PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Next generation sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 181 records · Page 10Linked to original sources
PubMed2026

Molecular characterization of CD36 deficiency in blood donors of Middle Eastern and African origin reveals transcript-level defects beyond genomic variants.

BACKGROUND: The increasing diversity of blood donor populations has created new challenges for transfusion services worldwide. The identification of donors lacking relevant high-prevalence antigens is becoming increasingly important to ensure compatible blood products for alloimmunized patients and to support the development of rare donor registries. CD36 (ISBT 045) is a glycoprotein expressed on platelets, monocytes, and erythroid precursor cells. CD36 deficiency has been reported across multiple populations and is of relevance due to its association with anti-CD36 isoantibodies, which may cause platelet transfusion refractoriness and fetal/neonatal alloimmune thrombocytopenia. STUDY DESIGN AND METHODS: We analyzed CD36 expression in 1250 blood donors of diverse ancestry using flow cytometry. CD36-negative samples underwent molecular characterization using Sanger sequencing and next-generation sequencing of genomic DNA, complemented by cDNA analysis and cloning to investigate transcript-level alterations. RESULTS: We identified CD36 deficiency in 27 donors (2.16%). Genomic sequencing revealed 18 distinct coding variants, including three novel variants, most in the heterozygous state. In one CD36-negative donor, cDNA analysis demonstrated a 52-bp deletion in exon four and complete skipping of exon 9, despite the absence of splice-site variants in genomic DNA. Cloning confirmed coexistence of aberrant and wild-type transcripts in this individual. CONCLUSION: Our findings demonstrate that CD36 deficiency can arise from transcript-level defects in the absence of detectable coding or splice-site variants. These results indicate that genomic sequencing alone may be insufficient to fully resolve CD36-negative phenotypes and highlight the importance of integrating transcriptomic approaches to improve molecular diagnostics and transfusion support in increasingly diverse donor populations.

CD36 deficiency↗
PubMed2026

Library strategies differentially shape microbial, functional, and host signals in clinical metagenomic sequencing.

Metagenomic next-generation sequencing (mNGS) is increasingly used in infectious disease diagnostics, yet how library preparation shapes the microbial, functional, and host signals recovered from clinical samples remains poorly defined. Here, we performed a within-sample parallel comparison of three mNGS library preparation strategies-DNA-based libraries (DNAlib), RNA-based libraries (RNAlib), and total nucleic acid-based libraries (TNAlib)-across a diverse range of clinical specimens spanning five sample types. Using a curated clinical infectome as a benchmark, we show that library strategies are not interchangeable but capture distinct biological dimensions of the same specimen. RNAlib provided the most comprehensive standalone recovery of the clinical infectome, with improved detection of RNA viruses and cellular pathogens, enhanced resolution of resistance and virulence signals, and preservation of infection-associated host immune signatures. DNAlib showed stronger baseline recovery of DNA viruses and broader host genome coverage, whereas the TNAlib workflow evaluated here largely behaved as an intermediate strategy rather than a consistent improvement over dedicated DNA- or RNA-based workflows. Together, these results establish that the library preparation protocol is a major determinant of how clinical mNGS data should be interpreted and provide a framework for selecting sequencing strategies according to specific diagnostic and biological questions.IMPORTANCEMetagenomic sequencing is increasingly used in infectious disease research and clinical diagnostics, but different library preparation strategies may recover fundamentally different biological signals from the same sample. These signals include not only pathogens but also background microbes, microbial functional activity, and host immune-response patterns. Here, we systematically compared DNA-, RNA-, and total nucleic acid-based metagenomic sequencing libraries using the same clinical samples processed in parallel. We found that the three strategies did not provide equivalent information. RNA-based sequencing generated the most informative single-library view of infection, particularly for RNA viruses, cellular pathogens, functional microbial signals, and host immune-response patterns. DNA-based sequencing was more effective for DNA virus and host genome recovery, whereas the total nucleic acid sequencing workflow evaluated here generally behaved as an intermediate strategy. These findings show that library preparation can substantially influence the interpretation of metagenomic data.

functional characterization↗
PubMed2026

Genome-Wide Single-Nucleotide Polymorphism (SNP)-based Profiling of Loss of Heterozygosity Reveals Distinct Molecular Subgroup-Specific Patterns in Gastrointestinal Stromal Tumors (GIST).

