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PubMed2025

Extraction, Purification, and Next-Generation Sequencing (NGS) Analysis of DNA and RNA from Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue.

Formalin fixed paraffin embedded (FFPE) tissues have long been used for immunohistological analyses. FFPE tissues can be stored at room temperature for several years enabling analyses to be performed later. Ease of storage and transport makes these tissues an attractive source of biological material. However, formalin fixation results in chemical modifications of proteins and nucleic acids that poses a major challenge to any type of analysis. Recovery of nucleic acids for quantitative assays is rendered difficult due to degradation resulting from fixation and long-term storage, producing low usable yields. Extensive efforts in the last 20 years have led to significant improvements in use of FFPE tissues for DNA and RNA analyses and resulted in development of sensitive assays for a wide range of applications, including next-generation sequencing. In this chapter, we describe the optimization of methods for sequential extraction of DNA and RNA from FFPE tissue and subsequent preparation of DNA-seq and RNA-seq libraries for use with the Illumina platform using commercially available reagents/kits.

Paraffin Embedding↗

PubMed2026

Systematic performance evaluation and application validation of an end-to-end NGS workstation.

Next-generation sequencing (NGS) library preparation is a core component of precision genomics, but it is commonly constrained by inefficiency, variability, and low throughput of manual protocols. To address these limitations, we developed and systematically evaluated a fully automated NGS workstations and further validated its performance across representative application scenarios. The automated system reduced total processing time from 8 to 10 to 4–6 h. At the same time, it maintained similar performance in pre-library metric, including DNA yield and fragment size, as well as post-capture sequencing metrics (Q30 > 90%, mapping rates > 95%, on-target rates 85–90%). The duplication rate was reduced to 5–8%, compared with 10–15% for manual methods, indicating increased library complexity. Bioinformatic evaluation of inter-species read mapping showed minimal cross-contamination, with a maximum contamination ratio of 0.0003%, indicating effective sample isolation in the automated workflow. High concordance in variant detection was observed between automated and manual workflows. Overall, this automated workstation provides a standardized and reproducible workflow that supports scalable precision genomics applications.

High-Throughput Nucleotide Sequencing↗

PubMed2026

[State Changes and Stability Grading of Driver Genes in Non-small Cell Lung Cancer Based on Repeated NGS Testing].

BACKGROUND: Next-generation sequencing (NGS)-based driver gene testing has become a routine component of molecular subtyping and precision therapy for non-small cell lung cancer (NSCLC). Dynamic genomic monitoring facilitates early detection of resistance-related molecular alterations and informs timely therapeutic adjustments. However, standardized criteria for evaluating the stability of serial NGS testing are currently lacking, and the applicability of NGS using formalin-fixed paraffin-embedded (FFPE) specimens for dynamic monitoring remains poorly defined. This study aims to establish a stability grading system for driver gene status alterations based on repeated NGS testing, and to provide evidence-based support for clinical repeat biopsy strategies. METHODS: Data from 1232 patients with NSCLC who underwent two or more NGS tests on FFPE tissue specimens at Beijing Chest Hospital between June 2019 and April 2026 were collected retrospectively. Patients with an interval of ≥4 months between the initial and last tests were included to ensure the representativeness of temporal analysis, resulting in a main analysis cohort of 942 patients. The Kappa consistency test was used to evaluate the state stability of nine core driver genes [epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1), mesenchymal‑epithelial transition factor (MET), rearranged during transfection (RET), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), erb‑b2 receptor tyrosine kinase 2 (ERBB2), and phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit alpha (PIK3CA)] and to construct a five‑level grading system. Paired variant allele frequency (VAF) differences were compared using the Wilcoxon signed‑rank test. Independent influencing factors for mutation accumulation were identified by binary Logistic regression. RESULTS: The state stability of the nine genes was classified into five levels: EGFR showed high stability (Kappa=0.838), ROS1/ALK/KRAS good stability, BRAF/PIK3CA/RET moderate stability, and ERBB2 low stability, and MET showed high instability. MET exhibited the highest rate of state change (9.3%) with a raw observed agreement of 90.7%. Its Kappa value (0.172) was influenced by the low prevalence (3.7%) compression effect and should therefore be interpreted alongside the observed agreement (90.7%) and the prevalence-adjusted and bias-adjusted Kappa (PABAK). The VAF of PIK3CA increased significantly (P=0.005). T790M positivity increased from 5.8% to 10.8%, and 30 new C797S mutations were detected at the last test (13 with T790M, 17 without). The overall rate of new driver gene variants in the main cohort was 18.0%. Binary Logistic regression showed that a lower number of initial mutated genes was the only independent predictor of new variants [odds ratio (OR)=0.399, P<0.001], while sex and detection interval showed no independent association. CONCLUSIONS: A five level stability grading system for state changes of driver genes in NSCLC based on repeated NGS testing has been established. MET showed the most frequent state changes, which should be interpreted in conjunction with the prevalence effect. The VAF increase of PIK3CA is an observational finding, and its clinical significance requires further prospective validation. A lower initial mutation burden may reflect tumor clonal complexity and was associated with a higher likelihood of subsequent acquisition of new variants. FFPE based NGS is applicable for repeated testing at clinical treatment decision nodes.

