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Results for “high-throughput genotyping”

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PubMed2025

Order among chaos: High throughput MYCroplanters can distinguish interacting drivers of host infection in a highly stochastic system.

The likelihood that a host will be susceptible to infection is influenced by the interaction of diverse biotic and abiotic factors. As a result, substantial experimental replication and scalability are required to identify the contributions of and interactions between the host, the environment, and biotic factors such as the microbiome. For example, pathogen infection success is known to vary by host genotype, bacterial strain identity and dose, and pathogen dose. Elucidating the interactions between these factors in vivo has been challenging because testing combinations of these variables quickly becomes experimentally intractable. Here, we describe a novel high throughput plant growth system (MYCroplanters) to test how multiple host, non-pathogenic bacteria, and pathogen variables predict host health. Using an Arabidopsis-Pseudomonas host-microbe model, we found that host genotype and bacterial strain order of arrival predict host susceptibility to infection, but pathogen and non-pathogenic bacterial dose can overwhelm these effects. Host susceptibility to infection is therefore driven by complex interactions between multiple factors that can both mask and compensate for each other. However, regardless of host or inoculation conditions, the ratio of pathogen to non-pathogen emerged as a consistent correlate of disease. Our results demonstrate that high-throughput tools like MYCroplanters can isolate interacting drivers of host susceptibility to disease. Increasing the scale at which we can screen drivers of disease, such as microbiome community structure, will facilitate both disease predictions and treatments for medicine and agricultural applications.

Arabidopsis↗

PubMed2025

Detection and characterization of neonatal cytomegalovirus through nanopore sequencing using flongle flow cells: Pilot study in Philadelphia, Pennsylvania.

BACKGROUND: Cytomegalovirus (CMV) remains a significant infection in neonates and its early detection can aid with further treatment (antiviral, audiology). However, current diagnostics do not provide genetic information. OBJECTIVE: We explored the use of the portable and comprehensive sequencing method from Oxford Nanopore Technologies, utilizing low-cost Flongle flow cells to detect and perform sequence-level characterization of neonatal urine samples that tested positive for CMV by PCR. STUDY DESIGN: We performed a pilot study based on a retrospective cohort study of neonates who were positive for CMV by PCR, who were admitted at two birth hospitals in Philadelphia, PA. We leveraged deep and long-read sequencing results to analyze the reads in two forms: by comparing them against a reference-based strain and by reconstructing the genome through de novo assembly with phylogenetic tree analysis. RESULTS: We assayed seven clinical samples, including a positive and negative control sample, from newborns ranging from 23 weeks' gestation to term, with testing performed for microcephaly, hearing test results, small gestational age, and thrombocytopenia. Each sample showed multiple differences compared to the reference strain, and the phylogenetic tree analysis of the de novo assembly depicted the genetic diversity of the samples. CONCLUSION: This pilot study shows that nanopore sequencing with low-cost Flongle flow cells can detect and characterize CMV strains from clinical neonatal urine samples. This, coupled with current screening and diagnostic criteria, could further our genomic understanding of neonatal CMV, such as viral genome diversity, genotype-phenotype associations, and spread of strains.

Humans↗

PubMed2009

An outlook on ovarian cancer and borderline ovarian tumors: focus on genomic and proteomic findings.

Among the gynaecological malignancies, ovarian cancer is one of the neoplastic forms with the poorest prognosis and with the bad overall and disease-free survival rates than other gynaecological cancers. Ovarian tumors can be classified on the basis of the cells of origin in epithelial, stromal and germ cell tumors. Epithelial ovarian tumors display great histological heterogeneity and can be further subdivided into benign, intermediate or borderline, and invasive tumors. Several studies on ovarian tumors, have focused on the identification of both diagnostic and prognostic markers for applications in clinical practice. High-throughput technologies have accelerated the process of biomolecular study and genomic discovery; unfortunately, validity of these should be still demonstrated by extensive researches on sensibility and sensitivity of ovarian cancer novel biomarkers, determining whether gene profiling and proteomics could help differentiate between patients with metastatic ovarian cancer and primary ovarian carcinomas, and their potential impact on management. Therefore, considerable interest lies in identifying molecular and protein biomarkers and indicators to guide treatment decisions and clinical follow up. In this review, the current state of knowledge about the genoproteomic and potential clinical value of gene expression profiling in ovarian cancer and ovarian borderline tumors is discussed, focusing on three main areas: distinguishing normal ovarian tissue from ovarian cancers and borderline tumors, identifying different genotypes of ovarian tissue and identifying proteins linked to cancer or tumor development. By these targets, authors focus on the use of novel molecules, developed on the proteomics and genomics researches, as potential protein biomarkers in the management of ovarian cancer or borderline tumor, overlooking on current state of the art and on future perspectives of researches.

