CD8+ TILs were sparse in most HGT cases. Conclusion: Our findings reveal novel gene alterations involved in the progression of HGT in SCs. Most HGT SCs patients cannot benefit from PD‐L1 blocking and may be approached with a distinct treatment strategy including the lymph node dissection and application of molecular target drugs in precision oncology. [ABSTRACT FROM AUTHOR]

Background: Laboratory rats, as model animals, have been extensively used in the fields of life science and medicine. It is crucial to routinely monitor the genetic background of laboratory rats. The conventional approach relies on gel electrophoresis and capillary electrophoresis (CE) technologies. However, the experimental and data analysis procedures for both of these methods are time consuming and costly. Results: We established a single-nucleotide polymorphism (SNP) typing scheme using multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to address the genetic background ambiguity in laboratory rats. This methodology involved three rounds of PCR and two rounds of magnetic bead selection to improve the quality of the sequencing data. We simultaneously analysed 100 laboratory rats (including rats of 5 inbred strains and 2 in-house closed colonies), and the sequencing depth varied from an average of 108.25 to 5189.89, with sample uniformity ranging from 82.5 to 97.5%. A total of 98.9% of the amplicons were successfully genotyped (≥ 30 reads). Genetic background analysis revealed that all 38 experimental rats from the 5 inbred strains were successfully identified (without a heterozygous allele). For the 2 in-house closed colonies, the average heterozygosity (0.162 and 0.169) deviated from the typical range of 0.5–0.7, indicating a departure from the ideal heterozygosity level. Additionally, we employed multiplex PCR-CE to validate the NGS-based method, which yielded consistent results for all the rat strains. These results demonstrated that this approach significantly improves efficiency, saves time, reduces costs and ensures accuracy. Conclusion: By utilizing NGS technology, our developed method leverages SNP genotyping for genetic background identification in laboratory rats, demonstrating advantages in terms of labour efficiency and cost-effectiveness, thereby rendering it well suited for projects involving extensive sample cohorts. [ABSTRACT FROM AUTHOR]

Background: Methotrexate (MTX) serves as the initial treatment for rheumatoid arthritis (RA). However, a substantial proportion of RA patients, estimated between 30% and 50%, do not respond positively to MTX. While the T-cell receptor (TCR) is crucial for the immune response during RA, its role in differentiating MTX responsiveness has not been thoroughly investigated. Methods: This study used next-generation sequencing to analyze the TCR β-chain complementary determining region sequences in peripheral blood mononuclear cells obtained from RA patients before MTX treatment. This study aimed to compare the characteristics of the TCR repertoire between the MTX responder and non-responder groups. Results: The study identified a significant difference in the TRBV6–6 gene (p =.003) concerning MTX treatment response. Additionally, a significant difference was found in the number of "3" nucleotide deletions at the 5'Jdels site (p =.023) in the VDJ rearrangement. Conclusion: These findings suggest distinct TCR repertoire characteristics between MTX responder and non-responder groups among RA patients. This discovery offers new insights into understanding the variable responses of RA patients to MTX therapy. [ABSTRACT FROM AUTHOR]

