Search

Summary Vacuoles, E1 enzyme, X‐linked, autoinflammatory, somatic (VEXAS) is a haemato‐inflammatory syndrome genetically defined by somatic mutations in the X‐linked UBA1 gene, typically Val/Thr/Leu substitutions at the Met41 hotspot. Clinical manifestations are heterogeneous and refractory to most haemato‐rheumatological treatments. To date, no guidelines exist for the management of VEXAS, and scarce is the evidence on methodology and clinical significance of longitudinal UBA1 clonal burden evaluation upon therapy. Here, we validated a method to quantify UBA1 clonal burden and explored its applicability in patients with VEXAS. Given the different treatment interactions, droplet digital polymerase chain reaction (ddPCR) may allow for informed therapeutic decisions and implementation of personalized strategies. [ABSTRACT FROM AUTHOR]

Proteoforms are proteins derived from highly related genes or post translational modifications (PTMs) of the same protein. They share extremely similar primary structures but have varying functions. Unfortunately, protein de novo sequencing including specific PTM/mutation detection is still challenging. Herein, a nanopore‐based technique is reported to resolve the amino acid order of amyloid‐β (Aβ1‐42) with site specificity. Subnanopores are sputtered in 5 nm‐thick inorganic membranes with a sensing depth of 0.66 nm inferred by finite element analysis. Denatured molecules at 0.45 ng mL−1 translocate through subnanopores while the current traces are sampled at 500 kHz with rms noise <15 pA. Hundreds of blockades are clustered using machine learning, and multiple blockades are averaged to establish current consensus. Consensus traces strongly correlate with a linear model of amino acid volume of Aβ1‐42 at single residue resolution, with Pearson Correlation Coefficients (PCCs) of 0.81 ± 0.03 and 0.92 ± 0.03 before and after dynamic time warping (DTW). A scrambled version of Aβ1‐42 is tested for validation purposes. Deep learning classification reveals that different polypeptides generate distinct translocation fluctuating patterns, but variations become imperceptible for the same species measured across nanopores (Area Under the Curve, AUC 0.93 ± 0.05 vs 0.64 ± 0.12). Lastly, important PTMs and mutations are site‐specifically located along the primary structure, implying new potential clinical applications. [ABSTRACT FROM AUTHOR]

Objectives: The studies on the spread of major animal diseases and its evolution of public opinion are of great significance to the improvement of epidemic prevention and public opinion guidance. With the development of Web 2.0 technology and the popularity of smart phones, various forms of social media platforms become important channels for obtaining, sharing and discussing hot topics. A large number of texts with geographical location information are generated, which have provided a new way for the research of animal epidemic and other emergencies. Methods: Taking Sina microblog data during African swine fever (ASF) spread in our country from 2018 to 2019 as the case study, the objective of this work is to establish the spread spatiotemporal characteristics analysis and public opinion mining model. First, the Mann-Kendall mutation detection method is introduced to objectively divide the epidemic transmission cycle and investigate the spatial distribution characteristics of different stages. Then, the latent Dirichlet allocation theme clustering model is used to describe the evolution of public opinion topics among different ASF epidemic stages. Finally, the primary factors influencing public opinion attention are explored based on the geographical detector method. Results: The results show that the spread of ASF in China showed a trend of spreading from northeast to southwest, and experienced four stages: Incubation, outbreak, fluctuation and recession. Conclusions: At each stage, public opinion around outbreak itself and the specific theme is derived, and with the development of epidemic derivative subject is more abundant, popular sentiment also from at each stage. Public opinion around outbreak itself and the specific derivative topics, and derivative topics become more abundant with the development of epidemic, public sentiment also gradually changes from negative to positive. Regional awareness of ASF is strongly influenced by pork consumption and production, rather than by local education and urbanization levels. [ABSTRACT FROM AUTHOR]