Ovarian cancer↗

PubMed2025

Deletion of the HLA-B Gene in One of the Inherited Haplotypes in a Northern European Family.

Targeted next generation sequencing-based HLA typing of a 17-year-old female transplant patient showed homozygosity for the HLA-B allele. The segregation analysis of HLA haplotypes of family members only allowed the conclusion that the B-allele was deleted in the haplotype inherited from the father and accordingly paternal grandfather, resulting in false homozygous genotyping. The subsequent whole-genome sequencing of the patient and her father confirmed an approximately 85 kb deletion at 6p21.33 from the 5' end of the HLA-B to the 3' end of the HLA-C gene extending telomeric to HLA-C.

Adolescent↗

PubMed2026

Deep soil layers show the most pronounced genetic variation in wheat root length.

Wheat is one of the most important cereals worldwide, yet significant gaps remain in our understanding of genetic variability in root traits, especially those associated with deeper rooting that support resource acquisition in challenging environments. Root traits are typically controlled by many genes with small effects and often display low heritability. Our aim was to develop a statistical approach to analyse root variation across soil depth and to determine where genetic differences in root intensity are most detectable. An experiment was conducted at the RadiMax semi-field facility, which is designed to measure deep root systems. Five years of phenotypic data recorded each June produced observations from 1500 rows. Each row captured root intensity across the soil profile from 0.6 m to 2.6 m, enabling detailed analysis of vertical root distribution. Across the five years, 513 winter wheat cultivars were grown in the facility, and among those 409 were genotyped with SNP chips. Depth-resolved regression models with random coefficients were used to quantify genetic and non-genetic variation in root intensity across soil depths, while accounting for spatial variation between rows. Random variation within rows was found to be constant across depths. The models showed that genetic variance for cumulative root intensity increased substantially below 1.1 m, with the deepest layers exhibiting the largest differences between wheat lines. Narrow-sense heritability of point measurements peaked at approximately 1.5 m ([Formula: see text]).

Genetic variability↗

PubMed2025

Missense variants pathogenicity annotation from homologous proteins.

MOTIVATION: High-throughput DNA sequencing has revealed millions of single nucleotide variants (SNVs) in the human genome, with a small fraction linked to disease. The effect of missense variants, which alter the protein sequence, is particularly challenging to interpret due to the scarcity of clinical annotations and experimental information. While using conservation and structural information, current prediction tools still struggle to predict variant pathogenicity. In this study, we explored the pathogenicity of homologous missense variants-variants in equivalent positions across homologous proteins-focusing on proteins involved in autosomal dominant diseases. RESULTS: Our analysis of 2976 pathogenic and 17 555 non-pathogenic homologous variants demonstrated that pathogenicity can be extrapolated with 95% accuracy within a family, or up to 98% for closer homologs. Remarkably, the evaluation of 27 commonly used mutation predictor methods revealed that they were not fully capturing this biological feature. To facilitate the exploration of homologous variants, we created HomolVar, a web server that computationally predicts the pathogenesis of missense variants using annotations from homologous variants, freely available at https://rarevariants.org/HomolVar. Overall, these findings and the accompanying tool offer a robust method for predicting the pathogenicity of unannotated variants, enhancing genotype-phenotype correlations, and contributing to diagnosing rare genetic disorders. AVAILABILITY AND IMPLEMENTATION: HomolVar is freely available at https://rarevariants.org/HomolVar.