Genetic sequencing technologies are evolving at a rapid pace with major implications for research and clinical practice. In this review, the authors provide an updated overview of next-generation sequencing (NGS) and emerging methodologies. NGS has tremendously improved sequencing output while being more time and cost-efficient in comparison to Sanger sequencing. The authors describe short-read sequencing approaches, such as sequencing by synthesis, ion semiconductor sequencing, and nanoball sequencing. Third-generation long-read sequencing now promises to overcome many of the limitations of short-read sequencing, such as the ability to reliably resolve repeat sequences and large genomic rearrangements. By combining complementary methods with massively parallel DNA sequencing, a greater insight into the biological context of disease mechanisms is now possible. Emerging methodologies, such as advances in nanopore technology, in situ nucleic acid sequencing, and microscopy-based sequencing, will continue the rapid evolution of this area. These new technologies hold many potential applications for hematological disorders, with the promise of precision and personalized medical care in the future. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor 2 (HER2) is known for its oncogenic activities in diverse cancers, including non-small cell lung cancer (NSCLC). However, the prevalence of HER2 alterations in Malaysian NSCLC patients remains unreported. This study examined the prevalence and characteristics of HER2 mutations and amplification in a Malaysian cohort. Additionally, a systematic review was conducted to evaluate the global prevalence of HER2 alterations in NSCLC, as well as the efficacy of HER2-targeted therapies observed in clinical trials. NSCLC tumour samples received from October 2019 to December 2022 for next-generation sequencing diagnostics were included in the retrospective analysis. In this patient cohort, HER2 alteration was present in 5.8% of patients; 3.9% had HER2 mutations, 1.5% had HER2 amplifications and 0.4% were both HER2-mutated and amplified. HER2 exon 20 insertions were the most common HER2 variants, detected in 47/59 (79.7%) of HER2-mutated patients. Among cases with HER2 exon 20 insertions, the Y772&lowbar;A775dup variant was found in 34 patient samples. HER2-mutated patients were significantly younger than non-HER2-mutants (61 versus 64 years old; p = 0.046) and were inclined to be female and never-smokers, albeit not statistically significant. Patients with HER2 amplification were more likely to have progressed post-tyrosine kinase inhibitor therapy (p = 0.015). The systematic review highlighted a global variation in the prevalence of HER2 alterations in NSCLC, ranging from 0.3% to 9.1% for mutations and 0.2% to 19% for amplification. Finally, phase II clinical trials involving HER2-altered NSCLC patients demonstrated promising treatment outcomes with trastuzumab deruxtecan, trastuzumab emtansine, pyrotinib, pyrotinib + apatinib and trastuzumab + pertuzumab + docetaxel. In conclusion, the prevalence of HER2 alteration among Malaysian NSCLC patients falls within the global range. A systematic review of clinical trials revealed promising treatment outcomes and Malaysian NSCLC patients with HER2 alterations are anticipated to similarly benefit from HER2-targeted therapies. [ABSTRACT FROM AUTHOR]

Background: Esophageal neoplasms rank as the 7th most common cancers in the world. Squamous cell carcinomas of esophagus (SCCE) are the predominant subset, linked to a number of genetic alterations. Gene-driven tumour pathways are being increasingly identified with the emerging role of next-generation sequencing. Case presentation: We report a case of an 82-year-old male patient who was diagnosed with SCCE involving the cervical region. He received definitive concurrent chemo-radiotherapy with Carboplatin and Paclitaxel. To trace the family history of malignancy, a genetic test was carried out which turned out to be a pathogenic BRCA1 variant. Conclusion: SCCE arising in the context of known BRCA1 mutation has been rarely reported to date. Testing for these mutations should be considered in patients who present with esophageal cancer, especially in the backdrop of familial neoplasms. [ABSTRACT FROM AUTHOR]

Producing alternative staple foods like millet will be essential to feeding ten billion people by 2050. The increased demand for millet is driving researchers to improve its genetic variation. Millets include protein, dietary fiber, phenolic substances, and flavonoid components. Its climate resilience makes millet an appealing crop for agronomic sustainability. Integrative omics technologies could potentially identify and develop millets with desirable phenotypes that may have high agronomic value. Millets’ salinity and drought tolerance have been enhanced using transcriptomics. In foxtail, finger, and pearl millet, proteomics has discovered salt-tolerant protein, phytohormone-focused protein, and drought tolerance. Metabolomics studies have revealed that certain metabolic pathways including those involving lignin, flavonoids, phenylpropanoid, and lysophospholipids are critical for many processes, including seed germination, photosynthesis, energy metabolism, and the synthesis of bioactive chemicals necessary for drought tolerance. Metabolomics integration with other omics revealed metabolome engineering and trait-specific metabolite creation. Integrated metabolomics and ionomics are still in the development stage, but they could potentially assist in comprehending the pathway of ionomers to control nutrient levels and biofortify millet. Epigenomic analysis has shown alterations in DNA methylation patterns and chromatin structure in foxtail and pearl millets in response to abiotic stress. Whole-genome sequencing utilizing next-generation sequencing is the most proficient method for finding stress-induced phytoconstituent genes. New genome sequencing enables novel biotechnological interventions including genome-wide association, mutation-based research, and other omics approaches. Millets can breed more effectively by employing next-generation sequencing and genotyping by sequencing, which may mitigate climate change. Millet marker-assisted breeding has advanced with high-throughput markers and combined genotyping technologies. [ABSTRACT FROM AUTHOR]