Abstract Pancreatic ductal adenocarcinoma (PDAC) presents challenges in detecting somatic mutations due to its complex cellular composition. This study investigated the utility of patient-derived organoids (PDOs) to overcome these obstacles and enhance somatic mutation identification. Surgically resected PDAC tumors and their paired PDOs from 21 patients were examined. Whole-exome sequencing (WES) of tumor tissue, organoids, and peripheral blood mononuclear cells was performed to identify somatic mutations. Our findings demonstrate that PDOs retained about 80% of the somatic mutations from the original tumors, showing high concordance in mutation types. PDOs exhibited increased tumor purity and uncovered key driver mutations, aiding in identifying clinically relevant genomic alterations. Moreover, eight cycles of FOLFIRINOX treatment did not significantly alter the mutational landscape at the DNA level, indicating the stability of the mutational profile after therapeutic pressure in patients. In conclusion, PDOs are potentially important tools for exploring the somatic mutational landscape of PDAC. While they can reveal mutations that may be challenging to detect through traditional biopsy sequencing due to the inherently low tumor purity of PDAC, it is important to note that PDOs may not always fully recapitulate all mutations found in primary tumors. Despite this limitation, PDOs can still offer critical insights into the genomic complexities of PDAC, which is crucial for the development of personalized vaccines and therapies for this disease.

The globally prevalent pests, Diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) and Beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), pose significant threats to cruciferous vegetables. They have rapidly developed resistance to a wide range of insecticides, leading to significant yield losses and increased control expenses. In this study, we have established an efficient approach utilizing amplicon sequencing to detect the frequency of 15 target resistance mutant sites in 6 molecular targets, acetylcholinesterase 1 (ACE1), chitin synthase 1 (CHS1), the γ-aminobutyric acid receptor (GABAR), glutamate-gated chloride channel (GluCl), voltage-gated sodium channels (NaV), and ryanodine receptor (RyR) in P. xylostella and the frequency of 11 mutations in 5 molecular targets (except GluCl) in S. exigua in China. Our findings indicate that P. xylostella exhibits remarkably high frequency (over 88.67%) in pyrethroid resistance-related mutations T929I and L1014F of NaV. In S. exigua , the frequencies of L659F mutation were ranging from 41.92% to 74.89%. In addition, the organophosphorus resistance-related mutations A298S and G324A of ACE1 were detected at frequencies ranging from 34.29% to 75.66%, and these 2 mutations occurred simultaneously (from 29.22% to 65.79%) in P. xylostella. An interannual variation in mutation frequency from 2019 to 2021 was found for P. xylostella in HNCS. The frequency of A298S and G324A mutations steadily increased while the frequency of G4946E and I4790M mutations continuously decreased. These results unveil a worrisome scenario of multiple resistance sites in these 2 pests in China and provide valuable insights for the practical application of pesticides in the field. [ABSTRACT FROM AUTHOR]

Genes, expressed as sequences of nucleotides, are susceptible to mutations, some of which can lead to cancer. Machine learning and deep learning methods have emerged as vital tools in identifying mutations associated with cancer. Thyroid cancer ranks as the 5th most prevalent cancer in the USA, with thousands diagnosed annually. This paper presents an ensemble learning model leveraging deep learning techniques such as Long Short-Term Memory (LSTM), Gated Recurrent Units (GRUs), and Bi-directional LSTM (Bi-LSTM) to detect thyroid cancer mutations early. The model is trained on a dataset sourced from asia.ensembl.org and IntOGen.org, consisting of 633 samples with 969 mutations across 41 genes, collected from individuals of various demographics. Feature extraction encompasses techniques including Hahn moments, central moments, raw moments, and various matrix-based methods. Evaluation employs three testing methods: self-consistency test (SCT), independent set test (IST), and 10-fold cross-validation test (10-FCVT). The proposed ensemble learning model demonstrates promising performance, achieving 96% accuracy in the independent set test (IST). Statistical measures such as training accuracy, testing accuracy, recall, sensitivity, specificity, Mathew's Correlation Coefficient (MCC), loss, training accuracy, F1 Score, and Cohen's kappa are utilized for comprehensive evaluation. [ABSTRACT FROM AUTHOR]