PURPOSE: Gastrointestinal stromal tumors (GIST) are molecularly heterogeneous neoplasms defined by mutually exclusive driver alterations (KIT, PDGFRA, SDH, BRAF, RAS, and NF1). However, driver mutations alone do not fully explain their biological and clinical variability. Chromosomal imbalances and loss of heterozygosity (LOH) may represent an additional layer of tumor characterization. We developed a single-nucleotide polymorphism (SNP)-based next-generation sequencing panel enabling genome-wide LOH assessment from formalin-fixed paraffin-embedded tissue. MATERIALS AND METHODS: Forty-nine GIST cases molecularly classified using targeted next-generation sequencing (KIT n = 19, PDGFRA n = 9, SDH-deficient n = 8, NF1 n = 7, quadruple wild-type n = 6) were analyzed. LOH was inferred from variant allele frequency patterns across 1826 genome-wide SNPs. RESULTS: Chromosome 14 was the most commonly affected (63%), followed by chromosomes 22 (45%), 15 (41%), 21 (27%), and 13 (20%). Loss of chromosome arm 1p occurred in 43% of tumors. Distinct subgroup-specific patterns emerged: KIT-mutant GIST exhibited the highest degree of genomic instability, whereas both SDH-deficient tumors and PDGFRA-mutant GIST displayed minimal chromosomal instability. NF1-mutant tumors showed recurrent single-arm chromosome 17 LOH. Quadruple wild-type GISTs were heterogeneous, including 1 case with extensive chromosomal instability. CONCLUSIONS: Genome-wide SNP-based LOH profiling reveals distinct, subgroup-specific patterns of chromosomal imbalance in GIST and may serve as a feasible complementary approach to driver mutation analysis for refined molecular characterization and potential future clinical utility.

Humans↗
PubMed2025

Enzymes in high-throughput RNA sequencing: Applications and challenges.

High-throughput RNA sequencing provides genome-wide information on the dynamics of RNA in each cell and how the dynamics responds to environmental changes. Next-generation sequencing by the Illumina platform currently provides the highest information output as compared to other platforms. A key component of next generation sequencing of each RNA is the successful end-to-end reverse-transcription into a cDNA strand. This can be highly challenging given the propensity of each RNA to adopt ordered structures and to contain post-transcriptional modifications. While many reverse transcriptase (RT) enzymes have been developed over the years to maximize read-through of an RNA, their processivity and efficiency varies, raising the question of how to select the RT for the experiment at hand. Here, we use tRNA as a model for genome-wide sequencing, as tRNA has a stable secondary and tertiary structure and has a high density and wide variety of post-transcriptional modifications, presenting one of the most challenging problems of sequencing RNA. We compare the efficiency of end-to-end cDNA synthesis of tRNA among several recent RT enzymes and provide a general sequencing workflow that is applicable to most of these enzymes.

High-Throughput Nucleotide Sequencing↗
PubMed2026

Genomic Sequencing in Neonatal Encephalopathy and Suspected Hypoxic-Ischaemic Encephalopathy: A Systematic Review.