Bovine viral diarrhoea (BVD) is one of the most economically damaging livestock enzootic diseases in the world. BVD aetiological agents are three pestiviruses (BVDV-1, -2 and HoBi-like pestivirus), which exhibit high genetic diversity and complex transmission cycles. This considerably hampers the management of the disease, which is why eradication plans have been implemented in several countries. In France, a national plan has been in place since 2019. Our understanding of its impact on the distribution of BVDV genotypes is limited by the availability of French genetic data. Here, we conducted a molecular epidemiology study to refine our knowledge of BVDV genetic diversity in France, characterise its international relationships, and analyse national spatio-temporal genotypic distribution. We collated 1037 BVDV-positive samples throughout France between 2011 and 2023, with a greater sampling effort in two major cattle production areas. We developed a high-throughput sequencing protocol which we used to complete the 5'UTR genotyping of this collection. We show that two main BVDV-1 genotypes, 1e and 1b, account for 88% of genotyped sequences. We also identified seven other BVDV-1 genotypes occurring at low frequencies and three BVDV-2 samples (genotype 2c). Phylogenetic analyses indicate different worldwide distribution patterns between the two main BVDV-1 genotypes. Their relative frequencies present no major changes in France since the 1990s and few variations at the national scale. We also found some degree of local spatial structuring in western France. Overall, our results demonstrate the potential of large-scale sequence-based surveillance to monitor changes in the epidemiological situation of enzootic diseases. [ABSTRACT FROM AUTHOR]

The impact of pollution on the Ologe Lagoon was assessed by comparing physicochemical properties, hydrocarbon concentrations and microbial community structures of the sediments obtained from distinct sites of the lagoon. The locations were the human activity site (OLHAS), industrial-contaminated sites (OLICS) and relatively undisturbed site (OLPS). The physicochemical properties, heavy metal concentrations and hydrocarbon profiles were determined using standard methods. The microbial community structures of the sediments were determined using shotgun next-generation sequencing (NGS), taxonomic profiling was performed using centrifuge and statistical analysis was done using statistical analysis for metagenomics profile (STAMP) and Microsoft Excel. The result showed acidic pH across all sampling points, while the nitrogen content at OLPS was low (7.44 ± 0.085 mg/L) as compared with OLHAS (44.380 ± 0.962 mg/L) and OLICS (59.485 ± 0.827 mg/L). The levels of the cadmium, lead and nickel in the three sites were above the regulatory limits. The gas chromatography flame ionization detector (GC-FID) profile revealed hydrocarbon contaminations with nC14 tetradecane > alpha xylene > nC9 nonane > acenaphthylene more enriched at OLPS. Structurally, the sediments metagenomes consisted of 43 phyla,75 classes each, 165, 161, 166 orders, 986, 927 and 866 bacterial genera and 1476, 1129, 1327 species from OLHAS, OLICS and OLPS, respectively. The dominant phyla in the sediments were Proteobacteria, Firmicutes, Actinobacteria, and Chloroflexi. The principal component ordination (PCO) showed that OLPS microbial community had a total variance of 87.7% PCO1, setting it apart from OLHAS and OLICS. OLICS and OLHAS were separated by PCO2 accounting for 12.3% variation, and the most polluted site is the OLPS. [ABSTRACT FROM AUTHOR]

Selection is a fundamental part of the plant breeding process, enabling the identification and development of varieties with desirable traits. Thanks to advances in genetics and biotechnology, the selection process has become more precise and efficient, resulting in faster breeding progress and better adaptation of crops to environmental challenges. Genetic parameters related to gene additivity and epistasis play a key role and can influence decisions on the suitability of breeding material. In this study, 188 rapeseed doubled haploid lines were assessed in field conditions for resistance to Leptosphaeria spp. Through next-generation sequencing, a total of 133,764 molecular markers (96,121 SilicoDArT and 37,643 SNP) were obtained. The similarity of the DH lines at the phenotypic and genetic levels was calculated. The results indicate that the similarity at the phenotypic level was markedly different from the similarity at the genetic level. Genetic parameters related to additive gene action effects and epistasis (double and triple) were calculated using two methods: based on phenotypic observations only and using molecular marker observations. All evaluated genetic parameters (additive, additive-additive and additive-additive-additive) were statistically significant for both estimation methods. The parameters associated with the interaction (double and triple) had opposite signs depending on the estimation method. [ABSTRACT FROM AUTHOR]

