The Novel HLA-C*07:01:130 Allele, Identified by Next-Generation Sequencing in a Greek Haematopoietic Stem Cell Donor.
The HLA-C*07:01:130 allele differs from the HLA-C*07:01:01:01 allele by a single nucleotide substitution in exon 3.
Humans↗
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Results for “High-throughput sequencing”
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Targeted sequencing reveals a distinct genetic alteration landscape in oral multiple primary squamous cell carcinomas.
OBJECTIVE: Oral multiple primary cancers (MPCs) are associated with poor clinical outcomes, yet their genomic characteristics remain insufficiently understood. DESIGN: Fifty-four formalin-fixed paraffin-embedded (FFPE) tumor samples from 30 patients with oral MPCs were analyzed using high-depth targeted sequencing of a customized 14-gene panel derived from prior whole-exome sequencing data. Detected alterations were analyzed after removal of synonymous mutations. RESULTS: Non-silent genomic alterations were identified in 59.3% (32/54) of samples, involving 19 patients. A total of 70 variant loci across 13 genes were detected. AKAP13 was the most frequently mutated gene at both the sample (22.2%, 12/54), with recurrent mutations observed across multiple patients. In contrast, TP53 mutations occurred at a substantially lower frequency (11.1%, 6/54). Marked inter- and intra-patient mutational heterogeneity was observed. CONCLUSIONS: FFPE-based targeted sequencing enabled an initial characterization of genomic alterations in oral MPCs. Recurrent alterations in AKAP13, GLI2, JMJD1C, and DNAH8, together with the relatively low frequency of TP53 alterations, identify candidate genomic features for further investigation and provide a basis for future studies of the molecular basis of oral MPCs.
Humans↗
Strategies for mosaic variant calling in brain disorders.
The human brain is a genomic mosaic, where postzygotic mutations arising from embryogenesis to senescence drive diverse neurodevelopmental and neurodegenerative diseases. Because of numerous sequencing artifacts at ultralow variant allele frequencies (VAFs), detecting these variants remains a significant analytical challenge. This review focuses on single-nucleotide variants and small indels, summarizing current strategies for aligning sampling methods, including bulk, laser capture microdissection, and single-cell genomics, with the expected clonal architecture of the brain. It emphasizes that mosaic detection sensitivity is fundamentally constrained by sequencing depth, since even the most advanced algorithms cannot identify variants not physically represented in the sequencing library. The review further recommends the selection of variant calling algorithms based on validated VAF detection performance, matching tools like MuTect2 and MosaicForecast to their optimal performance ranges. Furthermore, we discuss how multitissue sampling, as emphasized by the SMaHT project, addresses the matched-control dilemma and supports accurate variant classification via cross-tissue VAF gradients. Integrating these established pipelines with multiomics modalities, including transcriptomic and epigenetic data, could advance the field toward a functional understanding of how the somatic genome impacts human brain health and disease.
Humans↗
Microbial signal profiles and organism-level concordance between plasma metagenomic sequencing and blood culture in suspected bloodstream infection.
Plasma metagenomic next-generation sequencing (mNGS) and blood culture detect different components of the microbial signal and frequently produce discordant organism reports. We characterized microbial signal class, report-derived burden, organism-level concordance, and independent clinical attribution in a retrospective, single-center, episode-level cohort. Among 329 episodes with evaluable plasma mNGS reports, 315 had blood culture performed; 232 were mNGS positive/culture negative and 53 were positive by both methods. In the 232 discordant episodes, the recorded routine-care diagnosis classified 124 as bloodstream infection (BSI) and 108 as non-BSI. Nonviral signals were present in 78.2% and 42.6%, respectively (P < 0.001), and median maximum report-derived sequence counts were 98.5 and 11.5 (P < 0.001). Two laboratory physicians then independently reviewed source records using structured criteria while masked to the recorded BSI label and mNGS organism and sequence-count information. Initial agreement for the five-category BSI assessment was 97.6% (Cohen's kappa, 0.960). Within the mNGS-positive/culture-negative subgroup, adjudicated BSI likelihood showed a modest ordinal association with report burden (Spearman rho = 0.190; P = 0.004), while mNGS organisms were considered supported in 1 episode, plausible in 158, unlikely or contaminant in 72, and unresolved in 1. Among 53 dual-positive episodes, 33 (62.3%) shared at least one species, but only 5 (9.4%) had complete species-set concordance. Plasma mNGS and blood culture therefore frequently generated non-equivalent organism sets. Signal class and report burden contributed graded contextual evidence, but organism-level attribution required clinical review and orthogonal microbiology rather than binary positivity alone.