BACKGROUND: Neonatal encephalopathy (NE) is a major cause of neonatal mortality and long-term neurological disability. Although hypoxic-ischaemic encephalopathy (HIE) is the most common cause, several genetic disorders may mimic or coexist with hypoxic-ischaemic injury. Next-generation sequencing has emerged as a promising diagnostic tool in this setting. This systematic review evaluated the current evidence on genomic sequencing in NE. MATERIAL AND METHODS: A systematic review was conducted according to PRISMA 2020 guidelines and prospectively registered in PROSPERO. PubMed/MEDLINE, Embase, and Scopus were searched from inception to June 2026. Eligible studies included neonates (≤28 days) with NE, suspected or confirmed HIE, HIE mimics, or unexplained NE who underwent genomic sequencing. Whole-exome sequencing (WES), whole-genome sequencing (WGS), clinical exome sequencing (CES), rapid genomic sequencing, and targeted next-generation sequencing panels were considered. Study quality was assessed using the Newcastle-Ottawa Scale. RESULTS: Seven studies met the inclusion criteria. Considerable heterogeneity was observed regarding patient selection, sequencing strategies, and reported outcomes. Among diagnostic sequencing studies, diagnostic yield ranged from 23.5% to 53.1%. Pathogenic and likely pathogenic variants were identified in genes associated with developmental and epileptic encephalopathies, metabolic disorders, mitochondrial diseases, and neurodevelopmental syndromes, including SCN2A, KCNQ2, CACNA1A, STXBP1, PTPN11, BCOR, MMUT, COQ2, and GBE1. Genomic sequencing frequently refined or changed the initial diagnosis, improved prognostic assessment and genetic counselling, and, in selected cases, guided disease-specific treatment. One study investigated genetic susceptibility to hypoxic-ischaemic injury rather than diagnostic sequencing. CONCLUSIONS: Genomic sequencing provides clinically meaningful diagnoses in a substantial proportion of neonates with unexplained NE or atypical HIE presentations. Current evidence supports integrating genomic sequencing into the diagnostic evaluation of selected infants, although larger prospective studies are needed to define its optimal timing, clinical utility, and cost-effectiveness.

Humans↗
PubMed2025

Well-differentiated systemic mastocytosis: Genetics, mast cell immunophenotypes, and KIT autophosphorylation.

BACKGROUND: Well-differentiated systemic mastocytosis (WDSM) is a rare myeloid neoplasm where the genetic etiology is often unknown. OBJECTIVE: We aimed to assess WDSM patients for novel KIT variants, mast cell (MC) aberrant immunophenotypes, and KIT autophosphorylation patterns. METHODS: Next-generation sequencing, MC immunophenotyping, and KIT autophosphorylation studies were performed. RESULTS: Among 454 SM patients, there were 432 with KIT p.D816V+ SM and 4 with KIT p.D816Y+ SM-notably, none of these patients had WDSM. Of the remaining patients, we identified 7 with WDSM (1.5%) and 2 relatives with mastocytosis in skin. Next-generation sequencing revealed that 6 of 9 subjects carried known or novel germline KIT variants corresponding to regions outside of codon 816. Three patients had germline KIT p.K509I; 2 had germline KIT p.A533D; 1 had two germline KIT variants p.F681L and p.M541L; and 3 had no KIT mutation. Intracellular expression of CD2 and CD25 and less robust expression of CD30 was observed in MCs from WDSM patients. By developing a novel transient transfection assay in 293T cells, we found that unlike KIT p.D816F/V/Y variants that exhibit nearly exclusive intracellular localization and strong ligand-independent autophosphorylation (class II), WDSM-associated KIT variants showed enhanced ligand-dependent autophosphorylation relative to wild type (class I). CONCLUSIONS: Our study doubles the number of KIT variants identified in WDSM patients. No KIT p.D816V+ SM patient had WDSM. Intracellular CD2 and CD25 expression was more robustly detected in MCs from WDSM patients compared to CD30.

Humans↗
PubMed2025

Guidelines to Analyze ChIP-Seq Data: Journey Through QC and Analysis Considerations.

ChIP-Seq is used to study DNA-protein interactions, unraveling chromatin states and gene regulatory properties of transcription factors. ChIP-Seq involves immunoprecipitation followed by sequencing using Next-Generation sequencing approaches. The ENCODE consortium provides extensive guidelines for ChIP-Seq analysis. Meanwhile, appropriate QC metrics and knowledge to interpret outcomes are essential for a good ChIP-Seq experiment. This chapter outlines the various QC metrics and analytical tools for ChIP-Seq analysis to provide a better understanding of the results.

Chromatin Immunoprecipitation Sequencing↗
PubMed2025

Diagnostic and Monitoring Strategies for VEXAS Syndrome: Evaluating Sanger Sequencing, NGS, and the SWIM-Score.