Mutation, Missense↗

PubMed2026

From the Microscope to the Genome: A New Era in the Molecular Genetics of Epidermolysis Bullosa.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited disorders characterised by skin fragility, caused by pathogenic variants in genes encoding structural components of the dermo-epidermal junction. With the advent of next-generation sequencing (NGS), the diagnostic paradigm has shifted from a morphological to a genotype-oriented approach. This review summarises the genetic architecture of EB, the types of mutations and genotype-phenotype relationships, the challenges in interpreting variants of unknown significance (VUS), and therapeutic strategies targeting specific mutational mechanisms, including read-through approaches, exon skipping and genome editing. The role of modifier genes and epigenetic factors in clinical variability is also discussed. The focus is on the translational potential of genomics for personalized therapy in EB. Overall, this review synthesizes the molecular basis of all four major EB types across 16+ classical genes, highlights the paradigm shift where NGS achieves a diagnostic yield exceeding 90%, and critically assesses recent therapeutic milestones-ranging from the first FDA-approved topical gene therapy to precision RNA and genome-editing modalities.

Humans↗

PubMed2026

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↗

PubMed2025

Coalescing single-cell genomes and transcriptomes to decode breast cancer progression.

Understanding epithelial lineages of breast cancer and genotype-phenotype relationships requires direct measurements of the genome and transcriptome of the same single cells at scale. To achieve this, we developed wellDR-seq, a high-genomic-resolution, high-throughput method to simultaneously profile the genome and transcriptome of thousands of single cells. We profiled 33,646 single cells from 12 estrogen-receptor-positive breast cancers and identified ancestral subclones in multiple patients that showed a luminal hormone-responsive lineage, indicating a potential cell of origin. In contrast to bulk studies, wellDR-seq enabled the study of subclone-level gene-dosage relationships, which showed near-linear correlations in large chromosomal segments and extensive variation at the single-gene level. We identified dosage-sensitive and dosage-insensitive genes, including many breast cancer genes as well as sporadic copy-number aberrations in non-cancer cells. Overall, these data reveal complex relationships between copy number and gene expression in single cells, improving our understanding of breast cancer progression.

Breast Neoplasms↗

PubMed2025

Identification of intragenic variants in pediatric patients with intellectual disability in Peru.

BACKGROUND: Intellectual disability in Latin America can reach a frequency of 12% of the population, these may include nutritional deficiencies, exposure to toxic or infectious agents, and the lack of universal neonatal screening programs. In 90% of patients with intellectual disability, the etiology can be attributed to variants in the genome. OBJECTIVE: to determine intragenic variants in patients with intellectual disability between 5 and 18 years old at Instituto Nacional de Salud del Niño. METHODS: It is a descriptive cross-sectional study with convenience sampling. A total of 124 children diagnosed with intellectual disability were selected based on psychological test results and availability for whole exome sequencing. In addition, a chromosomal analysis of 6.55 M was performed on ten patients with a negative result in sequencing. Relative and absolute frequencies and measures of central tendency and dispersion were determined according to their nature. In addition, multiple linear regression and Poisson regression were used to determine the association between some clinical characteristics and the probability of occurrence in patients with positive results. RESULTS: The median age of the patients was 6.3 (IQR = 5.95), males accounted for 57.3%, and 91.9% of the cases had mild intellectual disability. Exome sequencing determined the etiology in 30.6% of patients with intellectual disability, of which 52.6% were autosomal dominant inheritance. The most frequent genes found were MECP2, STXBP1 and LAMA2. A broad genotype-phenotype correlation was identified, highlighting the genetic heterogeneity of intellectual disability in this population. The presence of dermatologic lesions, dystonia, peripheral neurological disorders, and fourth finger flexion limitation were observed more frequently in patients with intellectual disability with "positive results". CONCLUSIONS: This study shows that one-third of patients with intellectual disability exhibit intragenic variants, highlighting the importance of genetic analysis for accurate diagnosis. The identification of genes such as MECP2, STXBP1, and LAMA2 underscores the genetic heterogeneity of intellectual disability in the studied population. These findings emphasize the need for genetic testing in clinical management and the implementation of early detection programs in Peru.