Humans↗
Diagnostic value of plasma cell-free DNA metagenomic next-generation sequencing in patients with suspected infections and exploration of clinical scenarios-a retrospective study from a single center.
BACKGROUND: Plasma cell-free DNA metagenomic next-generation sequencing (mNGS) is a non-invasive comprehensive method for the etiological diagnosis of various infectious diseases. However, research on the early diagnosis and real-world clinical impact of plasma mNGS in patients with suspected infection are still limited. MATERIALS AND METHODS: This study retrospectively included 140 patients with suspected infections who underwent early plasma mNGS and conventional culture testing. Referring to the clinical diagnosis of infectious diseases, the diagnostic performance of plasma mNGS and culture tests was compared, and the application scenarios and clinical effects of plasma mNGS were evaluated. RESULTS: The positive rate of plasma mNGS was significantly higher than that of culture methods (55.71% vs 25.10%, p < 0.001) and blood cultures (55.71% vs 12.86%, p < 0.001). Regarding clinical diagnosis, the sensitivity of plasma mNGS was significantly higher than that of culture (58.27% vs 37.80%, p = 0.002). The combination of mNGS and culture achieved a higher detection sensitivity (69.29%), especially in patients with multi-site co-infections (73.68%) and blood infections (73.17%). Plasma mNGS demonstrated higher sensitivity in patients with procalcitonin (PCT) index > 5 ng/ml or human neutrophil lipocalin (HNL) index > 200 ng/ml. In terms of treatment, a total of 69 patients (54.33%) benefited from plasma mNGS. CONCLUSION: This study highlights the significant improvement in pathogen detection performance by combining conventional culture with plasma mNGS detection, especially in patients with multi-site co-infections and blood infections. Early use of plasma mNGS as an adjunct to culture can better guide clinicians to initiate appropriate anti-infective therapy.
Humans↗
Feasibility of routine clinical liquid-based cytology for lung cancer compact panel testing.
BACKGROUND: The Lung Cancer Compact Panel (cPANEL) is a recently approved highly sensitive multiplex gene panel in Japan that supports both DNA- and RNA-based next-generation sequencing. Although cytological specimens are acceptable for cPANEL, unfixed cell pellets or dedicated preservation tubes are typically recommended. However, evidence remains limited regarding whether residual liquid-based cytology (LBC) cell suspensions prepared for routine cytological diagnosis can be used directly for cPANEL testing without dedicated molecular preservation or additional preanalytical processing. In this study, we evaluated the feasibility of applying LBC specimens that are widely used in contemporary clinical practice to cPANEL. METHODS: We analyzed DNA and RNA quality in 69 clinical LBC specimens. Among these, 51 specimens containing non-small cell lung cancer cells with previously determined driver alteration status were subjected to cPANEL testing to evaluate assay concordance with clinical companion diagnostic results. RESULTS: DNA integrity was generally well preserved (DNA Integrity Number [DIN]: 6.2 ± 1.5). In contrast, RNA integrity showed greater variability (DV200: 16.4 ± 12.1%). ThinPrep-fixed specimens demonstrated lower DIN and DV200 values compared with CytoRich Red-fixed specimens. Although all samples successfully passed the DNA-based cPANEL assay, six cases (11.8%) failed the RNA-based assay, with RNA yield being a major contributing factor. Among the 46 evaluable specimens, concordance was 95.7% and sensitivity was 92.3%, or 88.9% including RNA module failures as cPANEL-negative. CONCLUSIONS: With appropriate fixative selection and adequate cellularity, cPANEL using clinical LBC specimens may serve as a practical diagnostic platform. We demonstrated that routine LBC specimens can be directly applied to cPANEL without special preanalytical processing.
Humans↗
Characteristics of p53 and Smad4 immunohistochemistry in pancreatic ductal adenocarcinoma and validation by next-generation sequencing.