VEXAS syndrome is an adult-onset autoinflammatory disorder caused by somatic UBA1 variants, but there are no standardized criteria for genetic testing or diagnostics. This study compared Sanger sequencing and next-generation sequencing (NGS) for detecting UBA1 variants in patients with suspected VEXAS, assessed the ability of Sanger sequencing to estimate variant allele fractions (VAFs), and evaluated the Maeda et al. scoring system for selecting patients for genetic testing in a primary cohort and a validation cohort. In the primary cohort of 104 patients, Sanger sequencing identified VEXAS variants in 12%, with no additional cases detected by NGS. Sanger sequencing accurately quantified VAFs ranging from 0.1 to 0.9. In a small longitudinal subset (n = 3), VAFs in blood correlated with CRP levels, increased over time despite various treatments, but decreased in two patients after initiation of Azacitidine treatment. The novel parameters, VAF in myeloid cells and VEXAS cell concentration, showed promise as exploratory markers for patient monitoring. The Maeda-score, requiring a threshold score of 2 for 100% sensitivity, exhibited low specificity-29% in the primary cohort and 41% in the validation cohort (n = 62, with 2 carrying VEXAS variants). In contrast, the simplified SWIM-score-based on Skin involvement, Weight loss, Inflammation, and Macrocytic anemia-achieved 100% sensitivity in both cohorts, with higher specificities of 47% and 65%, respectively. In conclusion, Sanger sequencing reliably detected UBA1 variants and quantified VAFs. Monitoring VAF and VEXAS cell concentration may track disease progression, and the SWIM-score demonstrated potential for accurately selecting patients for UBA1 testing.

Humans↗
PubMed2025

The first case of GOLGA5-RET fusion-positive malignant spindle cell sarcoma of the head and neck responsive to selpercatinib.

Soft-tissue sarcoma (STS) is a rare malignancy that accounts for less than 1% of all cancers, and recent advances in molecular biology have led to its classification based on genomic information. Some RET-rearranged neoplasms have been reported to present pathological features similar to Neurotrophic Tyrosine Kinase Receptor-rearranged spindle cell neoplasms. Here, we report the first case of head and neck spindle cell sarcoma with a GOLGA5-RET fusion that demonstrated a sustained clinical response to selpercatinib, identified through targeted next-generation sequencing (NGS). The patient was a 43 year-old man with a tumor in the arytenoid region that was resected and diagnosed as a malignant spindle cell tumor. Despite initial treatment with surgical resection alone, local recurrence was confirmed, requiring salvage therapy with total laryngectomy and bilateral cervical dissection. Surgical specimen revealed a spindle tumor with a patternless pattern and collagenous stroma. Immunohistochemistry (IHC) with positivity for CD34, bcl-2 (focally), S100, and weak nuclear staining for STAT6, with absence of expression of CK AE1/3, desmin, c-kit, smooth muscle actin, myogenin, synaptophysin, and SOX10. Trk A/B/C were also negative on IHC. Following confirmation of multiple lung metastases, the patient was treated with doxorubicin monotherapy. Targeted NGS identified GOLGA5-RET rearrangement, FGF14 amplification (equivocal), CDKN2B loss, and CDKN2A loss. GOLGA5-RET rearrangements were validated through fluorescence in situ hybridization. The patient subsequently was enrolled in a phase 1/2 trial for the selective RET inhibitor selpercatinib, resulting in a sustained partial response over 5 years. Although solitary fibrous tumor (SFT) was initially considered as a differential diagnosis based on immunohistochemical findings, the lack of strong and diffuse STAT6 expression made this diagnosis unlikely. Subsequent next-generation sequencing (NGS) revealed a RET fusion, leading to the diagnosis of an RET-rearranged spindle cell neoplasm. This case highlights the importance of genomic testing for certain spindle cell sarcomas and the potential benefit of RET-specific inhibitors against RET-altered sarcomas.

Next-generation sequencing↗
PubMed2026

A novel relationship between time offsets in capillary electrophoresis and DNA sequence variations in short tandem repeats.

