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PubMed2025

Targeted Next-Generation Sequencing in Rare Diseases.

Targeted next-generation sequencing (NGS) in rare disease focuses on genetic analysis of specific regions in genome that are linked to a rare disease. In addition to library preparation, sequencing, and data analysis, targeted NGS includes an additional step of target enrichment of selected genes and regions. It allows for more sensitive and profound sequencing, as it is a fast and cost-effective approach with less data burden and is therefore often a method of choice for identifying rare variants in known genes, especially in diagnostics of rare diseases. Several in silico tools address the pathogenicity predictions of rare variants of unknown significance (VUS) and can therefore facilitate clinical interpretation.

Rare Diseases↗
PubMed2026

Computed tomography-guided precision biopsy combined with metagenomic next-generation sequencing for etiological diagnosis in patients with blood culture-negative systemic infections.

ObjectiveTo evaluate the diagnostic efficacy of computed tomography-guided percutaneous biopsy combined with metagenomic next-generation sequencing in patients with blood culture-negative systemic infections and to assess the clinical impact of using this combined strategy for etiological confirmation and guidance of targeted antimicrobial therapy.MethodsThis single-center retrospective observational cohort study enrolled 78 patients who met the Sepsis-3 consensus criteria for suspected systemic infection and had negative conventional microbiological work-ups (at least two sets of blood cultures) between April 2022 and March 2025. All patients underwent computed tomography-guided biopsy of radiologically identified infectious foci, with specimens processed concurrently for conventional culture and metagenomic next-generation sequencing. Diagnostic performance was benchmarked against the final comprehensive clinical diagnosis, and the influence of metagenomic next-generation sequencing findings on antimicrobial therapy modification was analyzed. Sample size calculation, based on a prior study estimating an metagenomic next-generation sequencing detection rate of 85% (&#x3b1;&#x2009;=&#x2009;0.05, &#x3b2;&#x2009;=&#x2009;0.2), indicated a minimum of 68 cases; accordingly, 78 patients were enrolled.ResultsComputed tomography-guided biopsy was technically successful in all 78 patients (100%). The pathogen detection rate of metagenomic next-generation sequencing (91.0%, 71/78) was significantly higher than that of conventional culture (55.1%, 43/78; p&#x2009;<&#x2009;0.001). Using the final clinical diagnosis as the reference standard, metagenomic next-generation sequencing achieved a sensitivity of 94.7% (95% confidence interval: 86.9-98.5), specificity of 100.0% (95% confidence interval: 29.2-100.0), positive predictive value of 100.0% (95% confidence interval: 94.9-100.0), and negative predictive value of 42.9% (95% confidence interval: 9.9-81.6). Among the 35 culture-negative specimens, metagenomic next-generation sequencing established a definitive microbiological diagnosis in 28 cases (80.0%) and detected polymicrobial infections in 11 cases (14.1% of the cohort). Antimicrobial therapy was rationally adjusted based on metagenomic next-generation sequencing results in 69.2% (54/78) of the patients.ConclusionsThe integration of computed tomography-guided precision biopsy with metagenomic next-generation sequencing offers a highly effective diagnostic approach for blood culture-negative systemic infections. This synergistic strategy improves etiological diagnosis by providing high-yield target specimens that enable comprehensive, unbiased pathogen screening, facilitates differentiation between infectious and non-infectious etiologies, and supplies critical evidence for guiding precision antimicrobial therapy. These findings highlight the growing role of interventional radiology in the contemporary framework of precision infectious disease management.

Humans↗
PubMed2026

Employing Metagenomics Capture targeted next-generation sequencing for the etiological diagnosis of bloodstream infections.

BACKGROUND: Bloodstream infections (BSIs) represent a significant public health concern. Metagenomic Capture targeted next-generation sequencing technology, as a newly emerging method for pathogen detection, has been applied in the etiological diagnosis of various infectious diseases and demonstrates good diagnostic efficacy. However, there is relatively limited research on the diagnostic value of this technology for the etiological diagnosis of BSIs. METHODS: A comprehensive retrospective analysis was performed on patients suspected of having BSIs who were admitted to the Affiliated Guangdong Second Provincial General Hospital of Jinan University in 2024. These patients underwent both blood culture analysis and Metagenomic Capture targeted next-generation sequencing technology for diagnostic testing, and a detailed comparison of the results was conducted. RESULTS: It was found that the Metagenomic Capture-targeted next-generation sequencing method has a shorter time to result [1.33 (1.18 - 1.69) vs 2.73 (1.89 - 3.84) days, p&#xa0;<&#xa0;0.001], more pathogenic microbial species detected, higher positive detection rate and higher sensitivity than blood culture. CONCLUSIONS: Metagenomic Capture targeted next-generation sequencing technology is a promising tool for pathogen identification in BSIs, offering substantial methodological advantages in terms of turnaround time, detection breadth, and sensitivity. These diagnostic performance characteristics support its potential utility in clinical microbiology practice.