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

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

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↗

PubMed2026

Day + 30 detection of minimal residual FLT3-ITD by high-sensitivity PCR-NGS predicts relapse risk and guides post-transplant maintenance in AML.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has improved outcomes in patients with acute myeloid leukemia (AML) harboring FLT3-internal tandem duplication (FLT3-ITD) mutations. However, relapse still occurs in 15-35% of these patients after transplantation. Therefore, early and highly sensitive detection methods are required to identify patients at risk of relapse and enable timely post-transplant intervention. METHODS: In this NICHE cohort study, a total of 136 patients were included, then we evaluated whether high-sensitivity polymerase chain reaction (PCR)-next-generation sequencing (NGS) for FLT3-ITD (limit of detection: 5 × 10-6) on day + 30 post-HSCT could identify patients at a high risk of relapse and inform decisions regarding maintenance therapy. RESULTS: Among the 136 patients, 37 patients (27.2%) had detectable FLT3-ITD clones on day + 30. These patients exhibited a significantly higher cumulative incidence of post-HSCT multiparameter flow cytometry (MFC)-measurable residual disease (MRD) relapse (40.3% vs. 18.8%, p = 0.001). Notably, FLT3-ITD-positive patients who received FLT3 inhibitor maintenance therapy had no relapses, while 6 out of the 13 patients who did not receive maintenance therapy relapsed. Conversely, FLT3-ITD-negative patients without high-risk factors (2022 European LeukemiaNet adverse-risk group, relapsed/refractory AML, MFC-MRD positivity pre-HSCT) showed limited benefit from maintenance therapy (MFC-MRD-free survival: hazard ratio (HR) = 0.25 (0.03-2.11), p = 0.204; OS: HR = 0.20 (0.02-1.70), p = 0.142). CONCLUSIONS: This is the first study to demonstrate that detection of minimal FLT3-ITD clones at the fixed time point of day + 30 post-HSCT can reliably stratify relapse risk in AML patients and provide a rationale for individualized post-transplant maintenance therapy.

Humans↗

PubMed2025

A Comprehensive Bioinformatics Approach to Analysis of Variants: Variant Calling, Annotation, and Prioritization.

Next-Generation Sequencing (NGS), also known as high-throughput sequencing technologies, has enabled rapid and efficient sequencing of large amounts of DNA and RNA. These technologies have revolutionized the field of genomics, transcriptomics, and proteomics and have been widely used in cancer research, leading to advances in clinical diagnosis and treatment. Improvements in the NGS technologies enabled millions of fragments to be sequenced simultaneously in a time- and cost-effective manner and resulted in large amount of genomic data which require efficient analysis methods. Analysis of the genomic data requires both efficient computer resources and bioinformatics approaches. This chapter details a comprehensive computational approach and analysis steps for genomic data analysis.

Computational Biology↗

PubMed2026

Molecular characterization of salivary cancers: Patterns of genomic alterations and potential for impact on therapeutic choices.

BACKGROUND: Salivary cancers are rare malignancies with diverse histologies, molecular landscape, and limited effective systemic therapy options. Recent tumour genomics research has identified driver alterations in salivary gland cancers that have led to personalized therapy approaches. The primary objective was to perform molecular characterization using next generation sequencing (NGS) panel and evaluate the potential impact of results on clinical decision-making and treatment outcomes. METHODS: Patients with locally advanced or incurable metastatic salivary cancers suitable for systemic therapy underwent NGS tumour testing with an amplicon-based DNA/RNA NGS panel. Patient demographics, baseline characteristics, treatment and treatment outcomes were retrospectively collected. RESULTS: From 2021 to 2024, 58 advanced salivary cancer patients underwent molecular characterization of their tumour. Baseline characteristics at diagnosis: male 60%, median age 67, most common histologies; adenoid cystic 27%, salivary duct 19% and mucoepidermoid 12%. PIK3CA alterations were the most common molecular finding across all subtypes 22% (13/58) and were enriched in salivary duct carcinoma 73% (8/11). Other alterations identified were: ERBB2 (4), EGFR (2), HRAS (3), NTRK3 (2), BRAF p.V600E (1), and RET (1). Immunohistochemistry identified androgen receptor positivity across salivary cancer subtypes in 8/19 and HER2 positivity in 2/20 tested. Twenty-two patients received systemic therapy prior to NGS results for incurable/metastatic disease, first line treatments included 69% chemotherapy, 18% anti-androgen, 9% lenvatinib, 4% trial. CONCLUSION: Molecular characterization of salivary cancers identified targetable alterations in 37% of patients. The identification of potential therapeutic targets offers the opportunity for expanded treatment options to benefit salivary gland cancer patients.