Next-generation sequencing (NGS) provides increased discriminatory power in forensic DNA analysis due to the detection of isoalleles. Differences in sequences between alleles allow for a second layer of differentiation between DNA contributors beyond the number of short tandem repeat (STR) repeat units. However, because NGS is a more time and resource-intensive analysis than conventional capillary electrophoresis (CE), laboratories may benefit from indicators that suggest NGS is likely to provide added value. This study examined whether CE migration offsets, measured as residuals in the OSIRIS analysis software, can differ significantly among STR isoalleles. Residuals represent the time offset between a sample allele peak and its corresponding allelic ladder peak. Paired CE and NGS data from 95 single source samples were analyzed for CE-based residual differences, as the NGS data provided the sequence information of the corresponding isoalleles. Residual values differed significantly among isoalleles at several STR loci. Statistically significant differences were identified at D16S539 and D3S1358, as well as at specific allele lengths within D12S391, D13S317, and D8S1179. These findings demonstrate that CE residual variation can reflect underlying STR sequence differences between contributors. In practice, residual-based metrics could help laboratories to identify casework reference samples where NGS is likely to provide additional discrimination, without the need for processing outside of a routine CE workflow. Due to the potentially large number of isoalleles, community wide efforts to aggregate CE residual differences versus isoallele sequences may be useful in the validation and implementation of this approach to add value to forensic DNA analyses.

Electrophoresis, Capillary↗
PubMed2025

Genome-Wide Profiling of Histone Modifications in Fission Yeast Using CUT&Tag.

Eukaryotic DNA is organized in the nucleus in the form of chromatin. Nucleosomes, the fundamental unit of chromatin, are subject to many posttranslational modifications (PTMs) as well as compositional variations through incorporation of histone variants. These alterations play important roles in regulation of genome structure and activity. Genome-wide profiling of these regulatory features is essential for understanding of genome function. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) is a widely used method to assay genome-wide localization in fission yeast but suffers from the requirement for a large amount of input chromatin, antibodies, and a cumbersome experimental pipeline. New methods such as Cleavage Under Targets and Tagmentation (CUT&Tag), which combine the specificity of targeted cleavage and adapter insertion with the sensitivity of next-generation sequencing, enable identification and characterization of various epigenetic marks affording low input requirement as well as more streamlined protocols. However, these approaches have not been adapted for use in fission yeast, Schizosaccharomyces pombe. Here, we describe an adapted CUT&Tag protocol for epigenomic profiling in fission yeast using the heterochromatin-associated histone H3K9 methylation PTM for benchmarking.

Schizosaccharomyces↗
PubMed2025

Composition-on-composition regression analysis for multi-omics integration of metagenomic data.

MOTIVATION: Compositional data are frequently encountered in many disciplines, such as in next-generation sequencing experiments widely used in biomedical studies. Regression analysis with compositional data as either responses or predictors has been well studied. However, when both responses and predictors are compositional, the inventory of analysis tools is surprisingly limited, especially in the high-dimensional setting. Among the few existing methods, most of them rely on a log-ratio transformation to move compositional data from the simplex to real numbers. Yet, a serious weakness of these methods is their failure to handle the substantial fraction of zeroes observed in data collected from next-generation sequencing experiments. RESULTS: To investigate associations between two high-dimensional multi-omics compositions, we propose a composition-on-composition (COC) regression analysis method which does not require log-ratio transformations and hence can handle zeroes in the data. To account for high dimensionality, we estimate regression coefficients using a penalized estimation equation approach. Finally, inference procedures for COC regression are also proposed. Superior performance of COC is demonstrated through both comprehensive numerical simulations and case studies. AVAILABILITY AND IMPLEMENTATION: Source R codes to implement COC method is available at https://github.com/nrios4/COC.

Regression Analysis↗
PubMed2026

Validation of an integrated metagenomic pipeline combining optimized wet-lab processing and tiered reporting for CSF pathogen detection.

