Biology Methods and Protocols最新文献

{"title":"Novel reference method for precise determination of tryptophanase activity.","authors":"Marwa M Ali, Inas J Mahdi, Hayder Obayes Hashim, Asad M Hadwan, Mahmoud Hussein Hadwan, Roaa Altaee","doi":"10.1093/biomethods/bpag017","DOIUrl":"https://doi.org/10.1093/biomethods/bpag017","url":null,"abstract":"<p><p>Tryptophanase (TnaA; EC 4.1.99.1) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the β-elimination of L-tryptophan into indole, pyruvate, and ammonia. It plays a key role in bacterial signaling and biofilm formation and is industrially relevant for L-tryptophan biosynthesis. Existing assay methods for TnaA activity measurement, including Kovács reagent colorimetry, HPLC, and NADH-coupled spectrophotometry, each carry limitations in cost, complexity, or analytical sensitivity that restrict their routine use. The current study aims to develop and validate a simple, cost-effective spectrophotometric method for quantifying TnaA activity using 2,4-dinitrophenylhydrazine (2,4-DNPH) as a chromogenic reagent, targeting the pyruvate product of the enzymatic reaction. The DNPH-TnaA assay uses a stable hydrazone complex formed between pyruvate and 2,4-DNPH, which is measurable at 432 nm. It was applied to <i>Escherichia coli</i> lysates (ATCC 8739 and five clinical isolates). Performance was evaluated for linearity (5-500 µM), detection limits, intra- and inter-assay precision (CV%), spike recovery, selectivity against nine interference classes, and signal stability over 120 h. Validation included comparisons with Kovács colorimetric and HPLC methods, using Passing-Bablok and Bland-Altman analyses. The limits of detection (LOD) and quantification (LOQ) were 2.25 U/l and 6.7 U/l, respectively. Intra-assay coefficient of variation (CV) was 1.19% (mean 270.83 ± 3.23 U/l), inter-assay CV was 2.65% (mean 273.55 ± 7.25 U/l), both acceptable. Selectivity errors were within ±3.33%. The colored hydrazone was stable at 25°C for 48 h. Passing-Bablok regression showed <i>r</i> = 0.99 with near-unity slopes and negligible intercepts, and Bland-Altman analysis indicated minimal bias. ANOVA found no significant differences among the methods at the .05 level. The DNPH-TnaA assay is a precise, accurate, and robust method for measuring TnaA activity, comparable to Kovács and HPLC methods. Its simplicity, low cost, and compatibility with common lab equipment make it ideal for routine diagnostics and research applications.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag017"},"PeriodicalIF":1.3,"publicationDate":"2026-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13094545/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147783754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>LAMPrey</i>: a standardised method for analysing quantitative LAMP reactions using the inflection cycle threshold.","authors":"Adam Bates, Jiao Li, Sofia Vamos, Francisco Rivero, Katharina C Wollenberg Valero","doi":"10.1093/biomethods/bpag016","DOIUrl":"https://doi.org/10.1093/biomethods/bpag016","url":null,"abstract":"<p><p>Quantitative loop-mediated isothermal amplification (qLAMP) is a gene expression quantification method that has gained popularity in recent years, particularly in disease identification, including during the recent SARS-CoV-2 pandemic. Unlike conventional quantitative PCR (qPCR), qLAMP features reaction kinetics that may diverge from sigmoidal expectation, and may not include ROX dye in commercial kits. Determining cycle threshold (Ct or Cq) values through automatic thresholding may therefore produce inaccurate results, and the nature of these thresholds complicates comparability between studies and softwares. We introduce a new method for transforming sigmoidal amplification curves into inflection cycle threshold curves (iCt) to address issues with auto thresholds and analysis of qLAMP. This method is implemented as a set of R functions named <i>LAMPrey</i>, suitable for analysis of both qPCR and qLAMP reactions performed in the two most commonly used real-time thermocyclers. We simulate qLAMP amplification differences, demonstrate that iCt and Ct methods perform equivalently for conventional qPCR with an Illumina library quantitation kit, and show that iCt values outperform Ct and the sigmoid curve-fitting metric FDM for quantifying 2416 qLAMP reactions in of zebrafish embryos. All scripts developed for this article are available at https://github.com/dodged13/LAMPrey.