Abstract Structural variants (SVs) are one of the significant types of DNA mutations and are typically defined as larger-than-50-bp genomic alterations that include insertions, deletions, duplications, inversions, and translocations. These modifications can profoundly impact the phenotypic characteristics and contribute to disorders like cancer, response to treatment, and infections. Four long-read aligners and five SV callers have been evaluated using three Oxford Nanopore NGS human genome datasets in terms of precision, recall, and F1-score statistical metrics, depth of coverage, and speed of analysis. The best SV caller regarding recall, precision, and F1-score when matched with different aligners at different coverage levels tend to vary depending on the dataset and the specific SV types being analyzed. However, based on our findings, Sniffles and CuteSV tend to perform well across different aligners and coverage levels, followed by SVIM, PBSV, and SVDSS in the last place. The CuteSV caller has the highest average F1-score (82.51%) and recall (78.50%), and Sniffles has the highest average precision value (94.33%). Minimap2 as an aligner and Sniffles as an SV caller act as a strong base for the pipeline of SV calling because of their high speed and reasonable accomplishment. PBSV has a lower average F1-score, precision, and recall and may generate more false positives and overlook some actual SVs. Our results are valuable in the comprehensive evaluation of popular SV callers and aligners as they provide insight into the performance of several long-read aligners and SV callers and serve as a reference for researchers in selecting the most suitable tools for SV detection.

Structural variants (SVs) are one of the significant types of DNA mutations and are typically defined as larger-than-50-bp genomic alterations that include insertions, deletions, duplications, inversions, and translocations. These modifications can profoundly impact the phenotypic characteristics and contribute to disorders like cancer, response to treatment, and infections. Four long-read aligners and five SV callers have been evaluated using three Oxford Nanopore NGS human genome datasets in terms of precision, recall, and F1-score statistical metrics, depth of coverage, and speed of analysis. The best SV caller regarding recall, precision, and F1-score when matched with different aligners at different coverage levels tend to vary depending on the dataset and the specific SV types being analyzed. However, based on our findings, Sniffles and CuteSV tend to perform well across different aligners and coverage levels, followed by SVIM, PBSV, and SVDSS in the last place. The CuteSV caller has the highest average F1-score (82.51%) and recall (78.50%), and Sniffles has the highest average precision value (94.33%). Minimap2 as an aligner and Sniffles as an SV caller act as a strong base for the pipeline of SV calling because of their high speed and reasonable accomplishment. PBSV has a lower average F1-score, precision, and recall and may generate more false positives and overlook some actual SVs. Our results are valuable in the comprehensive evaluation of popular SV callers and aligners as they provide insight into the performance of several long-read aligners and SV callers and serve as a reference for researchers in selecting the most suitable tools for SV detection. [ABSTRACT FROM AUTHOR]

Background: The accurate and expeditious detection of SARS-CoV-2 mutations is critical for monitoring viral evolution, assessing its impact on transmission, virulence, and vaccine efficacy, and formulating public health interventions. In this study, a detection system utilizing micro temperature gradient gel electrophoresis (μTGGE) was developed for the identification of the D614 and G614 variants of the SARS-CoV-2 spike protein. Methods: The in vitro synthesized D614 and G614 gene fragments of the SARS-CoV-2 spike protein were amplified via polymerase chain reaction and subjected to μTGGE analysis. Results: The migration patterns exhibited by the D614 and G614 variants on the polyacrylamide gel were distinctly dissimilar and readily discernible by μTGGE. In particular, the mid-melting pattern of D614 was shorter than that of G614. Conclusions: Our results demonstrate the capability of μTGGE for the rapid, precise, and cost-effective detection of SARS-CoV-2 spike protein D614 and G614 variants without the need for sequencing. Therefore, this approach holds considerable potential for use in point-of-care mutation assays for SARS-CoV-2 and other pathogens. [ABSTRACT FROM AUTHOR]

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing. [ABSTRACT FROM AUTHOR]