B‐cell acute lymphoblastic leukaemia (B‐ALL) is the most prevalent hematologic malignancy in children and a leading cause of mortality. Managing B‐ALL remains challenging due to its heterogeneity and relapse risk. This study aimed to delineate the molecular features of paediatric B‐ALL and explore the clinical utility of circulating tumour DNA (ctDNA). We analysed 146 patients with paediatric B‐ALL who received systemic chemotherapy. The mutational landscape was profiled in bone marrow (BM) and plasma samples using next‐generation sequencing. Minimal residual disease (MRD) testing on day 19 of induction therapy evaluated treatment efficacy. RNA sequencing identified gene fusions in 61% of patients, including 37 novel fusions. Specifically, the KMT2A‐TRIM29 novel fusion was validated in a boy who responded well to initial therapy but relapsed after 1 year. Elevated mutation counts and maximum variant allele frequency in baseline BM were associated with significantly poorer chemotherapy response (p = 0.0012 and 0.028, respectively). MRD‐negative patients exhibited upregulation of immune‐related pathways (p < 0.01) and increased CD8+ T cell infiltration (p = 0.047). Baseline plasma ctDNA exhibited high mutational concordance with the paired BM samples and was significantly associated with chemotherapy efficacy. These findings suggest that ctDNA and BM profiling offer promising prognostic insights for paediatric B‐ALL management. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study is to report the results of the genetic analysis in a large and well‐characterized population with pediatric‐onset epilepsies and to identify those who could benefit from precision medicine treatments. Methods: In this retrospective observational study, we consecutively recruited patients with pediatric‐onset epilepsy observed at a tertiary neurological center over a time span of 7 years, collecting clinical and laboratory findings. Following in‐depth diagnostic process to exclude possible structural and metabolic causes of the disease, patients with a suspected genetically determined etiology underwent next‐generation sequencing (NGS) screening with panels for the analysis of target genes causative of epilepsy. Results: We detected likely pathogenic or pathogenic variants (classes IV and V) in 24% of the 562 patients who underwent genetic investigations. By the evaluation of patients' data, we observed that some features (onset of epilepsy before one year old, presence of neurological deficits, psychomotor delay/cognitive disability, and malformative aspects at brain MRI) were significantly associated with class IV or V variants. Moreover, statistical analysis showed that the diagnostic yield resulted higher for patients affected by Progressive Myoclonic Epilepsy (PME) and with early onset developmental and epileptic encephalopathies (DEE), compared with focal epilepsies, genetic generalized epilepsies, DEE with onset at/after 1 y.o., and unclassified epileptic syndromes. According to the results of the genetic screening, up to 33% of patients carrying class IV or V variants resulted potentially eligible for precision medicine treatments. Significance: The large‐scale application of NGS multigene panels of analysis is a useful tool for the molecular diagnosis of patients with pediatric‐onset epilepsies, allowing the identification of those who could benefit from a personalized therapeutic approach. Plain Language Summary: The analysis of patients with pediatric‐onset epilepsy using advanced technologies for the screening of all the implicated genes allows the identification of the cause of diseases in an ever‐increasing number of cases. Understanding the pathogenic mechanisms could, in some cases, guide the selection and optimization of appropriate treatment approaches for patients. [ABSTRACT FROM AUTHOR]