BACKGROUND: Mutations in four major driver genes -KRAS, CDKN2A, TP53, and SMAD4- are central to the pathogenesis of pancreatic ductal adenocarcinoma (PDAC) and critically inform diagnosis, therapeutic decision-making, and prognostic assessment. Although next-generation sequencing (NGS) is widely regarded as the gold standard for detecting these mutations, its clinical application is often limited by suboptimal analytical efficiency and substantial economic cost. Among these genes, immunohistochemical (IHC) staining for the proteins encoded by TP53 and SMAD4 has been extensively adopted in routine pathology practice. However, standardized IHC pattern classification schemes and rigorous validation of their predictive accuracy for underlying genomic alterations remain lacking in PDAC. METHODS: We retrospectively enrolled 63 PDAC patients and systematically characterized the typical IHC expression patterns of p53 and Smad4. Targeted NGS was subsequently performed on all available tumor specimens, and the resulting mutational profiles were correlated with corresponding IHC findings. Diagnostic performance including sensitivity, specificity and accuracy of p53 IHC for predicting TP53 mutations and of Smad4 IHC for predicting SMAD4 mutations was rigorously evaluated. RESULTS: Among the four canonical driver genes, co-occurring double- or triple-gene mutations were prevalent; within TP53 and SMAD4, missense mutations constituted the most frequent variant type. Using NGS as the reference standard, we validated the diagnostic utility of a three-tiered p53 IHC classification system, particularly in fine-needle biopsy (FNB) specimens. Furthermore, we proposed a novel, refined Smad4 IHC pattern classification that incorporates an "intermediate" category, thereby expanding upon conventional binary interpretation. This new scheme achieved markedly improved mutation prediction accuracy (0.76) compared with traditional approaches (0.57). CONCLUSION: Our study highlights the complementary diagnostic value of p53 and Smad4 IHC relative to molecular testing in PDAC, especially when tissue is limited, as commonly encountered in FNB specimens. The newly established Smad4 IHC classification system, which integrates an intermediate expression category into the conventional two-tier framework, demonstrates superior clinical utility and enhances predictive accuracy for SMAD4 genomic alterations.
Humans↗
Next-generation sequencing in head and neck sarcoma: a single-centre institutional experience and review of the literature.
Head and neck sarcomas (HNS) are rare, heterogeneous malignancies representing less than 1% of head and neck cancers. Their complex anatomy and overlapping morphologies pose significant challenges for traditional diagnosis. We aimed to evaluate the clinical utility of next-generation sequencing (NGS) within a tertiary referral centre and synthesise these findings with current global molecular standards. We conducted a retrospective review of an original, previously unpublished, cohort of 12 patients with histologically verified HNS treated at University College London Hospital (UCLH) between 2023 and 2024. Molecular profiling included targeted DNA (RMH200) and RNA-fusion panels. This was supplemented by a qualitative synthesis of 16 key studies (2010-2026) identified through a systematic search strategy. In the institutional cohort, NGS provided definitive diagnostic or therapeutic clarification in 66% of cases (8/12). Key findings included the identification of pathognomonic fusions (such as EWSR1::FLI1, PAX3::MAML3), a novel MAMLD1::VGLL3 fusion, and actionable variants such as BRAF V600E and MYOD1. Furthermore, the formal exclusion of Neurotrophic tropomyosin receptor kinase (NTRK) fusions in some cases allowed for therapeutic streamlining. Literature synthesis aligned these results and emphasised the need for NGS for more accurate diagnosis and adequate treatment. NGS is a clinical necessity in the management of ultra-rare HNS. By transitioning from traditional morphology to high-resolution molecular interrogation, clinicians can resolve diagnostic ambiguity and identify targeted therapeutic pathways. Integration with emerging 2026 standards, including epigenetic classification and liquid biopsy monitoring, represents the future of precision surgery in head and neck sarcoma.
Humans↗
Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.
Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.
CRISPR-Cas9↗
Plant cis-regulatory grammar: Decoding the multidimensional code of transcriptional regulation for programmable crop engineering.