Metastatic salivary gland cancer↗

PubMed2026

Detection of Tumor Suppressor Genes Rare Variants: Findings From Neuroblastoma Using Next-Generation Sequencing.

BACKGROUND/OBJECTIVES: Neuroblastomas (NB) influenced by genetic alterations, which plays significant role in disease progression. Tumor suppressor genes (TSGs) are crucial in regulating cell growth, suppressing replication, and inducing apoptosis to prevent cancer formation. However, mutations in TSGs can lead to loss of normal activity, contributing to cancer development. This study aimed to identify TSG variants in NB patients and assess their clinical significance. METHODS: One hundred two NB patients diagnosed and monitored according to the International Neuroblastoma Risk Group Staging System (INRGSS) protocol were included in this study. DNA was extracted from paraffin-embedded tissue samples, and Next-Generation Sequencing (NGS) was conducted using the Pillar ONCO/Reveal Multi-Cancer v4 panel. RESULTS: The most frequently recurring TSG variant detected was RB1, p.P793S (n = 21; 25%) followed by ATM, p.D1853N (n = 20, 19.6%).Stop-gain variants were identified in TP53 (p.R196*), FBXW7 (p.R367*), and PTEN (p.G129*). CONCLUSIONS: Our findings underscore the significance of specific TSG variants in NB, particularly in relation to disease progression and potential prognostic markers. Further research is needed to comprehensively assess the role of TSGs in NB, with an emphasis on germline variants and protein expression in larger patient cohorts.

Humans↗

PubMed2025

Molecular Characterization and Epidemiology of Human Noroviruses in the Sverdlovsk Region, Russian Federation.

Human noroviruses (HuNoVs) stand as the primary cause of acute viral gastroenteritis outbreaks worldwide, particularly impacting children under the age of five. In Russia, reports of norovirus gastroenteritis have surged, especially in the post-COVID-19 era starting in 2022, with elevated infection rates reported into 2024. These viruses exhibit significant mutational variability, leading to the emergence of recombinant strains that can evade immune responses. A comprehensive examination of the complete genome is crucial for understanding the evolution of norovirus genes and for predicting potential outbreaks. This research focuses on analyzing the genotypic composition of HuNoVs circulating in the Sverdlovsk region during 2024, using Sanger sequencing and next-generation sequencing (NGS). Biological samples were collected (n = 384) from patients diagnosed with norovirus infection within the region. Bioinformatics analysis targeted the nucleotide sequences of the ORF1/ORF2 fragment and the assembly of complete genomes for the GII.4 and GII.7 genotypes. In total, 220 HuNoVs were characterized, representing 57.3% of the collected samples. The main capsid variants forming the predominant genotypic profile included GII.4 (n = 88, 40%), GII.7 (n = 86, 39%), and GII.17 (n = 14, 6%). Using NGS, we successfully assembled 8 out of 10 complete genomes for noroviruses GII.4[P16] and GII.7[P7]. Non-synonymous substitutions appeared at amino acid sites corresponding to the subdomains of VP1 in these strains. This molecular-genetic analysis provides contemporary insights into the genotypic composition, circulation patterns, and evolutionary dynamics associated with the dominant genovariants GII.4[P16] and GII.7[P7].

Norovirus↗

PubMed2026

URMD-Seq: A high-throughput method for scalable detection of ultra-rare mutations in the human mitochondrial genome.

The study of mitochondrial genetics has long been limited to polymorphisms and high frequency mutations owing in part to technical and technological limitations in reliably detecting and quantifying rare somatic mutations. Over the past decade or so, the study of rare somatic mitochondrial DNA (mtDNA) variants has expanded and continues to garner increasing interest in a wide range of research fields. Here, we describe Ultra-Rare Mutation Detection-Sequencing (URMD-Seq), a high-throughput method that combines unique molecular identifier (UMI)-based library preparation and Next Generation Sequencing (NGS) for the accurate and scalable detection of ultra-rare mutations in the mtDNA control region. Our method exploits degenerate primers to label individual mtDNA molecules. This is followed by several purification, quantification and amplification steps, to obtain high quality amplicons for sequencing on the Illumina MiSeq platform. Our approach enables the use of total genomic DNA extract as starting point for the assay, overcoming the need for organelle isolation and/or mtDNA enrichment, hence broadening the type of specimen that can be studied, while offering cost and time benefits. The assay described herein has been demonstrated to reliably measure variants present at on average 0.09%, but as low as 0.03%, variant allele frequency in a variety of tissues, including fresh and frozen biobanked specimens. Using this protocol, library preparation of 300 specimens can be completed by a single individual with general nucleic acid handling experience in approximately 20 days. Given its flexibility and scalability, URMD-Seq is particularly well suited for epidemiological studies using a large number of specimens.