UNLABELLED: Metagenomic next-generation sequencing (mNGS) in the infectious disease diagnostic space has been gaining traction and is popular for aiding in the diagnosis of central nervous system infections. However, many challenges and obstacles remain in making this technology a gold standard for infectious disease diagnostic testing. One major challenge is being able to distinguish between the clinically relevant organisms from background contamination. We performed a validation study for mNGS on cerebrospinal fluid (CSF) that utilized positive clinical samples and contrived samples that incorporated a bioinformatics pipeline that can better distinguish between background contamination and clinically relevant organisms and used a three-tiered reporting algorithm meant to decrease the inherent subjectivity that comes with interpreting and reporting data from clinical metagenomic sequencing. The validation of this assay and category-based reporting pipeline revealed an overall concordance of 91.8%, with a sensitivity of 100% and a specificity of 72.4%. In addition, we improved the detection of clinically relevant RNA viruses to almost 100% in the CSF by modifying the wet lab processing of the sample. This bioinformatics pipeline with a category-based reporting algorithm will provide more confidence in reporting microorganisms detected with this technology, mNGS, and improving patient care. IMPORTANCE: Metagenomic next-generation sequencing (mNGS) can offer a broad, unbiased approach for the detection of infectious pathogens and has shown promise in diagnosing central nervous system infections. Despite its potential, clinical implementation remains limited by challenges in distinguishing clinically relevant organisms from background contamination. This study validated an mNGS assay for cerebrospinal fluid that incorporates an optimized bioinformatics pipeline with a three-tiered reporting algorithm designed to reduce subjectivity and enhance diagnostic confidence. The assay also has improved detection of clinically relevant RNA viruses through modified wet-lab processing. These findings support the clinical utility of a structured, category-based reporting approach for mNGS, advancing its reliability as a diagnostic tool in infectious disease testing.

Metagenomics↗
PubMed2024

Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Humans↗
PubMed2026

Clinical impact of 16S rRNA RC-PCR NGS on infectious disease management.

16S rRNA metagenomics provides a culture-independent method for diagnosing infections with fastidious or uncultivable organisms, guiding targeted therapy, and detecting polymicrobial communities. This study utilizes reverse complement (RC)-PCR next-generation sequencing (NGS) to accurately identify bacterial pathogens from clinical specimens and assess its impact on clinical decision-making, setting it apart from conventional 16S sequencing approaches. A retrospective analysis of an ISO 15189 accredited 16S RC-PCR NGS diagnostic workflow targeting the V1-6 and V9 regions of the 16S rRNA gene was conducted over a 2-year period, including 390 clinical specimens from 316 patients. 16S RC-PCR NGS results were discussed in a multidisciplinary consultation and subsequently reported to the clinic. In total, 1,283 RC-PCR results were analyzed, of which 517 were from clinical specimens, 284 were negative controls, 66 were positive controls, and 416 were from wet lab and bioinformatic pipeline validation. 16S RC-PCR NGS assay detected bacterial taxa in 179/390 (45.9%) of clinical specimens, while 201/390 (51.5%) were negative, and 10/390 (2.6%) yielded uninterpretable results. The specimen types pus, pleural fluid, and heart valves exhibited the highest positivity rate (68% to 70%). Overall, 16S RC-PCR NGS influenced diagnostic decision making in 145/282 (51.4%) clinical cases and guided therapeutic management in 77/282 (27.3%) cases. Results providing definite evidence for either the presence or absence of bacterial infection were considered clinically valuable. Integration of 16S RC-PCR NGS pathogen detection with multidisciplinary consultation markedly improved clinical management, directly impacting diagnosis and treatment of complex clinical cases in a tertiary care setting. The effect was most pronounced in brain abscess patients, where RC-PCR results guided treatment decisions in 9/13 (69.2%) of cases.IMPORTANCETimely and accurate diagnosis is essential for managing serious infections, yet clinicians often face situations where routine laboratory tests do not provide clear answers. This study demonstrates that next-generation sequencing (NGS) of the bacterial 16S rRNA gene can decisively resolve these uncertainties. By revealing whether bacteria are present in clinical specimens, this approach influenced clinical reasoning and supported treatment decisions across a variety of challenging cases. 16S reverse-complement PCR was especially powerful for brain abscesses and infections where the causative microorganism was unclear, providing clarity that directly improved patient care. These findings show that integrating advanced sequencing with expert clinical interpretation can enhance the management of complex infections and support more confident, evidence-based therapy.

Humans↗
PubMed2026

Longitudinal surveillance of antibiotic resistance and virulence evolution in Clostridioides difficile: a 4-year retrospective study of hospitalized patients in a tertiary hospital in China.