Humans↗
PubMed2024

The use of next-generation sequencing in personalized medicine.

The revolutionary progress in development of next-generation sequencing (NGS) technologies has made it possible to deliver accurate genomic information in a timely manner. Over the past several years, NGS has transformed biomedical and clinical research and found its application in the field of personalized medicine. Here we discuss the rise of personalized medicine and the history of NGS. We discuss current applications and uses of NGS in medicine, including infectious diseases, oncology, genomic medicine, and dermatology. We provide a brief discussion of selected studies where NGS was used to respond to wide variety of questions in biomedical research and clinical medicine. Finally, we discuss the challenges of implementing NGS into routine clinical use.

High-throughput sequencing↗
PubMed2025

The Use of Next-Generation Sequencing in Personalized Medicine.

The revolutionary progress in development of next-generation sequencing (NGS) technologies has made it possible to deliver accurate genomic information in a timely manner. Over the past several years, NGS has transformed biomedical and clinical research and found its application in the field of personalized medicine. Here we discuss the rise of personalized medicine and the history of NGS. We discuss current applications and uses of NGS in medicine, including infectious diseases, oncology, genomic medicine, and dermatology. We provide a brief discussion of selected studies where NGS was used to respond to wide variety of questions in biomedical research and clinical medicine. Finally, we discuss the challenges of implementing NGS into routine clinical use.

Humans↗
PubMed2026

Metagenomic next-generation sequencing for tuberculosis diagnosis: enhanced performance and cost-effectiveness.

UNLABELLED: Metagenomic next-generation sequencing (mNGS) is a promising tool for diagnosing challenging infections like tuberculosis (TB). However, previous studies largely focused on case-specific application of mNGS in TB diagnosis. Thus, we conducted a retrospective observational study to first systematically evaluate the diagnostic performance and cost-effectiveness of mNGS for TB diagnosis. We retrieved a total of 16,776 results of the seven TB diagnostic assays, including mNGS, tuberculosis IgG antibody, TB interferon-&#x3b3; release assay (TB-IGRA), TB-DNA, Xpert MTB/RIF (Xpert), culture, and acid-fast bacilli staining (AFS) from 3,757 participants with suspected TB infection at Sichuan Provincial People's Hospital from September 2021 to July 2024. Diagnostic metrics were compared against a composite reference standard. Microbial composition and a cost-utility analysis were performed. Among seven TB assays studied, the World Health Organization (WHO)-recommended assays AFS, culture, and Xpert, as well as TB-IGRA, were requested most frequently for TB diagnosis, whereas mNGS ranked last. mNGS demonstrated the highest specificity (100%), accuracy (72.3%), and area under the curve (AUC) (0.795). Its sensitivity in bronchoalveolar lavage fluid and tissue was 71.0% and 72.7%, respectively. Sequential use of mNGS after initial WHO-recommended tests (Xpert/Culture/AFS) significantly improved diagnostic performance (sensitivity, 70.4%; AUC, 0.823). Microbial analysis associated Candida albicans with TB. Cost-utility analysis showed sequential mNGS became cost-effective at higher willingness-to-pay thresholds (>200,000 RMB per correct diagnosis). mNGS offers superior specificity for TB diagnosis. A sequential strategy applying mNGS to conventional-test-negative cases provides enhanced diagnostic performance and is cost-effective at higher healthcare investment values, supporting its utility for diagnostically challenging TB. IMPORTANCE: This study systematically assesses the diagnostic performance and cost utility of metagenomic next-generation sequencing (mNGS) for tuberculosis (TB) in a large real-world cohort of 3,757 suspected patients, comparing it against six conventional assays (tuberculosis IgG antibody, TB interferon-&#x3b3; release assay, TB-DNA, Xpert, culture, and acid-fast bacilli staining). mNGS demonstrated the highest specificity (100%), accuracy (72.3%), and area under the curve (AUC) (0.795), with sensitivities of 71.0% in bronchoalveolar lavage fluid and 72.7% in tissue. Notably, sequential use of mNGS after the World Health Organization-recommended tests significantly improved sensitivity to 70.4% and AUC to 0.823. Candida albicans showed significant differences among the three groups. The sequential mNGS strategy was cost-effective compared with no mNGS, and its cost-effectiveness increased with a rising willingness-to-pay threshold. Overall, these results highlight mNGS as a valuable supplementary tool for challenging TB cases, especially when conventional tests are inconclusive, and provide strong evidence for integrating it into diagnostic algorithms to optimize clinical decision-making and resource allocation.