A 'classical' and a 'basal‐like' subtype of pancreatic cancer have been reported, with differential expression of GATA6 and different dosages of mutant KRAS. We established in situ detection of KRAS point mutations and mRNA panels for the consensus subtypes aiming to project these findings to paraffin‐embedded clinical tumour samples for spatial quantitative analysis. We unveiled that, next to inter‐patient and intra‐patient inter‐ductal heterogeneity, intraductal spatial phenotypes exist with anti‐correlating expression levels of GATA6 and KRASG12D. The basal‐like mRNA panel better captured the basal‐like cell states than widely used protein markers. The panels corroborated the co‐existence of the classical and basal‐like cell states in a single tumour duct with functional diversification, i.e. proliferation and epithelial‐to‐mesenchymal transition respectively. Mutant KRASG12D detection ascertained an epithelial origin of vimentin‐positive cells in the tumour. Uneven spatial distribution of cancer‐associated fibroblasts could recreate similar intra‐organoid diversification. This extensive heterogeneity with functional cooperation of plastic tumour cells poses extra challenges to therapeutic approaches. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Abstract Background The SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages. Methods The presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2–positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2–negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing. Results All genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2–positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages. Conclusion The developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Background: The SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages. Methods: The presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2–positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2–negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing. Results: All genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2–positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages. Conclusion: The developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform. [ABSTRACT FROM AUTHOR]

The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor's temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes. [ABSTRACT FROM AUTHOR]

Abstract Background Blood plasma, one of the most studied liquid biopsies, contains various molecules that have biomarker potential for cancer detection, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA). As the vast majority of cell-free nucleic acids in circulation are non-cancerous, a laboratory workflow with a high detection sensitivity of tumor-derived nucleic acids is a prerequisite for precision oncology. One way to meet this requirement is by the combined analysis of cfDNA and cfRNA from the same liquid biopsy sample. So far, no study has systematically compared the performance of cfDNA and cfRNA co-purification to increase sensitivity. Results First, we set up a framework using digital PCR (dPCR) technology to quantify cfDNA and cfRNA from human blood plasma in order to compare cfDNA/cfRNA co-purification kit performance. To that end, we optimized two dPCR duplex assays, designed to quantify both cfDNA and cfRNA with the same assays, by ensuring that primers and probes are located within a highly abundant exon. Next, we applied our optimized workflow to evaluate the co-purification performance of two manual and two semi-automated methods over a range of plasma input volumes (0.06–4 mL). Some kits result in higher nucleic acid concentrations in the eluate, while consuming only half of the plasma volume. The combined nucleic acid quantification systematically results in higher nucleic acid concentrations as compared to a parallel quantification of cfDNA and cfRNA in the eluate. Conclusions We provide a framework to evaluate the performance of cfDNA/cfRNA co-purification kits and have tested two manual and two semi-automated co-purification kits in function of the available plasma input amount and the intended use of the nucleic acid eluate. We demonstrate that the combined quantification of cfDNA and cfRNA has a benefit compared to separate quantification. We foresee that the results of this study are instrumental for clinical applications to help increase mutation detection sensitivity, allowing improved disease detection and monitoring.

