Characterisation of the Novel HLA-DQA1*05:06:02 Allele by Next-Generation Sequencing.
HLA-DQA1*05:06:02 shows one single nucleotide substitution at position 6 C > T in exon 1 compared with HLA-DQA1*05:06:01:01.
Humans↗
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Results for “High-Throughput Nucleotide Sequencing”
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Molecular diagnostic yield and barriers in inherited retinal diseases: a retrospective cohort study.
OBJECTIVE: To evaluate the diagnostic yield of panel-based genetic testing for inherited retinal diseases (IRDs) and identify barriers to molecular resolution. DESIGN: Retrospective cohort. PARTICIPANTS: A total of 404 patients with clinically confirmed IRDs who were evaluated at the Adult Inherited Retinal Dystrophy Service, Ontario, Canada (October 2021-September 2024). METHODS: Patients underwent targeted massive parallel sequencing panel testing. Diagnostic yield was calculated, and unresolved cases were reviewed. Associations between yield, phenotype, ethnicity, and sex were assessed using χ² analysis. RESULTS: Of 685 referrals, 570 had confirmed IRDs. After we excluded 140 pending results and 26 patients who declined testing, 404 patients were analyzed. At referral, 94 patients (23.2%) had a previous molecular diagnosis, and 138 (34.0%) were diagnosed through clinic-initiated testing, giving an overall yield of 57.4%. Yield varied significantly by phenotype (χ², P = 1.4 × 10⁻⁶), from 94.4% in vitelliform macular dystrophies to 25.0% in vitreoretinopathies, with no sex association (P = 1.0). Disease-causing variants were identified in 83 IRD-associated genes, most frequently ABCA4, USH2A, and BEST1. Of 172 unresolved cases, 62 (36.0%) had negative panels, and 110 (63.9%) were inconclusive, including 30 with unphased pathogenic variants in recessive genes and 10 with high-suspicion variants of uncertain significance. Key barriers included limited family availability for phasing, restricted access to functional assays, and lack of public coverage for whole-exome or whole-genome sequencing. CONCLUSIONS: Massive parallel sequencing-based panel testing achieved a 57% diagnostic yield in this IRD population. Success was strongly phenotype-dependent with substantial heterogeneity. Whole-exome sequencing, whole-genome sequencing, family segregation, and functional genomics could improve diagnostic outcomes and management.
Humans↗
The HLA-DPB1*1469:01 Allele Identified by Next-Generation Sequencing.
HLA-DPB1*1469:01 shows a non-synonymous mutation in codon 205 within Exon 4 compared with HLA-DPB1*25:01.
Humans↗
Identification of the Novel HLA-DQB1*02:02:41 Allele by Next-Generation Sequencing.
HLA-DQB1*02:02:41 differs from HLA-DQB1*02:02:01:01 by one synonymous nucleotide substitution at Codon 39 in Exon 2.
Humans↗
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↗
Characterisation of the Novel HLA-A*03:01:133 Allele by Next-Generation Sequencing in Brazil.
The novel HLA-A*03:01:133 allele differs from A*03:01:01:01 by a change of C ➔ T in exon 4.
Humans↗
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↗
Molecular characterization of a new emaravirus infecting Clerodendrum thomsoniae plants.
High-throughput sequencing revealed a new emaravirus named clerodendrum yellow blotch virus (ClYBV), infecting Clerodendrum thomsoniae plants in São Paulo State, Brazil. Four genomic RNA segments (RNA1, RNA3, RNA4, and RNA5) were recovered, whereas RNA2 remained undetected. Phylogenetic analysis and pairwise comparisons based on amino acid sequences showed ClYBV is most closely related to redbud yellow ringspot-associated virus (RYRaV, Emaravirus cercidis). No ClYBV particles were observed by transmission electron microscopy in leaf tissues from infected plants. These results support the identification of ClYBV as a new Emaravirus member, though the undetectable RNA2 suggests the isolate may be defective, lacking this segment and unable to produce virus particles. The binomial species name "Emaravirus clerodendri" is proposed.
Phylogeny↗
Pervasive noise in human pre-mRNA splice site selection.