Cis-regulatory elements (CREs) orchestrate the spatiotemporal precision of gene expression that underlies plant development, adaptation, and domestication. Decoding the cis-regulatory grammar of plant genomes remains a central challenge in modern biology, with profound implications for programmable crop engineering. Here, recent conceptual and technological advances are synthesized to reshape our understanding of plant CREs. This review first argues that CRE function is not only an intrinsic property of DNA sequence alone but also emerges from a multidimensional context, including chromatin accessibility, histone modifications, three-dimensional genome topology, and cell type-specific regulatory landscapes. Furthermore, the convergence of single-cell epigenomics, high-throughput functional assays, and CRISPR-based dissection has begun to unravel this contextual grammar, revealing the computational principles governing transcriptional regulation. Critically, we propose that artificial intelligence (AI) platforms are catalyzing an ongoing transition from descriptive discovery to predictive engineering, wherein these platforms outperform natural evolution in designing synthetic CREs. Finally, a roadmap is outlined toward a plant regulatory grammar foundation model, which will enable truly predictive engineering of gene expression when fine-tuned for specific tasks. Collectively, the integration of single-cell resolution maps, precise genome editing, AI-driven design, and regulatory-compliant delivery systems promises to transform our ability to reprogram plant gene regulation for next-generation agriculture, bridging the gap between foundational regulatory biology and tangible crop improvement.
artificial intelligence↗
Decoding sequence recognition code of nucleic acid-binding proteins of human-infecting DNA viruses.
Human-infecting DNA viruses remain major health threats, yet the DNA-recognition mechanisms of their nucleic acid-binding proteins (NBPs) are poorly understood. Here, we systematically profiled 103 viral NBPs from human-infecting DNA viruses, with three NBPs from non-human-infecting DNA viruses as controls, using high-throughput screening. This analysis identified diverse DNA-binding motifs and specificity modules, including convergent recognition of a conserved CCACC motif across phylogenetically distant viruses. Notably, viral NBP binding-site distributions varied with genome size, and several NBPs from small-genome viruses showed enrichment on mitochondrial DNA. Functional assays further supported their mitochondrial association and effects on mitochondrial membrane potential. By integrating an ivTRT-based ssDNA-SELEX workflow, we further found that ssDNA viral NBPs recognize dimer-like and inverted-repeat sequences with potential to form stem-loop structures. Collectively, this study constructs a comprehensive viral NBP DNA-recognition atlas, offering a fundamental resource for elucidating viral genome recognition mechanisms, virus-mitochondria interactions, and developing future antiviral strategies.
Letter↗
High-throughput recovery of integron cassettes for gene discovery screens.
Integrons capture functional genes in mobile genetic elements called integron cassettes, which represent an untapped source of genes of biotechnological interest. Here we present two tools, cassette gatherer and cassette hunter, that enable high-throughput establishment of gene libraries either from genetically tractable strains or directly from DNA. We re-engineered a class 1 integron into counterselection markers on a plasmid or on the chromosome of a naturally competent Vibrio cholerae, which enabled capture of single cassettes in a sequence- and function-independent manner. When applied to Vibrio strains and genomic libraries, our tools recovered hundreds of single cassettes per assay with more than 99% specificity. We further subjected the library of cassettes generated by the hunter and gatherer tools to screens against phages ICP2 and T4, and identified nine phage-defence systems, including five previously undescribed. These tools enable rapid and large-scale recovery of integron cassettes that could be leveraged for functional gene discovery.
Journal Article↗
Herd-level heterogeneity of antimicrobial resistance in commensal Escherichia coli: A nationwide high-throughput survey of Australian pig herds.
Antimicrobial resistance in commensal Escherichia coli provides a useful indicator for overall antimicrobial resistance burden. We applied this approach to assess antimicrobial resistance within and between commercial pig herds across Australia. A high-throughput robotic workflow was used to isolate 2730 E. coli colonies from rectal contents collected in 2022 from healthy slaughter pigs (n = 300) representing 30 herds (∼70% of national production). Up to 94 isolates per herd underwent antimicrobial susceptibility testing using the Robotic Antimicrobial Susceptibility Platform. Isolate- and herd-level antimicrobial resistance indices were calculated, weighting antimicrobials by their human health importance. Resistance to first-line agents was widespread: ampicillin 77% and tetracycline 79%. By contrast, resistance to critically important antimicrobials was rare (ciprofloxacin 0.11%; extended-spectrum cephalosporins 0.04%), and no clinical resistance to carbapenems or colistin was detected. Overall, 56.9% of isolates were multi-class resistant. Herd-level antimicrobial resistance within indices ranged from 1.51 to 5.76, revealing substantial between-herd heterogeneity. Three herds carried critically important antimicrobials-resistant isolates that would likely have been missed using conventional, lower-density sampling approaches. Whole-genome sequencing identified fluoroquinolone-resistant isolates belonging to ST10 and ST69 (both qnrS1), and ST744 (Quinolone Resistance Determining Region mutations plus blaCTX-M-27). By testing approximately tenfold more isolates than conventional surveys, we uncovered considerable antimicrobial resistance with heterogeneity within and between animals and herds, including farm-specific variability. This expanded sampling also enabled detection of critically important antimicrobial resistance at very low prevalence. In conclusion, high-throughput, high-density testing offers a practical early-warning system and herd-level benchmark to inform surveillance and targeted interventions.
