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Meningiomas are the most frequent primary intracranial tumors. The considerable variety of histological subtypes has been expanded by the definition of molecular alterations, which can improve both diagnostic accuracy and determination of individual patient's outcome. According to the upcoming WHO classification of brain tumors, the in‐time analysis of frequent molecular events in meningiomas may become mandatory to define meningioma subtypes. We have compiled a custom‐made amplicon‐based next generation sequencing (NGS) meningioma panel covering the most frequent known recurrent mutations in 15 different genes. In an unselected consecutive meningioma cohort (109 patients) analyzed over a period of 12 months, we detected mutations in 11 different genes, with most frequent alterations in NF2 (43%), AKT1E17K (15%), and TRAF7 (13%). In 39 tumors (36%), two different mutations were detected, with NF2 and SUFU (n = 5) and KLF4 and TRAF7 (n = 5) being the most frequent combinations. No alterations were found in POLR2A, CDKN2A, CDKN2B, and BAP1, and no homozygous CDKN2A/B deletion was detected. NF2 mutations were found in tumors of all WHO grades, whereas mutations in KLF4, TRAF7, and SMO were restricted to WHO grade I meningiomas. In contrast, SMARCE1 and TERT mutations were associated with WHO grade II meningiomas (according to the WHO classification 2016). The distribution of mutations across histological subtypes or tumor localization was in line with the existing literature, with typical combinations like KLF4K409Q/TRAF7 for secretory meningiomas and preferential skull base localization of meningiomas harboring SMO and AKT1E17K mutations. Thus, we present a custom‐made NGS meningioma panel providing a time and cost‐efficient reliable detection of relevant somatic molecular alterations in meningiomas suitable for daily routine. Description of a novel custom‐made NGS panel for rapid molecular characterization of meningiomas.

Background: Synovial sarcoma (SS) is a malignant soft tissue tumor of mesenchymal origin that frequently occurs in young adults. Translocation of the SYT gene on chromosome 18 to the SSX genes on chromosome X leads to the formation of oncogenic fusion genes, which lead to initiation and proliferation of tumor cells. The detection and quantification of circulating tumor DNA (ctDNA) can serve as a non-invasive method for diagnostics of local or distant tumor recurrence, which could improve survival rates due to early detection. Methods: We developed a subtype-specific targeted next-generation sequencing (NGS) approach specifically targeting SS t(X;18)(p11;q11), which fuses SS18 (SYT) in chromosome 18 to SSX1 or SSX2 in chromosome x, and recurrent point mutations. In addition, patient-specific panels were designed from tumor exome sequencing. Both approaches were used to quantify ctDNA in patients’ plasma. Results: The subtype-specific assay allowed detection of somatic mutations from 25/25 tumors with a mean of 1.68 targetable mutations. The minimal limit of detection was determined at a variant allele frequency of 0.05%. Analysis of 29 plasma samples from 15 tumor patients identified breakpoint ctDNA in 6 patients (sensitivity: 40%, specificity 100%). The addition of more mutations further increased assay sensitivity. Quantification of ctDNA in plasma samples (n = 11) from one patient collected over 3 years, with a patient-specific panel based on tumor exome sequencing, correlated with the clinical course, response to treatment and tumor volume. Conclusions: Targeted NGS allows for highly sensitive tumor profiling and non-invasive detection of ctDNA in SS patients, enabling non-invasive monitoring of tumor dynamics.

Precision oncology strategies guided by tumor molecular profiling often target key genomic aberrations in patients. Herein, we assembled National Cancer Center–Clinical Diagnostics Knowledgebase, compiling clinical targeted sequencing data from 6935 tumor tissues and matched normal samples, along with available pathological and clinical information. Comprehensive genomic profiling was conducted to characterize tumor type‐specific somatic alterations, and comparative analyses were performed across distinct cohorts. Key genomic characteristics included high‐frequency alterations in TP53 (57.8%), APC (22.6%), KRAS (21.3%), and EGFR (17.5%), among which EGFR mutations were significantly enriched in lung adenocarcinoma patients. In this cohort, 70.2% of the samples harbored at least one clinically actionable genomic aberration. 14.9% of patients showed high tumor mutational burden (TMB > 10 mutations/Mb), and the TMB level was significantly higher in patients with microsatellite instability‐high than in those with microsatellite stability. We also correlated next‐generation sequencing (NGS) results with conventional molecular pathology assays. We found high consistency between ERBB2 focal amplification cases determined by NGS and clinically targetable ERBB2 amplification/HER2 overexpression cases. In conclusion, this study constructed a large‐scale real‐world genomic dataset representative of Chinese cancer patients, spanning multiple tumor types. Moreover, our findings underscore the clinical value of NGS in identifying patients with ERBB2 amplification who may potentially benefit from targeted treatments, particularly in non‐small‐cell lung cancer cases where NGS panel testing is prioritized. We assembled National Cancer Center–Clinical Diagnostics Knowledgebase, a clinical genomic knowledgebase of 6935 tumors with matched normal samples, revealing key somatic alterations and actionable variants (70.2% of the cohort). Enrichment of certain different gene mutations was observed between Chinese and American populations, along with a strong concordance between ERBB2 focal amplification detected by next‐generation sequencing (NGS) and results from conventional HER2 testing. Through a multi‐platform validation framework, this real‐world dataset underscores the clinical utility of NGS in identifying targetable alterations. Created in BioRender. Li, H. (2025) https://BioRender.com/qoyylfh