Humans↗

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

An open-source clinical bioinformatics pipeline for real-world NGS implementation: translating genomic variants into actionable treatment strategies in oncology.

BACKGROUND: Next-Generation Sequencing (NGS) has become a cornerstone technology in clinical practice, yet its adoption presents significant challenges. Physicians and oncologists must manage vast amounts of genome-scale data and transform it into actionable insights for complex decision-making. While commercial systems exist to synthesize data from NGS experiments into clinical reports, many are hindered by limitations such as closed-source designs that restrict transparency and customization. Additionally, some fail to leverage publicly available genomic databases, missing opportunities to integrate valuable external data. Furthermore, the rigidity of many tools in accommodating diverse NGS panels limits their applicability across varied clinical scenarios. METHODS: To address these limitations, we developed OncoReport, an open-source tool that generates comprehensive reports from NGS analyses. By integrating publicly accessible databases, OncoReport provides a robust, user-friendly environment equipped with essential tools for NGS analysis. This design aims to enhance data interpretation and support informed clinical decision-making. RESULTS: Rigorous testing has demonstrated OncoReport’s effectiveness in producing detailed, actionable reports that are clear and easy to use. By automating key aspects of the workflow, the tool significantly reduces manual effort and expedites the synthesis and interpretation of NGS results, making genomic insights more accessible to clinicians. CONCLUSION: OncoReport offers a transparent, flexible, and efficient framework for clinicians to analyze and apply genomic data in patient care. By streamlining workflows and leveraging open-source principles, it empowers healthcare professionals to make informed, data-driven decisions. OncoReport is freely available at https://oncoreport.atlas.dmi.unict.it, with source code and issue tracking on GitHub: https://github.com/knowmics-lab/oncoreport .

Humans↗

PubMed2026

Trastuzumab Deruxtecan in Metastatic Urothelial Carcinoma with NGS-Detected ERBB2 Amplification: A Four-Patient Real-World Case Series.

Background: Next-generation sequencing (NGS)-detected ERBB2 amplification occurs in a subset of urothelial carcinomas, but its role as a treatment-selection marker for trastuzumab deruxtecan (T-DXd) remains uncertain. Methods: We retrospectively reviewed four patients with metastatic urothelial carcinoma treated with T-DXd in routine practice from 2024. Treatment selection was based on NGS-detected ERBB2 amplification because HER2 immunohistochemistry and in situ hybridization were unavailable. Results: Four men aged 65-76 years received T-DXd: one in the second line and three in the fourth or fifth line. The best radiological responses, abstracted from contemporaneous radiology reports and oncology medical records, were complete response in one patient, partial response in one, and stable disease in two. Three patients had previously received enfortumab vedotin. Documented adverse events included fatigue, anemia, diarrhea, rash, and leukopenia. No interstitial lung disease or pneumonitis was documented in the available records. Conclusions: These observations are descriptive and hypothesis-generating. They do not establish the efficacy or safety of T-DXd or validate ERBB2 amplification as a predictive biomarker, but they support prospective evaluation of genomic ERBB2 amplification when standard HER2 testing is unavailable.

Humans↗

PubMed2025

Genome-wide profiling the integration patterns with T7-PCR.

Integration of exogenous gene fragments into the host genomes is a widely used and powerful method for studying gene functions, advancing molecular breeding, and conducting gene therapy. Accurately identifying the integration sites is essential for ensuring both the safety and efficacy of genome engineering efforts. However, current mapping techniques are constrained by high costs and a low signal-to-noise ratio. In this study, we developed an innovative tool for mapping integration sites, leveraging T7 polymerase-mediated in vitro transcription (T7-IVT) to capture the junction fragments surrounding integration loci. This approach converts genomic flanking sequences into RNA, enabling the simultaneous enrichment of junction fragments and the elimination of background genomic DNA, thereby significantly enhancing the signal-to-noise ratio. We have validated the efficiency of this method, named T7-PCR, across yeast, plant, and human cells under diverse integration scenarios. T7-PCR outperforms current next-generation sequencing (NGS)-based mapping strategies in terms of efficiency and accuracy, with minimal positional effects. This method is highly applicable for high-throughput transgene screening and also supports the development of next-generation tools for targeted integration of large fragments.

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↗

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↗