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag016"},"PeriodicalIF":1.3,"publicationDate":"2026-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13099408/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147783826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing propagation and purification efficiency of M13 bacteriophage for improved phage display applications.","authors":"Md Monir Hossain, Foong Yong, Vipul Bansal, Ravi Shukla","doi":"10.1093/biomethods/bpag011","DOIUrl":"10.1093/biomethods/bpag011","url":null,"abstract":"<p><p>The global biosensors market is rapidly growing, driven by increasing demand for quick, affordable, and portable diagnostic tools across sectors, including healthcare, environmental monitoring, food safety, and biomedical research. The key to biosensor function is the biological recognition element (BRE), which determines the specificity, sensitivity, and reliability of the device. Traditional BREs, such as antibodies and enzymes, face significant limitations, including instability, high costs, and variability. Phage display technology offers a strong alternative, providing durable, stable, and highly specific peptides as BREs. However, it requires effective amplification and purification methods to produce high-quality peptide libraries. This study examines key factors affecting the amplification of filamentous M13 bacteriophage, highlighting the negative impact of high multiplicity of infection (MOI) caused by superinfection exclusion and reduced phage adsorption efficiency. Our findings indicate that the bacterial growth phase is the most important determinant of M13 amplification efficiency. Furthermore, post-infection PEG/NaCl precipitation followed by high-speed centrifugation significantly outperforms traditional filtration methods in purifying phages, maximizing recovery and viability. These findings present an optimized, reproducible, and scalable approach to M13 phage amplification, improving the effectiveness of phage display for developing advanced biorecognition elements. Ultimately, this research provides a foundational framework for more efficient biosensing and therapeutic applications, filling critical gaps in the current biosensor development landscape.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag011"},"PeriodicalIF":1.3,"publicationDate":"2026-03-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13006207/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147515459","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigating the association of NBN gene polymorphisms with multiple cancers through statistical meta-analysis and bioinformatics insights.","authors":"Md Harun-Or-Roshid, Md Selim Reza, Md Ariful Islam, Md Humaun Farid, Md Mostafizur Rahman, Saroje Kumar Sarkar, Md Nurul Haque Mollah","doi":"10.1093/biomethods/bpag012","DOIUrl":"10.1093/biomethods/bpag012","url":null,"abstract":"<p><p>Several individual genetic association studies, including meta-analyses, have investigated the association of two SNPs (rs1805794 and rs709816) of NBN gene with multiple cancer risks. However, their findings were inconsistent, making it challenging to use NBN gene as a diagnostic and prognostic biomarker. This study aims to provide an improved reliability on the association between NBN polymorphisms and multiple cancers through the extended statistical meta-analysis. We collected a comprehensive dataset comprising 58 individual SNP-cancer association studies, including 23 494 cases and 29 592 controls for rs1805794, and 5325 cases and 11 149 controls for rs709816 polymorphisms, using a systematic search strategy across online databases. The collected data were analyzed using statistical meta-analysis to investigate the association between two SNPs and cancers. This meta-analysis revealed that the <i>C</i> allele of rs1805794 and rs709816 polymorphisms is not significantly associated with overall cancer risk in each ethnic population. However, sub-group analysis based on cancer types showed that rs1805794 is significantly associated with the increased risk of bladder cancer under three, and nasopharyngeal cancer (subtype of head and neck cancer) under four genetic models. Also, it was seen that rs1805794 is partially associated with brain cancer risk under allelic model, while rs709816 is significantly linked to breast cancer. Notably, rs1805794 exhibited a trend toward increased cancer risk, while rs709816 showed a protective tendency. Besides, bioinformatics analysis results also supported the meta-analysis results from different viewpoints including expression analysis of NBN gene from TCGA database, disease-gene and gene-regulator network analysis, and gene ontology and pathway enrichment analysis, and indicate the NBN gene directly/indirectly associated with cancer risks. Meta-analysis results, supported by bioinformatics analyses, indicate potential associations between NBN gene variants and susceptibility to bladder, brain, breast, and nasopharyngeal cancers. However, these findings are exploratory and indicate biological relevance rather than established diagnostic or prognostic utility. Systematic review registration: https://www.crd.york.ac.uk/prospero/; identifier: CRD420251034651.