Background Drug-resistant tuberculosis has emerged as a major public health issue that requires immediate attention. NTS is an innovative method that allows for the direct detection of clinical samples without the need for culture. It could provide more accurate, reliable, and comprehensive information on drug resistance. Methods We collected clinical data retrospectively from patients suspected of having drug-resistant tuberculosis who visited the tuberculosis department at the Second Hospital of Nanjing in Jiangsu Province, China, from December 2023 to December 2024. The diagnostic efficiency of NTS for different types of drug-resistant tuberculosis and antimicrobial resistance was calculated. The relationship between resistance genes, mutated amino acids, and mutation sites was demonstrated. Results In this study, a total of 107 patients with drug-resistant tuberculosis were included, comprising 43 cases of mono-drug resistant tuberculosis, 20 patients with poly-drug resistant tuberculosis, 22 cases of multidrug-resistant tuberculosis, 21 cases of pre-extensively drug-resistant tuberculosis and 1 case of extensively drug-resistant tuberculosis. The accuracy of NTS in diagnosing drug-resistant tuberculosis ranged from 42.9 to 93.0%. Except for second-line injectable drugs, NTS achieved a sensitivity of over 70% for other anti-tuberculosis drugs. Serine was identified as the most frequently mutated amino acid in both the rpoB gene (66.2%, 49/74) and the katG gene (86.3%, 44/51). Additionally, the most frequently mutated amino acids in the embB gene, rpsL gene, and gyrA gene were methionine (94.7%, 44/51), lysine (100%, 28/28), and aspartic acid (66.7%, 20/30), respectively. Conclusion NTS could effectively and precisely deliver comprehensive drug resistance results, assisting medical professionals to create more personalized treatment plans. Besides, it would encourage the development of new anti-tuberculosis drugs to broaden clinical treatment options for drug-resistant tuberculosis.

Background In the Tunisian population, the molecular analysis of hearing impairment remains based on conventional approaches, which makes the task laborious and enormously expensive. Exploration of the etiology of Hearing Impairment and the early diagnosis of causal mutations by next‐generation sequencing help significantly alleviate social and economic problems. Methods We elaborated a custom SureSelectQXT panel for next‐generation sequencing of the coding sequences of 42 genes involved in isolated hearing impairment or along with defects of the retina, the thyroid, and the kidneys. Results We report eight pathogenic variants, four of which are novel in patients with isolated hearing impairment, hearing impairment, and renal tubular acidosis, Usher syndrome and Pendred syndrome. Functional studies using molecular modeling showed the severe impact of the novel missense mutations on the concerned proteins. Basically, we identified mutations in nuclear as well as mitochondrial genes in a Tunisian family with isolated hearing impairment, which explains definitely the phenotype detected since 2006. Conclusion Our results expanded the mutation spectrum and genotype‒phenotype correlation of isolated and syndromic hearing loss and also emphasized the importance of combining both targeted next‐generation sequencing and detailed clinical evaluation to elaborate a more accurate diagnosis for hearing impairment and related phenotypes especially in North African populations. Exploration of the etiology of Hearing Impairment and the early diagnosis of causal mutations by next‐generation sequencing help significantly alleviate social and economic problems. We elaborated a custom SureSelectQXT panel for Next Generation Sequencing of the coding sequences of 42 genes involved in isolated hearing impairment or along with defects of the retina, the thyroid, and the kidneys. We report eight pathogenic variants four of which are novel in patients with isolated hearing impairment, hearing impairment and Renal tubular acidosis, Usher syndrome, and Pendred syndrome. Our results expanded the mutation spectrum and genotype‒phenotype correlation of isolated and syndromic hearing loss and also emphasized the importance of combining both targeted next‐generation sequencing and detailed clinical evaluation to elaborate a more accurate diagnosis for hearing impairment and related phenotypes especially in North African populations.