Humans↗

PubMed2026

Integrating Next-Generation Sequencing into von Willebrand Disease Diagnostics: Insights from the PCM-EVW-ES Multicenter Project.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder, caused by quantitative or qualitative defects in von Willebrand factor (VWF). Diagnosis is challenging and requires integrating bleeding history, VWF antigen and activity measurements, FVIII assays, and specialized phenotyping. Genetic testing is increasingly recognized as a key component. Here, we review current concepts in VWD diagnostics and highlight the Spanish Clinical and Molecular Profile of von Willebrand Disease (PCM-EVW-ES) project as a model for genomics-enabled precision medicine. PCM-EVW-ES is a multicenter initiative involving 48 hospitals, centralized phenotypic testing, and next-generation sequencing of the VWF coding region, enabling definitive classification in 730 individuals with VWD to date. Harmonized recruitment criteria and standardized workflows improve subtype assignment, uncover complex genotypes, refine genotype-phenotype correlations, and facilitate the identification of asymptomatic carriers. The PCM-EVW-ES variant spectrum highlights recurrent disease-causing variants in Spain and underscores the value of coordinated national registries for variant curation. Building on these data, we propose a diagnostic algorithm in which bleeding assessment and first-line VWF/FVIII assays, combined with, early VWF molecular testing increases diagnostic accuracy and guides targeted second-line investigations to confirm and refine VWD subtype classification. We also outline persisting challenges, including the interpretation of variants of uncertain significance and patients without identifiable pathogenic VWF variants, and future directions integrating third-generation sequencing, expanded gene panels, functional studies, and artificial-intelligence-driven multiomic approaches. Together, these advances illustrate how robust multicenter studies can bridge the gap between complex diagnostics and clinical practice in VWD.

Humans↗

PubMed2026

UPDhmm: detecting uniparental disomy from NGS trio data.

SUMMARY: Uniparental disomies (UPDs) are copy-neutral chromosomal alterations that occur when both copies of a chromosome pair (entire or segmental) come from one parent. UPDs, including isodisomies (identical parental chromosome) and heterodisomies (two different homologs from the same parent), reflect meiotic and/or mitotic aberrations of chromosomal segregation that can be associated with congenital or acquired disease. Despite their relevance, current methods to detect UPDs using sequence data (exomes or genomes) have limited sensitivity for small events, cannot precisely determine the UPD sub-type or coordinates, and perform poorly when including individuals or populations with consanguinity. We present UPDhmm, a novel tool that uses trio-based sequence data (proband and parents) and models inheritance patterns. UPDhmm predicts the most likely inheritance scenario, normal Mendelian inheritance versus UPD event, based on genotype combinations using a Hidden Markov Model (HMM). We validated the method using simulations on exome and genome data from 1000-Genomes projects. UPDhmm overperformed currently available methods in detecting simulated UPD events in both data types. We applied UPDhmm to a collection of nearly 2400 families with a proband with autism spectrum disorder (Simons Simplex Collection Project) and identified UPD events in two affected individuals, one of them previously unreported. These two events, a paternal isodisomy of chr8 and a maternal heterodisomy of chr22, can be genetic causes of the disease, demonstrating the clinical utility of UPDhmm. Thus, UPDhmm can facilitate the incorporation of UPD detection into clinical pipelines of genomic analysis. AVAILABILITY AND IMPLEMENTATION: UPDhmm is implemented in R and is available in the Bioconductor package (version 1.5.0): https://www.bioconductor.org/packages/release/bioc/html/UPDhmm.html. The source code can be found at https://github.com/martasevilla/UPDhmm under the MIT license.

Uniparental Disomy↗

PubMed2025

Diagnostic and phylogenetic perspectives of the 2023 Murray Valley encephalitis virus outbreak in Australia: an observational study.