Comprehensive investigation of gut microbiota composition is important for understanding digestive physiology in mammals. The hog deer (Axis porcinus) is a small mammal at high risk of extinction in the wild. In the present study, we sequenced the V4 region of 16S rRNA gene and compared the gut microbiota composition among three ages of captive hog deer, including one infant (Z1, six months old), three young (Z2, ten months old) and 12 adult (Z3, 4-5 years old). A total of 26 phyla (15, 22 and 25 in Z1, Z2 and Z3) and 310 genera (153, 254 and 304 in Z1, Z2 and Z3) were identified. In Z1, Z2 and Z3, Firmicutes (63.65%, 62.07% and 61.96%) was the predominant phylum, followed by Bacteroidetes (29.08%, 29.62% and 30.02%) and Tenericutes (3.08%, 3.24% and 2.78%). The alpha diversity (Shannon Index and observed species) of Z2 was significantly higher than that of Z3 (P < 0.05). The Shannon Index of Z1 was higher than that of Z2. At the genus level, Christensenellaceae_R-7_group and Ruminiclostridium_5 had higher abundance in Z2 compared with Z3, whereas other genera, such as Fibrobacter, Lachnospiraceae_AC2044_group and Oscillibacter were enriched in Z3. In conclusion, our results reveal significant microbiota composition changes that occur with age in captive hog deer. [ABSTRACT FROM AUTHOR]

Metabolic dysfunction-associated fatty liver disease (MAFLD) is the most common liver disease worldwide. The risk of developing MAFLD varies among individuals, due to a combination of environmental inherited and acquired genetic factors. Genome-wide association and next-generation sequencing studies are leading to the discovery of the common and rare genetic determinants of MAFLD. Thanks to the great advances in genomic technologies and bioinformatics analysis, genetic and epigenetic factors involved in the disease can be used to develop genetic risk scores specific for liver-related complications, which can improve risk stratification. Genetic and epigenetic factors lead to the identification of specific sub-phenotypes of MAFLD, and predict the individual response to a pharmacological therapy. Moreover, the variant transcripts and protein themselves represent new therapeutic targets. This review will discuss the current status of research into genetic as well as epigenetic modifiers of MAFLD development and progression. [ABSTRACT FROM AUTHOR]

Background: Spindle cell carcinoid tumor (SCCT) is a rare variant of lung carcinoid tumor consisting predominantly or exclusively of spindle cells. To the authors' knowledge, this is the first study to date investigating the molecular characteristics of SCCTs. Methods: Eighty‐five carcinoid tumors initially diagnosed by fine‐needle aspiration over a period of 10 years were reviewed. The final diagnostic classification was based on resection specimens. Six SCCTs were identified and characterized based on cytomorphology, and immunohistochemical and molecular features. Results: Most patients with SCCT were Caucasian (100.0%), women (83.3%), asymptomatic (66.7%), and nonsmokers (83.3%). The median age at diagnosis was 78.0 years (range, 58.2–80.3 years). A higher proportion of patients who had SCCT were diagnosed with distant metastasis. The smears were cellular and demonstrated clean backgrounds without necrosis or mitotic activity. SCCTs comprised of bipolar‐to‐elongated cells with finely granular chromatin, inconspicuous nucleoli, scant cytoplasm, and minimal atypia or pleomorphism. The tumor cells sometimes appeared boomerang‐shaped and might mimic granulomas or blood vessels. SCCTs showed strong expression for pan‐cytokeratin, synaptophysin, chromogranin, and CD56, with weak TTF‐1 and a very low Ki‐67 proliferation index. All SCCTs had low tumor mutational burden and were microsatellite‐stable. One case showed multiple whole‐gene losses in chromosome 11, whereas another harbored duplication in ARID1A. Two cases demonstrated gains in chromosomes 17, one of which also showed gains in chromosome 18. None had a single nucleotide mutation. Conclusions: SCCT is a rare subset of lung carcinoid tumors. These tumors harbor unique cytologic, prognostic, and molecular features that may have significant diagnostic and clinical implications. The current study is the first to investigate the molecular characteristics of spindle cell carcinoid tumors. This rare subset of carcinoid tumors harbored unique cytologic, prognostic, and molecular features that may have significant diagnostic and clinical implications. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is a genetic disorder characterized by chronic microbial colonization and inflammation of the respiratory tract (RT), leading to pulmonary exacerbation (PEx) and lung damage. Although the lung bacterial microbiota has been extensively studied, the mycobiome remains understudied. However, its importance as a contributor to CF pathophysiology has been highlighted. The objective of this review is to provide an overview of the current state of knowledge regarding the mycobiome, as described through NGS-based studies, in patients with CF (pwCF). Several studies have demonstrated that the mycobiome in CF lungs is a dynamic entity, exhibiting a lower diversity and abundance than the bacterial microbiome. Nevertheless, the progression of lung damage is associated with a decrease in fungal and bacterial diversity. The core mycobiome of the RT in pwCFs is mainly composed of yeasts (Candida spp., Malassezia spp.) and molds with lower abundance. Some fungi (Aspergillus, Scedosporium/Pseudallescheria) have been demonstrated to play a role in PEx, while the involvement of others (Candida, Pneumocystis) remains uncertain. The “climax attack” ecological model has been proposed to explain the complexity and interplay of microbial populations in the RT, leading to PEx and lung damage. NGS-based studies also enable the detection of intra- and interkingdom correlations between fungi and bacteria. Further studies are required to ascertain the biological and pathophysiological relevance of these correlations. Finally, with the recent advent of CFTR modulators, our understanding of the pulmonary microbiome and mycobiome in pwCFs is about to change. [ABSTRACT FROM AUTHOR]