Animals↗
Exome sequencing and large-scale analysis of electronic medical record-linked biobank data identify candidate deafness genes.
INTRODUCTION: Rapid advances in whole-exome sequencing (WES) have enabled large-scale detection of pathogenic variants. Although hundreds of genes are implicated in hearing loss, up to half of inherited cases remain unsolved, limiting eligibility for gene therapy trials that require genetic diagnosis. Biobanks and electronic medical records (EMRs) offer opportunities to integrate genomic and clinical data at scale and expand the spectrum of hearing loss genes. Despite clinical value, EMRs often lack key information such as inheritance patterns, posing challenges for accurate interpretation. METHODS: WES was performed on DNA samples from 1038 hearing-impaired patients enrolled in the Maccabi Research and Innovation Center Tipa Biobank. Clinical data were extracted from EMRs. Audiograms were available for all cases, although data on age of onset, family history and mode of inheritance were mostly unavailable. We applied a scalable bioinformatics analysis strategy for high-throughput annotation, filtering and prioritisation of WES variants across more than 1000 patients, designed to accommodate incomplete and heterogeneous clinical records. RESULTS: Using this approach, 15% of cases were solved or potentially solved through known or novel variants in established deafness genes. Homozygous variants in novel candidate genes were identified in 3% of cases. Functional characterisation was performed for promising candidate genes to validate their role in the ear. CONCLUSION: These findings demonstrate that WES can determine disease aetiology in large, genetically heterogeneous populations, even in the context of incomplete clinical data. This approach supports large-scale genetic screening and provides a framework for identifying patients who may benefit from emerging gene-based therapies.
Genetic Testing↗
A streamlined protocol for small-scale protoplast generation and CRISPR/Cpf1-mediated genome editing in Fusarium oxysporum.
Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomic analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR-Cas9 limitations, such as restricted protospacer adjacent motif (PAM) sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using filter column-based methods that greatly reduce enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with shorter guide RNA design and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development.
CRISPR/Cpf1↗
2-Mercaptoethanol/DMSO Workflow Enables Highly Reproducible Quantitative Proteomics.
Proteomics provides a systematic and high-throughput approach to comprehensively characterize protein networks, enabling insights into cellular functions and disease mechanisms. Carbamidomethylation using iodoacetamide (IAA), a common method for cysteine alkylation, is known to cause nonspecific modifications that increase spectral complexity in mass spectrometry and reduce quantitative accuracy. Here, we established a reproducibility-focused 2-mercaptoethanol (2-ME)/dimethyl sulfoxide (DMSO) workflow and systematically evaluated its quantitative performance at the proteome-wide level. Mouse liver proteomes were processed using either 2-ME/DMSO or conventional IAA treatment, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The optimized 2-ME treatment increased the number of cysteine-modified peptides by 1.6- to 1.9-fold. Although total protein identifications were comparable, 77% of proteins exhibited improved sequence coverage with the optimized 2-ME treatment. Quantitative reproducibility was also enhanced, with the peptide quantified CV ≤ 20% increasing from 61.4% with IAA treatment to 86.1% with 2-ME treatment, and protein quantified CV ≤ 20% increasing from 80.6% with IAA treatment to 93.5% with 2-ME treatment. Application of this new workflow to ovarian clear cell carcinoma reliably detected cisplatin-induced alterations. The 2-ME/DMSO workflow offers a simple and highly reproducible proteomics strategy for accurate quantitative proteomics.