BACKGROUND: An outbreak of Murray Valley encephalitis virus (MVEV), the largest since 1974, was observed in Australia between Jan 1 and July 31, 2023. This study aims to characterise the utility of diagnostic platforms, testing algorithms, and genomic characteristics of MVEV to facilitate a comprehensive framework for MVEV testing and surveillance in the outbreak setting. METHODS: In this observational study, we assessed flavivirus diagnostics for all patients with suspected Murray Valley encephalitis in Australia from Jan 1 to July 31, 2023. We included all patients with confirmed Murray Valley encephalitis, probable Murray Valley encephalitis, or acute unspecified flavivirus infection using the Communicable Diseases Network Australia case definition. Cases were excluded if an alternative diagnosis was identified. We collected blood, serum, cerebrospinal fluid, brain tissue, urine, or a combination of these samples, as appropriate and at the discretion of the treating clinician. We conducted multimodal diagnostic testing, which included flavivirus-specific serological and nucleic acid amplification testing. Metagenomic next-generation sequencing, including next-generation deep sequencing, target-enrichment, and targeted amplification, was conducted on human and representative mosquito-derived samples obtained from established mosquito population surveillance programmes for phylogenetic analysis. FINDINGS: 27 patients with encephalitis were assessed for MVEV between Jan 1, 2023, and July 31, 2023, 23 (85%) of whom fulfilled national case definitions for confirmed Murray Valley encephalitis. Patient ages ranged from 6 weeks to 83 years (median 62·0 years [IQR 31·0-67·5]) and patients were mostly male (21 [78%] male patients and six [22%] female patients). Incidence varied widely by geographical region and was highest in the Northern Territory (32·0 per 1 000 000 population). Diagnostic specimen collection generally occurred promptly (median 6·0 days [IQR 4·0-14·5] from symptom onset to diagnostic specimen collection). In seven patients, case assignation relied on convalescent serum samples to assess for seroconversion or an appropriate rise in antibody titre (to four times the initial value or greater), or both. MVEV-specific IgM was detectable in serum samples of 17 (81%) of 21 patients tested by day 7 and MVEV IgG or total antibody (TAb) were detected in 18 (100%) of 18 patients tested by day 30. MVEV-specific IgM (or TAb) and MVEV RNA were detected in cerebrospinal fluid collected within 14 days of symptom onset in nine (39%) of 23 patients and seven (28%) of 25 patients, respectively. Phylogenetic analysis revealed two circulating MVEV genotypes, G1A and G2, in mosquitoes and humans in 2023. In southeast Australia, only G1A was detected and probably introduced from enzootic foci in northern Australia. INTERPRETATION: This study provides a comprehensive overview of the diagnostic workflows and phylogenetic evaluations used during the 2023 MVEV outbreak in Australia, emphasising the importance of a multimodal approach for accurate and timely confirmation of flavivirus infection. Further One Health surveillance for MVEV and other zoonotic flaviviruses is key, given potential expanded ecological niches in the context of episodic climatic events. FUNDING: None.

Humans↗

PubMed2026

Deep tissue sequencing improves genetic diagnostic yield in focal cortical dysplasia.

Focal cortical dysplasias (FCDs) are malformations of cortical development associated with drug-resistant focal epilepsy. We analyzed surgical tissue from 25 consecutive cases recruited from adult and pediatric epilepsy surgery programs. We performed high-depth sequencing of lesional tissue, validated somatic variants using droplet digital PCR or amplicon sequencing, and investigated genotype-phenotype correlations. A pathogenic or likely pathogenic variant was detected in 64% (n = 16/25) of cases. Of these, five cases with FCDIIa or FCDIIb had germline variants in NPRL3 (n = 3) or DEPDC5 (n = 2). Somatic variants were identified in 44% (n = 11/25) of cases. The genetic yield for FCDIIb was 77% of cases having a pathogenic mTOR pathway variant detected (n = 10/13), and for FCDIIa 66% (n = 6/9). High depth sequencing approaches allowed detection of somatic variants with very low (down to 0.4%) variant allele fractions (VAFs). No pathogenic variants were detected in 3 cases with FCDI. 62% (n = 15/24) of the cases with ≥12 months follow up experienced a favourable seizure outcome (Engel 1-2) following surgery. Of note, n = 9 patients required repeat surgery to resect residual dysplasia. Determining a genetic diagnosis reveals aetiology and paves the way to precision therapies that may benefit those with FCD who do not respond to current treatments.