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag012"},"PeriodicalIF":1.3,"publicationDate":"2026-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12967219/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147378807","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"One size does not fit all: A comparative framework for optimizing ribonucleic acid transfection across acute myeloid leukemia cell lines.","authors":"Monika M Kojic, Carmen Spencer, Olivia Kovecses, Maureen McKeague","doi":"10.1093/biomethods/bpag014","DOIUrl":"https://doi.org/10.1093/biomethods/bpag014","url":null,"abstract":"<p><p>Robust transient knockdown strategies are critical to oligonucleotide therapeutic development, yet ineffective and variable RNA delivery across experimental models continues to limit target validation. Efficient delivery of nucleic acids into acute myeloid leukemia (AML) cells is particularly challenging due to their immature, suspension phenotype. Here, we performed a head-to-head benchmarking comparison of commonly used chemical transfection reagents (Lipofectamine 3000, Lipofectamine 2000, RNAiMAX, INTERFERin) alongside a physical electroporation approach (Lonza nucleofection) across four genetically distinct AML cell lines (THP-1, OCI-AML3, MV4-11, MOLM-14). To establish recommended RNA delivery strategies, we used small-interfering RNA (siRNA) and quantified functional knockdown by RT-qPCR, with protein-level validation and paired assessment of post-transfection viability. Lipid-based formulations were most effective in more differentiated AML cell lines (for example, THP-1 and OCI-AML3), whereas more immature lines (MV4-11 and MOLM-14) were poorly responsive to chemical transfection but efficiently transfected by electroporation. A short serum-free incubation period enhanced lipid-mediated delivery in permissive lines and produced measurable gains in more resistant models. Transfection-associated cytotoxicity was strongly method-dependent, with lipid-based reagents producing minimal to modest viability losses and nucleofection causing substantially greater short-term reductions in viable cell numbers. Based on this systematic comparison, most chemical reagents supported efficient delivery in THP-1 and, to a lesser extent, OCI-AML3, while MV4-11 and MOLM-14 demonstrated strict dependence on electroporation for meaningful intracellular uptake. Together, our results define a concise, qPCR-guided workflow, validated at the protein level, that provides a replicable, decision-oriented framework for selecting efficient and fit-for-purpose short RNA delivery strategies in AML cell lines.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag014"},"PeriodicalIF":1.3,"publicationDate":"2026-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12987767/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147469535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"UniClo technology exploits methylation for universal scarless DNA assembly.","authors":"Carol N Flores-Fernández, Da Lin, Katherine Robins, Chris A O'Callaghan","doi":"10.1093/biomethods/bpag013","DOIUrl":"https://doi.org/10.1093/biomethods/bpag013","url":null,"abstract":"<p><p>Several Golden Gate-based DNA assembly techniques have been developed previously with different limitations including the requirement for domestication of sequences with internal sites for the type IIS restriction enzymes used, insertion of persistent scars in assembled DNA and the need for multiple assembly vectors and overhang sequences. We developed UniClo, which overcomes all these problems. Sequences with internal type IIS sites can be assembled, it allows fully scarless hierarchical assembly and requires only three assembly vectors and two universal overhang sequences. This is achieved by three key elements: (i) Recombinant methylases are used <i>in vitro</i> to methylate, and thus inactivate, any sites in fragments to be assembled for the type IIS restriction enzyme used in the assembly as well as the outer sites of the assembly vectors. (ii) A CRISPR-dCas9 molecule is used to protect the type IIS restriction enzyme sites required for the assembly from methylation, thus preserving the activity of these sites. (iii) A set of engineered vectors is used to trim overhangs that would otherwise generate scars. Here, we present a detailed protocol for performing DNA assembly using UniClo and describe the methylation-protection of the fragments to be assembled, methylation of the scarless vectors, the assembly reaction, and analysis of the final assembled DNA molecule. UniClo offers substantial flexibility in the assembly design and enables the assembly of any DNA molecule regardless of its sequence, nature and application.