Adult↗
PubMed2026

The diagnostic potential of combined quantitative polymerase chain reaction and next-generation sequencing using the same primers for periprosthetic joint infection.

Next-generation sequencing (NGS) enables the detection of specific pathogens unidentifiable by conventional cultures, but its application in orthopedics remains inconsistent due to background contamination and irreproducible findings. This study evaluated the diagnostic performance of a novel workflow combining broad-range 16S rRNA gene quantitative PCR (qPCR) screening with downstream NGS, focusing on bacterial biomass thresholds. The qPCR assay demonstrated excellent intrarater reliability, with an intraclass correlation coefficient (ICC) of 0.961 (95% confidence interval, 0.881 to 0.997). Based on serially diluted positive controls, a quantitative threshold of 10&#x2075; CFU/mL was established as the minimum concentration required for the consistent detection of fastidious taxa, such as Escherichia coli. When evaluated against conventional cultures using 95 sonicate fluid and 276 pre/intraoperative tissue samples, the qPCR assay achieved a sensitivity of 80% and a specificity of 72%. Subsequent NGS sequencing of 26 clinical samples and 9 controls showed concordance in 4 of 6 culture-positive infected cases with NGS taxonomy, whereas the remaining discrepancies were likely attributable to culture-based phenotypic misidentification. Notably, among the qPCR-positive cases, three were culture-negative, including two hip prosthesis loosening cases exhibiting polymicrobial profiles, and one post-traumatic osteoarthritis case harboring low-level Staphylococcus. Crucially, this post-traumatic patient developed delayed periprosthetic joint infection (PJI) 2 years post-surgery, with cultures identifying Staphylococcus previously detected by the initial NGS analysis. Integrating qPCR screening with targeted NGS effectively refines pathogen identification, filters environmental artifacts, and overcomes the diagnostic limitations of culture-negative infections in orthopedic practice.IMPORTANCENext-generation sequencing (NGS) enables the detection of specific pathogens in clinical samples that are not identifiable by conventional methods. However, NGS applications in orthopedics have not been quantitatively evaluated, and findings have been inconsistent owing to contaminants and the presence of non-credible causative organisms. These factors primarily stem from the failure to evaluate low-biomass samples and the absence of proper controls, such as negative controls or mock community DNA samples. This study demonstrates that interpreting results from low-biomass samples requires careful consideration because NGS relies on relative bacterial abundances; distinguishing likely pathogens from contaminants is particularly challenging when bacterial loads are low. We demonstrated that combining NGS with quantitative PCR (qPCR) and applying a Cq cutoff can reduce false positives.

Humans↗
PubMed2025

Next-generation sequencing in breast cancer: current clinical applications and future directions.

INTRODUCTION: Breast cancer is a heterogeneous disease that claims 670,000 lives by 2022. Omic technologies, particularly next generation sequencing (NGS) offers promising avenues for precision medicine. American Society of Clinical Oncology (ASCO) outlines genomic testing's utility, emphasizing prognostic and diagnostic potential. OBJECTIVES: This review succinctly explores NGS's evolution and clinical applications of NGS in breast cancer, thereby guiding future research to enhance patient care. METHODS: Comprehensive literature searches were conducted using databases such as PubMed, Google Scholar, and ResearchGate, focusing on keywords including breast cancer, HER-2 low breast cancer, circulating tumour DNA, single-cell RNA sequencing, and next-generation sequencing. Peer-reviewed, high-quality articles published in English were selected for inclusion. RESULTS: Previous studies have explored the evolution of NGS technology and its clinical applications in breast cancer, including genomic and transcriptomic characterization, treatment guidance, and resistance prediction. Molecular profiling of challenging entities such as early-onset breast cancer and HER-2 low tumours was summarized, with key findings highlighted. This review also discusses emerging technologies, including circulating DNA and single-cell sequencing, as promising avenues for discovery. CONCLUSION: NGS has revealed the genomic and transcriptomic diversity of breast cancer, identifying actionable alterations associated with chemotherapy response and resistance to therapies such as trastuzumab, TKIs, and CDK4/6 inhibitors. Circulating tumour DNA (ctDNA) shows potential for diagnosis, prediction, prognosis, and monitoring, despite tumour heterogeneity. Single-cell analysis enables exploration of individual cell transcriptomes, though high costs and low throughput remain barriers to widespread adoption. HER2-low tumours continue to pose significant research challenges.