Humans↗

PubMed2025

Columba: fast approximate pattern matching with optimized search schemes.

MOTIVATION: Aligning sequencing reads to reference genomes is a fundamental task in bioinformatics. Aligners can be classified as lossy or lossless: lossy aligners prioritize speed by reporting only one or a few high-scoring alignments, whereas lossless aligners output all optimal alignments, ensuring completeness and sensitivity. RESULTS: This paper introduces Columba, a high-performance lossless aligner tailored for Illumina sequencing data. Columba processes single or paired-end reads in FASTQ format and outputs alignments in SAM format. By utilizing advanced search schemes and bit-parallel alignment techniques, Columba achieves exceptional speed. Columba is available in two variants. The first, based on the bidirectional FM-index, prioritizes speed. The second, Columba RLC, uses run-length compression using a bidirectional move structure, significantly reducing memory usage for large, repetitive datasets like pan-genomes. Benchmarks on the human genome, as well as bacterial and human pan-genome datasets, demonstrate that Columba is much faster than existing lossless aligners and even competitive with lossy tools. We integrated Columba into the OptiType HLA genotyping pipeline, where it substantially reduced computational time while maintaining accuracy. These results position Columba as a versatile, state-of-the-art tool for high-sensitivity genomic analyses. AVAILABILITY AND IMPLEMENTATION: The source code of Columba is available at https://github.com/biointec/columba under AGPL license. Scripts to reproduce the benchmarks and analyses are available at https://doi.org/10.5281/zenodo.15849246.

Software↗

PubMed2026

A de novo algorithm for allele reconstruction from Oxford nanopore amplicon reads, with application to CYP2D6.

MOTIVATION: The Oxford Nanopore Technologies' sequencing platform offers a path towards bedside genomics, producing long reads that can completely cover a gene of interest, and detect any known or novel variant the gene contains. However, the analysis of these long reads to identify actionable genotypes remains challenging and typically requires customization depending on the target gene. RESULTS: Here, we describe a generic algorithm to accurately reconstruct allele sequences derived from long-reads of amplicon-based data. Rather than calling variants directly from these long-reads, our method takes a "sequence-first" approach, performing an unbiased reconstruction of the underlying amplicon sequences to generate high-confidence reconstructed allele sequences. This is done without user input of the target gene, allowing for any source amplicon to be reconstructed. These high-confidence reconstructed allele sequences are then compared to the genomic reference sequence of the gene to infer the specific diplotype present in the sample. This approach is agnostic towards the number of genes and alleles present and readily detects novel variants. We demonstrate our approach using three independent data sets for CYP2D6, a diverse and complex gene with over 175 known alleles of clinical significance. We show how our approach can accurately recover validated CYP2D6 diplotypes from 20 Coriell samples covering 14 distinct alleles, using different amplicons, flow cell versions, and depths. This includes inferring occurrences of allele duplication events from relative abundances of each allele, a critical factor for ascribing functional effects to a diplotype. Further, we demonstrate our approach's utility for other genomic regions, including HLA. AVAILABILITY: Custom code is available at the following GitHub repository, along with instructions for use and test data: https://github.com/scottdbrown/allele-reconstruction-long-read-amplicon-data. A snapshot of the code at the time of publication is available on Zenodo.org; doi 10.5281/zenodo.19716004. Raw .fastq sequence data for our three sequencing runs is available at the SRA under Bioproject PRJNA1357883 (https://www.ncbi.nlm.nih.gov/bioproject/1357883).

Alleles↗

PubMed2025

A universal, high-quality, and high-yield DNA purification method for mycobacteria, including Mycobacterium tuberculosis: large-scale assessment of the chloroform-bead method.