Purposes Rapid and accurate identification of non-tuberculous mycobacteria (NTM) is crucial yet challenging, promoting the development of novel molecular techniques such as amplification-based targeted high-throughput sequencing and metagenomic unbiased high-throughput sequencing. We aimed to evaluate the diagnostic value of these molecular techniques for NTM infection. Methods A total of 115 clinical specimens from patients with confirmed NTM infection were subjected to multiplex polymerase chain reaction detection techniques (multi-PCR), metagenomic Next-Generation Sequencing (mNGS), targeted Next-Generation Sequencing (tNGS), and targeted Nanopore sequencing (tNanopore). Positivity rates and species identification were compared among these techniques. Results The sensitivity of mNGS, tNGS, and multi-PCR in NTM-infection diagnosis was 44.3%, 42.6%, and 36.5%, respectively, while the sensitivity of the three methods in combination increased to 54.8%. The pathogen identification results of mNGS, tNGS and multi-PCR were matched in 80.6% (25/31) samples at the species level, among which 14 samples (45.2%) was completely matched at the subspecies level. The results of tNanopore, tNGS and mNGS at the species level were completely matched in 73.3% (22/30) samples. Conclusions These molecular assays demonstrated comparable performance in precisely identifying NTM species in clinical specimens, showing their promising potential as efficient and alternative tools for the rapid diagnosis of NTM disease.

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

Abstract Background Antimicrobial resistance (AMR) of Neisseria gonorrhoeae has spread worldwide. Rapid and comprehensive methods are needed to describe N. gonorrhoeae AMR profiles accurately. A method based on multiplex amplicon sequencing was developed to simultaneously sequence 13 genes related to AMR in N. gonorrhoeae directly from clinical samples. Methods Nine N. gonorrhoeae strains were used for the establishment and validation of the method. Eleven urethral swabs and their corresponding cultured isolates were matched as pairs to determine the accuracy of the method. Mock samples with different dilutions were prepared to determine the sensitivity of the method. Five nongonococcal Neisseria strains and 24 N. gonorrhoeae negative clinical samples were used to evaluate the cross-reactivity. Finally, the method was applied to 64 clinical samples to assess its performance. Results Using Sanger sequencing as a reference method, sequences recovered from amplicon sequencing had a base accuracy of over 99.5% and the AMR sites were correctly identified. The limit of detection (LOD) was lower than 31 copies/reaction. No significant cross-reactivity was observed. Furthermore, target genes were successfully recovered from 64 clinical samples including 9 urines, demonstrating this method could be used in different types of samples. For clinical samples, the results can be obtained within a time frame of 7 h 40 min to 10 h 40 min, while for isolates, the turnaround time was approximately 2 h shorter. Conclusions This method can serve as a versatile and convenient culture-free diagnostic method with the advantages of high sensitivity and accuracy.

Long-read RNA sequencing is a powerful technology for transcriptomics, but low throughput and high cost pose challenges. Adaptive sampling, a feature of Oxford Nanopore Technologies, offers real-time enrichment by selectively ejecting non-target molecules. We evaluate adaptive sampling for human transcriptome analysis. Adaptive sampling modestly enriches target transcripts (1.3 × for cDNA sequencing, 1.9 × for direct RNA sequencing) while preserving gene expression and splicing profiles, but is significantly less effective than cDNA hybridization capture. Short read lengths and low sequencing quality limit performance. Adaptive sampling on direct RNA sequencing can boost target yield (~ 20%) within fixed run times, potentially aiding time-constrained applications.

Lower respiratory tract infections are characterized by high morbidity and mortality, the latter associated with the low sensitivity and long turnaround time (TAT) of traditional diagnostic methods. Advances in next-generation sequencing (NGS) offers a promising solution, but in the face of so many different NGS products, how to use them appropriately remains a great challenge for clinicians. This study included 205 patients with suspected lower respiratory tract infections from the department of respiratory and critical care medicine, and collected their lower respiratory tract samples for metagenomic NGS (mNGS) and two different targeted NGS (tNGS), amplification-based tNGS and capture-based tNGS. We analyzed their microorganisms reported, and evaluated their detection performance based on the comprehensive clinical diagnosis. Compared to the two tNGS, mNGS showed significant higher cost ($840) and longer TAT (20 h). Conversely, it identified the highest number of species, totaling 80, compared to 71 species identified by capture-based tNGS and 65 species by amplification-based tNGS. When benchmarked against the comprehensive clinical diagnosis, the capture-based tNGS demonstrated significantly higher diagnostic performance than the other two NGS, with an accuracy of 93.17% and a sensitivity of 99.43%. However, it showed lower specificity compared to the amplification-based tNGS in identifying DNA virus (74.78% vs. 98.25%). The amplification-based tNGS exhibited a poor sensitivity for both gram-positive (40.23%) and gram-negative bacteria (71.74%). Moreover, tNGS was able to identify genotypes, antimicrobial resistance genes and virulence factors. In conclusion, mNGS is suited for the detection of rare pathogens; the capture-based tNGS is preferable for routine diagnostic testing; the amplification-based tNGS can be an alternative in situations requiring rapid results and constrained by limited resources.