UNLABELLED: Clostridioides difficile (C. difficile) is the primary pathogen responsible for nosocomial infectious diarrhea and pseudomembranous colitis. In China, metronidazole and vancomycin are the preferred treatments for C. difficile infection (CDI). This study aimed to investigate the evolution of vancomycin (VA) and metronidazole (MTZ) resistance, as well as the longitudinal changes in virulence over time, using next-generation sequencing, drug susceptibility tests, and analysis of resistance and virulence genes. Additionally, we monitored the emergence of the highly virulent C. difficile strain RT027 and the spread and potential outbreak of C. difficile in the hospital setting. A random stratified sampling method was used to select 114 fecal samples from inpatients at Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, between 2021 and 2024. Clinical data from the enrolled patients were also collected. We conducted antigen and toxin protein detection for C. difficile, strain isolation and identification, drug sensitivity tests, whole genome sequencing, and bioinformatics analysis. This included comparisons of drug resistance genes, detection of toxin genes, and the construction of phylogenetic trees based on pan-genome analysis to investigate the resistance and toxin gene variations in C. difficile. Among the 114 samples collected from Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, no vancomycin- or metronidazole-resistant strains were identified. However, the average minimum inhibitory concentration (MIC) of C. difficile to vancomycin increased annually (H = 33.208, P < 0.05). The average MIC of C. difficile to metronidazole was highest in 2022 but decreased in 2023 and 2024 (H = 41.990, P < 0.05). Notably, in 2024, one C. difficile strain exhibited an MIC for metronidazole at the resistance threshold (2.00 &#x3bc;g/mL). Further Spearman correlation analysis of the strain years with drug sensitivity results revealed a positive correlation between strain years and the MIC levels of vancomycin and metronidazole (r = 0.528, P < 0.05; r = 0.377, P < 0.05). The proportion of toxin-producing strains increased annually, with 100% of strains in 2024 producing toxins, representing the highest proportion compared to the previous three years (X&#xb2; =11.75, P < 0.05). Both vancomycin and metronidazole remain effective for the treatment of CDI in clinical practice. However, the sensitivity of C. difficile to these two drugs is gradually decreasing, and the rate of toxin gene carriage is also rising in clinical cases. No hospital outbreaks of C. difficile infections were identified in this study. IMPORTANCE: Clostridioides difficile has developed resistance to multiple antibiotics, including cephalosporins, clindamycin, and fluoroquinolones. This has exacerbated the global antibiotic resistance crisis. In China, according to current treatment guidelines, vancomycin and metronidazole are the preferred first-line drugs for treating C. difficile infections. However, there are reports indicating the emergence of new resistance to both vancomycin and metronidazole. Although there is extensive research on the long-term antibiotic resistance of C. difficile abroad, research on the continuous monitoring of antibiotic resistance and potential outbreaks of C. difficile in China is relatively limited. To fill this gap, we studied positive C. difficile strains from a tertiary general hospital in China. Through Next-Generation Sequencing (NGS), drug sensitivity testing, and analysis of drug resistance and virulence genes, we revealed the evolution of C. difficile's resistance to vancomycin and metronidazole, as well as changes in virulence, and monitored the spread within the hospital and potential outbreaks of C. difficile.

Humans↗
PubMed2025

Mapping Active RNA Polymerases in Proliferating and Quiescent Fission Yeast Cells Using Precision Run-On Sequencing.

The development of next-generation sequencing (NGS) approaches to investigate the functioning of RNA polymerases has led to groundbreaking advances in the field of transcriptional regulation. One powerful method, Precision nuclear Run-On sequencing (PRO-seq), maps the locations of RNA polymerase active sites genome-wide at high resolution. PRO-seq provides a snapshot of strand-specific transcriptional activity and does not rely on immunoprecipitation of the polymerase of interest. Notably, this technique has been utilized to investigate the control of the RNA polymerase II transcription cycle in a variety of model systems. However, the initially published PRO-seq method required significant amounts of starting sample and was technically challenging, both of which were deterrents for its broader use. Recently, an improved and simplified version called qPRO-seq that reduced the length of the experiment and the quantity of necessary input sample was developed for human and Drosophila cell lines. Here we provide an updated, step-by-step protocol in which we have validated and optimized qPRO-seq for the fission yeast Schizosaccharomyces pombe. Importantly, we have implemented this method for assessing RNA polymerase activity in nutrient-limiting conditions, for both proliferating and nitrogen-depleted quiescent cells.

Schizosaccharomyces↗