UNLABELLED: Genomic analysis of mycobacteria has become increasingly crucial for understanding drug-resistance mechanisms, molecular epidemiology, and pathogenesis. However, efficient extraction of high-molecular-weight genomic DNA from these organisms remains challenging because of their thick mycolic acid-rich cell walls. In this study, we report the chloroform-bead method, a universal DNA extraction protocol that combines chemical and mechanical disruptions to overcome these challenges. Multi-laboratory evaluation (16 sites) demonstrated the chloroform-bead method's superiority over conventional methods for Mycobacterium tuberculosis (DNA yield: 17.9 vs 1.9 µg, purity A260/A230: 1.86 vs 1.22, both P < 0.001). Single-facility assessment extended these findings to >32 nontuberculous mycobacterial species (n = 1,058), showing performance comparable to M. tuberculosis (n = 1,000), with both achieving median yields of 22.2 µg DNA and consistent quality metrics. The chloroform-bead method significantly reduced the processing time from 2 to 3 days to 2 h while ensuring complete sample sterilization, eliminating the need for species-specific optimization. This streamlined and universally applicable protocol represents a practical advancement in mycobacterial DNA extraction methodology, ideal for high-throughput genomic studies and routine clinical diagnostics. IMPORTANCE: Mycobacterial genomics is crucial for understanding pathogenesis and drug resistance; however, DNA extraction remains a significant challenge because of its unique cell wall. Traditional methods rely on enzymatic treatments, resulting in complex and time-consuming protocols with variable results. The chloroform-bead method introduces a paradigm shift by chemically and mechanically disrupting the mycolic acid layer and eliminating the need for enzymatic treatment. This standardized approach ensures consistent, high-quality DNA extraction across diverse mycobacterial species, thereby enhancing research capabilities and clinical applications.

Chloroform↗

PubMed2026

Prospective clinical validation of targeted long-read sequencing for preimplantation genetic testing of α-thalassaemia.

BACKGROUND: Preimplantation genetic testing for monogenic disorders (PGT-M) can prevent transmission of severe α-thalassaemia, but conventional workflows remain limited by family-specific assay design for direct variant detection, dependence on additional family samples for haplotype construction, and labour-intensive multi-step procedures across several platforms. Targeted long-read sequencing-based PGT-M for α-thalassaemia (tlrPGT-α-thal) integrates direct variant detection and haplotype linkage analysis within a single assay, but prospective clinical validation is lacking. METHODS: This prospective clinical study enrolled 103 families at high risk of transmitting α-thalassaemia at a reproductive medicine centre between August 2024 and March 2025. All families underwent blinded parallel analysis using both conventional NGS-based PGT-M (comparator) and tlrPGT-α-thal. RESULTS: In the primary concordance analysis, tlrPGT-α-thal was fully concordant with conventional NGS-based PGT-M (507/507, 100.0%; exact 95% CI, 99.3-100.0). Direct variant detection was successful in 501/507 embryos (98.82%; 95% CI, 97.4-99.6), haplotype linkage was established in 505/507 embryos (99.61%; 95% CI, 98.6-100.0), and one meiotic recombination event was identified. Among 93 families proceeding to embryo transfer, 57 pregnancies underwent invasive prenatal diagnosis, and all were concordant with the corresponding tlrPGT-α-thal results. Of the 26 comparator-inconclusive embryos, tlrPGT-α-thal resolved 6 complex cases, including cases with incomplete pedigrees or insufficient informative SNPs. Among the remaining 20 embryos with HBA-region aneuploidies, genotype and parental origin could be determined in 12. CONCLUSIONS: The findings show that tlrPGT-α-thal enables direct detection of diverse α-thalassaemia-causing variants together with efficient haplotype linkage analysis within a single workflow, without requiring family-specific assay design or additional family samples. The method demonstrated high diagnostic accuracy while providing added value in complex scenarios. Taken together, tlrPGT-α-thal represents a simplified and broadly applicable strategy for α-thalassaemia PGT-M.

Humans↗