Humans↗
PubMed2026

Clinical impact of metagenomic next-generation sequencing for pathogen identification and guided therapy in pediatric intensive care unit patients with severe pulmonary infections.

UNLABELLED: To explore the diagnostic efficiency, clinical concordance, and precision treatment value of metagenomic next-generation sequencing (mNGS) for severe pulmonary infections in children in the pediatric intensive care unit (PICU), and to provide evidence for improving microbiological diagnosis and optimizing anti-infective strategies. A retrospective cohort study included 89 children with severe pneumonia in the PICU in 2024. All underwent routine microbiological testing and mNGS of bronchoalveolar lavage fluid (BALF). Detection rates, pathogen composition, co-infection identification, diagnostic concordance, and treatment impact were analyzed. Metagenomic next-generation sequencing demonstrated high diagnostic sensitivity in the PICU setting, achieving a positive detection rate of 90.0% (80/89) and identifying a diverse spectrum of 103 pathogens, including 50.5% viruses, 43.7% bacteria, 38.8% co-infections (vs 11.6%), and 86.3% diagnostic concordance (vs 55.8%, P < 0.01). Among 46 patients included in the therapeutic outcome analysis (22 in the mNGS-guided group), 21 patients in the mNGS-guided group improved. Multivariate logistic regression analysis, adjusting for confounding factors (age, underlying diseases, PaO2/FiO2 ratio, PRISM III score, and preoperative antibiotic use duration), confirmed that mNGS-guided therapy was an independent protective factor for achieving the primary outcome (OR = 5.23, 95% CI: 1.87-14.61, P = 0.002) and secondary outcomes (C-reactive protein reduction &#x2265;50%: OR = 4.89, 95% CI: 1.72-13.93, P = 0.003; oxygenation improvement: OR = 5.67, 95% CI: 1.98-16.21, P = 0.001). Metagenomic next-generation sequencing demonstrated high diagnostic sensitivity in the PICU setting, guiding precision therapy, and improving prognosis. IMPORTANCE: It supports metagenomic next-generation sequencing (mNGS) as a supplementary tool for pediatric intensive care unit (PICU) refractory infections, guides anti-infective adjustments, and informs tiered diagnostic pathways for resource-limited settings to optimize cost-effectiveness.

Humans↗
PubMed2026

Identification and full genome sequencing of previously unknown sandfly-borne phleboviruses using a newly established capture-based next-generation sequencing approach.

Sandfly-borne phleboviruses cause febrile illness and neuroinvasive disease in humans. While infections are reported in the Mediterranean region, the discovery of previously unknown phleboviruses in sandflies from Kenya suggests a wider geographic distribution. Detection and characterization of novel phleboviruses are often hindered by low-quality and low-viral-load samples. We developed a capture-based target enrichment next-generation sequencing approach that showed a 99%-100% fold enrichment of viral genomes from primary material and provides a robust tool for generating complete genomes of both known and previously unknown viruses. From a collection of 15,652 sandflies in Kenya, we recovered seven complete coding sequences of Embossos, Bogoria, and Kiborgoch viruses, and of two previously unknown phleboviruses, which were named Sosoik and Shable viruses. Sosoik virus shared 83% amino acid identity in its RdRp gene with that of Bogoria virus, while Shable virus shared ca. 88% amino acid identity with viruses of the Salehabad serocomplex. Additionally, a reassortant of Shable virus was detected that possessed an M segment from an undescribed Ponticelli-like virus. DNA barcoding of blood-fed sandflies revealed several potentially novel Sergentomyia species and evidence of host-feeding on humans, livestock, and reptiles, suggesting possibilities for zoonotic transmission. Overall, our findings increase the known genetic diversity of Old World sandfly-borne phlebovirus species from 18 to 25 (by 38.9%), including the detection of viruses from all pathogenic sandfly-borne phlebovirus serocomplexes in East Africa, opening new horizons in disease ecology research.IMPORTANCEKnowledge of the genetic diversity of circulating pathogens is crucial for providing appropriate diagnostics and disease management. This study established a novel capture-based target enrichment next-generation sequencing approach that enabled the near-complete viral genome recovery from primary samples, while native NGS yielded negative or poor-quality results. In addition to the five recently discovered sandfly-borne phleboviruses in Kenya, two previously unknown phleboviruses were detected in sandflies from the same region. The viruses were detected in several sandfly species, which showed diverse host-feeding behaviors, including mixed feeding on humans and chickens. The study significantly advances the understanding of sandfly-borne phleboviruses by uncovering their broader geographic distribution and genetic diversity, particularly in East Africa, highlighting the importance of expanding surveillance efforts beyond traditionally studied regions.