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag013"},"PeriodicalIF":1.3,"publicationDate":"2026-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12996903/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147487758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A multi-dimensional CNN-Bi-GRU for IoT-based brain-computer interface in early epileptic seizure detection.","authors":"Biplov Paneru","doi":"10.1093/biomethods/bpag010","DOIUrl":"https://doi.org/10.1093/biomethods/bpag010","url":null,"abstract":"<p><p>The study focuses on seizure detection using EEG data from Mendeley. An early-alert IoT-BCI system is designed to simulate real-time support for patients during seizures. The proposed Multi-Dimensional CNN-Bi-GRU (MDCBG) outperforms hybrid deep learning models, achieving 97.43% accuracy, surpassing baseline EEGNet (92.17%) and CTNET (85.11%), along with models evaluated through ablation studies on seizure vs. non-seizure prediction. The proposed model, along with other models like Bi-GRU with attention, Bi-LSTM-GRU, and XGBoost, also performs well on classifying various types of seizures. SHAP analysis shows Channel 5 contributes most to predictions. An IoT-based automation system is simulated on seizure detection for triggering micro devices near the patient's environment. This approach supports early seizure warning and guides home-automation strategies to assist patients.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag010"},"PeriodicalIF":1.3,"publicationDate":"2026-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13049591/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147624033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Linking patient records at scale with a hybrid approach combining contrastive learning and deterministic rules.","authors":"Cheng Cao, Jay Pillai, Sara Daraei, Sina Ghadermarzi","doi":"10.1093/biomethods/bpag009","DOIUrl":"https://doi.org/10.1093/biomethods/bpag009","url":null,"abstract":"<p><p>Linking patient records across disparate healthcare systems is essential to create comprehensive views of patient health, yet this task is complicated by inconsistent identifiers and data quality issues. Although traditional deterministic and probabilistic record linkage (RL) methods have long been used for this purpose, deterministic approaches are brittle in the presence of noisy personally identifiable information (PII), while probabilistic approaches are often difficult to scale. As a result, large-scale linkage commonly relies on restrictive matching strategies that limit recall. This work presents a hybrid RL approach that integrates a deep embedding model with deterministic rules, leveraging both the flexibility and noise robustness of soft embeddings and the reliability and predictable baseline performance of deterministic rules. Using a large-scale real-world dataset, a BERT-based embedding model is fine-tuned in a Siamese network with contrastive loss to encode PII fields as numeric vectors. De-duplicated identifiers (Fuzzy IDs) are then obtained through a blocking-and-clustering step using the embedding vectors. The approach is evaluated using multiple signals (social security number, phone, and email) and is shown to outperform baseline methods. A postprocessing step based on deterministic rules allows embedding-based linkage to be overridden in a subset of cases where high-confidence rules apply, such as when a high-quality identifier is available. The system is deployed on a commercial database consisting of more than 200 million PII records, demonstrating scalability in a real-world healthcare setting.