Animals↗
Identification and characterization of ectopic chromosomal amplifications in acute myeloid leukemia cell limes using high-throughput chromosome conformation capture screening.
Despite advanced molecular diagnostics, improving outcomes for refractory acute myeloid leukemia (AML) remains challenging. Although many cancer-related genes are identified, their molecular mechanisms are not fully elucidated. Amplification is a mechanism of cancer-associated gene activation, and ectopic gene amplification may have particularly high pathological significance. However, research on ectopically amplified cancer-associated genes in leukemia remains limited. Here, we evaluated the usefulness of high-throughput chromosomal conformation capture (Hi-C) as a screening method for ectopic gene amplification and assessed whether ectopic amplification of cancer-associated genes may represent a general phenomenon in AML. We screened the U-937 and NB-4 cell lines using in situ Hi-C. Regions appearing as "high-intensity bands" in Hi-C contact maps were identified and validated using fluorescence in situ hybridization (FISH). Additionally, copy number variation analysis was performed using whole-genome sequencing (WGS) to extract cancer-associated genes with ectopic amplification. In the U-937, three genomic regions showing "high-intensity bands" were identified and confirmed as ectopic amplifications-including PDCD1LG2 (PD-L2), CD274 (PD-L1), and JAK2; that is, four copies were detected by WGS, and amplification signals were observed by FISH. In the NB-4, four such regions were detected, including MYC and KRAS, with expression level of 498 transcripts per million (TPM) and 34 TPM, respectively. Copy number variation analysis further identified multiple cancer-associated genes with ectopic amplification. Overall, these findings demonstrate the presence of ectopic amplification of cancer-associated genes in AML cell lines and support the usefulness of Hi-C as a screening method for detecting such genomic alterations.
Acute myeloid leukemia↗
Development of a PCR-based technique for genotyping UGT1A1 gene and distribution of rs3064744 alleles in the Russian population.
BACKGROUND: Accurate determination of tandem thymine-adenine (TA) repeat numbers in the UGT1A1 promoter region (rs3064744) is essential for diagnosing Gilbert's syndrome and personalizing therapy with toxic agents like irinotecan and atazanavir. However, traditional polymerase chain reaction (PCR) assays face severe limitations due to the AT-rich sequence and overlapping melting temperatures (Tm) of the highly homologous 7TA and 8TA alleles. In this context, melting curve analysis (MCA) employing fluorophore-quencher systems has emerged as a promising alternative. The purpose of this study was to develop a novel genotyping approach combining optimized aPCR-MCA analysis with an automated classifier to overcome the limitations posed by the differentiation of highly homologous alleles and to demonstrate its practical application, providing the distribution of rs3064744 genotypes across four regional cohorts of the Russian population. METHODS: A specialized Dual Head 1D-convolutional neural network (1D-CNN) ensemble with Test-Time Augmentation (TTA) was developed. The model was trained and internally validated on 1,620 engineered plasmid samples, and independently evaluated on an external clinical test set of 440 unique patient genomic DNA specimens. Real-time PCR was performed on CFX96 and DTprime platforms. Additionally, population-wide screening was conducted on 997 archival clinical samples from Moscow, Sakha (Yakutia), Dagestan, and Rostov regions. RESULTS: While 5TA and 6TA alleles were easily separated, absolute Tm distributions of 7TA and 8TA alleles overlapped significantly, and non-uniform Tm shifts of 0.8 °C-1.4 °C occurred across platforms. Conventional absolute Tm thresholding was therefore inadequate. By assessing relative morphological curve divergence against co-amplified 7TA/7TA and 7TA/8TA reference anchors, the 1D-CNN ensemble neutralized instrument noise. It achieved 100% accuracy on internal validation and 100% concordance (440/440) with clinical reference pyrosequencing. Population screening revealed that Dagestan, Yakutia, and Rostov cohorts closely align with the European population. Rare 5TA and 8TA alleles were detected at low frequencies in Yakutia and Moscow. CONCLUSION: Combining LNA-modified aPCR-MCA with a comparative 1D-CNN model successfully circumvents thermodynamic limitations and eliminates human operator bias. This integrated system offers an accessible, high-throughput, and clinically valid solution for routine UGT1A1 pharmacogenetic testing.
1D-CNN↗