RNA splicing has historically been thought to be highly efficient and accurate, with little opportunity for deviation from regulated alternative splicing. This dogma has been challenged by recent observations that biological noise may contribute substantially to transcriptome diversity. However, quantitative understanding of stochastic splicing variation is challenging because these transcripts are likely subject to rapid degradation. Here, we use deep sequencing across RNA compartments to track splicing intermediates in human cells and see abundant cryptic splicing associated with genomic features that promote splicing noise. We observe pervasive usage of low-fidelity splice sites, likely due to stochasticity in recruitment or binding of the spliceosome. These sites are turned over quickly and show evidence for nuclear and cytoplasmic degradation, suggesting widespread surveillance and rapid quality control of non-productive transcripts. Our findings provide insights into the propensity for error in RNA processing mechanisms and regulation of alternative splice sites across a gene.
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↗
Genetic determinants of gestational diabetes mellitus in thai pregnant women: role of GCKR, CDKAL1, TCF7L2, NEDD1, and CMIP variants.
BACKGROUND: Gestational diabetes mellitus (GDM) has a high global prevalence and arises from complex interactions between genetic predisposition and environmental factors. GDM is associated with metabolic disturbances and chronic low-grade inflammation, both of which contribute to its pathogenesis. This study aimed to investigate the association between GDM and 135 single-nucleotide polymorphisms (SNPs) across 20 genes related to metabolic traits. METHODS: In this case-control study, 152 pregnant women with GDM and 684 pregnant women with normal glucose tolerance (NGT) who underwent antenatal examination at Siriraj Hospital, Bangkok, were enrolled. Clinical data and blood samples were collected from all participants. Genomic DNA was isolated and subjected to whole-genome sequencing using the DNBSEQ-T7RS high-throughput sequencing platform. Genotype analyses were performed using R software, and haplotype analyses were conducted using the online SNPStats software. RESULTS: After adjusting for maternal age and pre-pregnancy body mass index, polymorphisms in TCF7L2 (rs34872471, rs7901695, rs4506565, rs7903146, rs12243326, and rs12255372), NEDD1 (rs10431408, rs11830756, rs249579, rs249585, and rs4762339), CMIP (rs2306115 and rs201681534), CDKAL1 (rs4710942), GCKR (rs2293572 and rs2293571), and GCK (rs5883890) were significantly associated with the risk of GDM. Haplotype analysis demonstrated that the TCF7L2 rs12243326-rs12255372 CA haplotype was associated with a decreased risk of GDM (OR = 0.44, 95% CI: 0.23-0.81), while the NEDD1 rs249579-rs249585-rs4762339 GGT haplotype was associated with an increased risk of GDM (OR = 1.40, 95% CI: 1.08-1.82). CONCLUSIONS: These findings suggest that genetic variations in TCF7L2, NEDD1, CMIP, CDKAL1, GCK, and GCKR contribute to GDM susceptibility in the Thai population.
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An Amplicon Panel for High-Throughput and Low-Cost Genotyping of Yesso Scallop Mizuhopecten yessoensis.
The Yesso scallop Mizuhopecten yessoensis was imported from Japan to western Canada in the late 1980s to establish an economically viable scallop aquaculture industry. Since this time, the industry in Canada has operated with existing genetic diversity within the broodstock, which is considerably limited relative to wild populations. The sector has not been able to realise its full potential in part due to idiopathic hatchery failures and farm stock collapses due to disease outbreaks associated with the intracellular bacterial pathogen Francisella halioticida. To support Yesso scallop production and breeding, here we generate a low-density, genotyping-by-sequencing amplicon panel using single nucleotide polymorphism (SNP) markers that are evenly spaced across the M. yessoensis genome and that show high heterozygosity in Canada and Japan. The panel can also exploit the high genetic polymorphism of the M. yessoensis genome, with de novo SNP calling identifying over 2,500 high quality SNPs within the 579 sequenced amplicons. We demonstrate the utility and versatility of this new genotyping tool for breeding applications including parentage assignment, low density family-based genome-wide association study, trait heritability evaluation to determine potential for genomic selection, and species differentiation (against the weathervane scallop Patinopecten caurinus). We did not find any genomic regions significantly associated with F. halioticida resistance but did identify potential for genomic selection. We could separate the two species based on genotypes, and did not see evidence of a past M. yessoensis x P. caurinus hybridization event within the M. yessoensis breeding population at Vancouver Island University. This low-cost genotyping panel is expected to accelerate selective breeding improvements for M. yessoensis in Canada and elsewhere.
Animals↗