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag009"},"PeriodicalIF":1.3,"publicationDate":"2026-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12952525/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147348947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DisSNPNet: Predicting disease-associated single-nucleotide polymorphisms using linkage disequilibrium, disease similarity, and 1000 Genomes Project datasets with evidence-based validation.","authors":"Duc-Hau Le","doi":"10.1093/biomethods/bpag004","DOIUrl":"10.1093/biomethods/bpag004","url":null,"abstract":"<p><p>Identifying disease-associated single-nucleotide polymorphisms (SNPs) is fundamental to understanding complex disease genetics, yet genome-wide association studies (GWAS) remain costly and data-intensive. Network-based approaches provide a complementary strategy by exploiting linkage disequilibrium (LD) structure- and disease-relatedness to prioritize candidate variants. We present DisSNPNet, a heterogeneous network-based framework that integrates chromosome-specific SNP LD networks derived from 1000 Genomes Project Phase 1 and Phase 3 data, a MeSH-based disease similarity network, and known disease-SNP associations from CAUSALdb. Random walk with restart was applied to rank SNPs for each disease. Predictive performance was evaluated using disease-wise 3-fold cross-validation with AUROC and AUPR. Biological plausibility was assessed by querying top-ranked SNPs in GWAS resources and by disease-specific KEGG pathway enrichment. A chromosome-matched random baseline was constructed to contextualize external GWAS evidence. DisSNPNet consistently outperformed SNP-only LD networks, with heterogeneous networks yielding higher AUROC and AUPR across chromosomes. Strong LD networks (<i>r</i> ² ≥ 0.8) improved precision, particularly in imbalanced settings. Top-ranked SNPs showed significantly greater GWAS evidence than random expectation across all chromosomes, indicating nonrandom enrichment. Disease-specific pathway enrichment revealed biologically coherent mechanisms across immune, metabolic, cardiovascular, and structural diseases. DisSNPNet provides a robust and interpretable framework for prioritizing disease-associated SNPs. While not a substitute for GWAS, it offers a scalable, evidence-supported approach for SNP prioritization and hypothesis generation, complementing experimental and population-based studies.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag004"},"PeriodicalIF":1.3,"publicationDate":"2026-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12895193/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146203128","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Resolving haplotypes of the glucose-6-phosphate dehydrogenase gene using long-range polymerase chain reaction and Oxford Nanopore sequencing.","authors":"Kamonwan Chamchoy, Beatriz Aira C Jacob, Usa Boonyuen","doi":"10.1093/biomethods/bpag008","DOIUrl":"https://doi.org/10.1093/biomethods/bpag008","url":null,"abstract":"<p><p>Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy and poses a major concern on safe administration of oxidative drugs, including antimalarials such as primaquine and tafenoquine. Common diagnostic approaches, such as enzyme assays, polymerase chain reaction (PCR)-based methods, and short-read genotyping, often fail to identify heterozygous female carriers and are unable to determine the phase of compound heterozygous mutations. To address these limitations, a workflow combining long-range PCR with Oxford Nanopore Technologies (ONT) sequencing was developed, enabling comprehensive analysis of the entire <i>G6PD</i> locus with direct haplotype resolution. The accuracy of the developed method was independently validated by Sanger sequencing for exonic variant detection and by adaptive sampling-based ONT sequencing for phasing accuracy. A total of 24 samples (20 females, 4 males) were analyzed using two long-range amplicons (∼12 and ∼14 kb) with an 8.2 kb overlap spanning both variant-rich and variant-sparse regions. ONT sequencing revealed 36 distinct variants across exonic, intronic, and regulatory regions. The design consistently captured multiple informative heterozygous sites, markedly improving haplotype reconstruction in females. Nanopore sequencing generated long reads (N50 ∼11 kb) with deep coverage (>700-fold), supporting accurate variant detection and phasing. These findings demonstrate the feasibility and robustness of a nanopore-based long-read approach for comprehensive <i>G6PD</i> haplotyping, integrating variant detection and phasing within a single analytical workflow, and providing a foundation for future studies on carrier detection and other X-linked genes.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag008"},"PeriodicalIF":1.3,"publicationDate":"2026-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12915576/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146229260","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}