Phlebovirus↗
PubMed2024

Reflective Evaluation of Next-Generation Sequencing Data during Early Phase Detection of the Delta Variant.

During the SARS-CoV-2 pandemic, next-generation sequencing (NGS) technologies like the Ion Torrent S5 and Illumina MiSeq, alongside advanced software, improved genomic surveillance in South Africa. This study analysed anonymized samples from the Eastern Cape using Genome Detective and NextClade, showing Ion Torrent S5 and Illumina MiSeq success rates of 96% and 94%, respectively. The study focused on genomic coverage (above 80%) and mutation detection (below 100), with the Ion Torrent S5 achieving 99% coverage compared to Illumina MiSeq's 80%, likely due to different primers used in amplification. The Ion Torrent S5 was more effective in sequencing varied viral loads, whereas Illumina MiSeq had difficulties with lower loads. Both platforms were adept at identifying clades, successfully differentiating between Beta (<45%) and Delta variants (<30%), despite minor discrepancies in assignments due to Illumina MiSeq's lower coverage, leading to a failure rate of up to 6%. Manual library preparation showed similar sample processing and clade identification capabilities for both platforms. However, differences in sequencing duration (3.5 vs. 36 hours), automation level, genomic coverage (80% vs. 99%), and viral load compatibility were noted, highlighting each platform's unique advantages and challenges in SARS-CoV-2 genomic surveillance. In conclusion, the Illumina MiSeq and Ion Torrent S5 platforms are both efficacious in executing whole-genome sequencing (WGS) via amplicons, facilitating precise, accurate, and high-throughput examinations of SARS-CoV-2 viral genomes. However, it is important to note the existence of disparities in the quality of data produced by each platform. Each system offers unique benefits and limitations, rendering them viable choices for the genomic surveillance of SARS-CoV-2.

Illumina MiSeq↗
PubMed2026

Application of metagenomic next-generation sequencing in children with pneumonia of unknown etiology.

OBJECTIVE: To investigate the pathogen spectrum and clinical application value of metagenomic next-generation sequencing (mNGS) in lower respiratory tract specimens from children with pneumonia of unknown etiology. METHODS: A retrospective analysis was conducted on children hospitalized in the intensive care unit (ICU) and respiratory department ward of Children's Hospital of Chongqing Medical University from January 2025 to December 2025. All enrolled cases presented negative results for conventional respiratory pathogen tests and received mNGS testing of lower respiratory tract specimens for etiological identification. The mNGS findings and clinical data of the included children were analyzed. RESULTS: A total of 92 children were enrolled, including 54 males and 38 females, with ages ranging from 2 months to 13 years and 8 months. Causative pathogens were detected in 77 cases (83.7%). The clinically adjudicated etiological diagnosis rates of bacteria, viruses, fungi and atypical pathogens were 75.0% (69/92), 37.0% (34/92), 13.0% (12/92) and 5.4% (5/92), respectively. Thirty-eight cases were complicated with polymicrobial infection, among which bacterial-viral infection was predominant, accounting for 23.1% (24/92). Children with immunocompromised conditions exhibited higher incidences of clinically adjudicated bacterial, fungal and polymicrobial infection than immunocompetent patients. The most common clinically confirmed causative pathogens in immunocompromised children were Streptococcus pneumoniae, human cytomegalovirus, Haemophilus influenzae, Stenotrophomonas maltophilia and Enterococcus faecalis. Treatment regimens were adjusted in 58 cases (63.0%) based on mNGS findings, switching to pathogen-targeted anti-infective therapy. CONCLUSION: For pediatric pneumonia with negative conventional etiological tests, mNGS of lower respiratory tract specimens significantly enhances pathogen detection rates, effectively identifies polymicrobial infection and opportunistic pathogens. Immune status serves as a critical stratification factor influencing pathogen spectrum and infection patterns, with immunocompromised children being more susceptible to opportunistic infections. Adjustment of anti-infective regimens based on mNGS results can effectively facilitate personalized anti-infective therapy.

Humans↗