Context The search for somatic mutations in adrenals resected from patients with primary aldosteronism (PA) is performed by Sanger sequencing, often implemented with immunohistochemistry (IHC)-guidance focused on aldosterone-producing (CYP11B2-positive) areas. Objective To investigate the impact of double IHC for CYP11B1 and CYP11B2 on Sanger and next-generation sequencing (NGS). Methods We investigated 127 consecutive adrenal aldosterone-producing adenomas from consenting surgically cured PA patients using double IHC for CYP11B1 and CYP11B2, by Sanger sequencing and NGS. Results Double IHC for CYP11B2 and CYP11B1 revealed 3 distinct patterns: CYP11B2-positive adenoma (pattern 1), mixed CYP11B1/CYP11B2-positive adenoma (pattern 2), and adrenals with multiple small CYP11B2-positive nodules (pattern 3). Sanger sequencing allowed detection of KCNJ5 mutations in 44% of the adrenals; NGS revealed such mutations in 10% of those negative at Sanger and additional mutations in 61% of the cases. Importantly the rate of KCNJ5 mutations differed across patterns: 17.8% in pattern 1, 71.4% in pattern 2, and 10.7% in pattern 3 (χ2 = 22.492, P <.001). Conclusion NGS allowed detection of mutations in many adrenals that tested negative at Sanger sequencing. Moreover, the different distribution of KCNJ5 mutations across IHC patterns indicates that IHC-guided sequencing protocols selecting CYP11B2-positive areas could furnish results that might not be representative of the entire mutational status of the excised adrenal, which is important at a time when KCNJ5 mutations are suggested to drive management of patients with aldosterone-producing adenomas. [ABSTRACT FROM AUTHOR]

Environmental pollution, driven by expanding industrial activities, is one of the most pressing issues today. Many fungi possess resistance mechanisms against heavy metals (HM), making them a sustainable approach for remediation processes. This study focuses on assessing HM concentrations in root samples taken from Pinus brutia in the Samsun Organized Industrial Zone (OIZ), a region heavily affected by environmental pollutants, and a nearby forested area (Adalar), while also examining the fungal diversity in the root endosphere. The fungal microbiota of the samples was determined using next-generation sequencing technology. Genera such as Aspergillus, Alternaria, Fusarium, Cladosporium, Trichoderma, Thelebolus, Plectosphaerella, Halokirschsteiniothelia, Exophiala, and Xenopolyscytalum were detected in the roots of P. brutia plants grown in the OIZ area, which is contaminated with HMs. The results indicate that higher levels of HM contamination may lead to changes in fungal diversity and an increase in the ratios of resistant taxa. This study aims to contribute to bioremediation strategies for HMs by elucidating the relationships between environmental pollutants and fungal microbiota. This research represents the first metagenomic analysis conducted in Samsun, Türkiye, to determine the impact of HMs on root fungal microbiota. [ABSTRACT FROM AUTHOR]