Objective: Hemophilia A (HA) is an X-linked recessive bleeding disease caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII). Available treatment to replenish the missing factor may not reach a good outcome for all patients because of potential complications that include the development of inhibitor antibodies directed against factor VIII. Therefore, the prevention of transmitting pathogenic mutations to the next generation is the best solution for this disease. Preimplantation genetic testing for a monogenic disorder (PGT-M) has become a standard method to prevent the transmission of monogenic heritable disease. The gold standard of the molecular technique used for PGT-M nowadays is the co-amplification of the polymorphic microsatellite linkage markers that use microsatellite DNA technique that overcomes the limitation of other methods. The important issue of this technique is the definition of markers that are specific for each allele on different loci. Each gene locus needs a characteristic design to allow accurate diagnosis that can be applied on PGT-M due to the limited quantity of DNA available. Here we present our study of four specific self-designed linked polymorphic markers applied on screening the embryos before implantation for hemophilia A families in Vietnam. Material and methods: In this study, we investigated the feasibility of application and diagnostic value of 4 STR loci (FXS1108, DXS9897, F8int22, DXS9901) in the intragenic or neighbouring regions of the F8 gene. 35 hemophilia A families were recruited for STR analysis to define at least two characteristic heterologous markers for each family and 12 cases of pre-implantation genetic testing (PGT-M) for carrier mothers were performed. Result: All 4 of these loci (FXS1108, DXS9897, F8int22, DXS9901) were found practical and useful for preimplantation genetic testing (PGT-M). All 12 cases of PGT-M using the method had informative STR results and correct diagnosis was achieved. 9 out of the 12 mothers (75%) were implanted with 1–2 thawed embryos after the biopsy resulting in the birth of 5 healthy babies (55%). Conclusion: We conclude that specific 4 STR markers for rapid pre-implantation genetic testing of hemophilia A can be successfully applied with high confidence and accuracy in clinical settings. The results of the study provide solid evidence confirming that the microsatellite DNA technique is a highly reliable method, suitable for hemophilia A families wishing to determine carrier status or having healthy babies.

In the context of colorectal cancer (CRC), circulating tumor DNA (ctDNA) is frequently used to monitor the minimal residual disease (MRD). ctDNA has become an excellent biomarker to predict which patients with CRC are likely to relapse due to the persistence of micrometastases. MRD diagnosis via analysis of ctDNA may allow much earlier detection of relapse compared with conventional diagnosis during follow-up. It should lead to an increased rate of curative-intended complete resection of an asymptomatic relapse. Besides, ctDNA can provide crucial information on whether and how intensively adjuvant or additive therapy should be administered. In the present case, analysis of ctDNA gave us a crucial hint to the use of more intensive diagnostics (MRI and Positron emission tomography–computed tomography PET-CT) which led to earlier detection of CRC relapse. Metastasis detected early are more likely to be completely resectable with curative intent. [ABSTRACT FROM AUTHOR]

The most common cause of mortality and disability globally right now is cholangiocarcinoma, one of the worst forms of cancer that may affect people. When cholangiocarcinoma develops, the DNA of the bile duct cells is altered. Cholangiocarcinoma claims the lives of about 7000 individuals annually. Women pass away less often than men. Asians have the greatest fatality rate. Following Whites (20%) and Asians (22%), African Americans (45%) saw the greatest increase in cholangiocarcinoma mortality between 2021 and 2022. For instance, 60–70% of cholangiocarcinoma patients have local infiltration or distant metastases, which makes them unable to receive a curative surgical procedure. Across the board, the median survival time is less than a year. Many researchers work hard to detect cholangiocarcinoma, but this is after the appearance of symptoms, which is late detection. If cholangiocarcinoma progression is detected at an earlier stage, then it will help doctors and patients in treatment. Therefore, an ensemble deep learning model (EDLM), which consists of three deep learning algorithms—long short-term model (LSTM), gated recurrent units (GRUs), and bi-directional LSTM (BLSTM)—is developed for the early identification of cholangiocarcinoma. Several tests are presented, such as a 10-fold cross-validation test (10-FCVT), an independent set test (IST), and a self-consistency test (SCT). Several statistical techniques are used to evaluate the proposed model, such as accuracy (Acc), sensitivity (Sn), specificity (Sp), and Matthew's correlation coefficient (MCC). There are 672 mutations in 45 distinct cholangiocarcinoma genes among the 516 human samples included in the proposed study. The IST has the highest Acc at 98%, outperforming all other validation approaches. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein receptor with intracellular tyrosine kinase activity. Mutations in the EGFR gene, including deletions in exon 19 and the mutation L858R, induce responsiveness of non-small cell lung cancer (NSCLC) to a group of drugs known as tyrosine kinase inhibitors. Here, we report the development of the CRISPR-based fluorescent reporter (CBFR) assay including a two-step strategy combining PCR amplification and Cas12a-driven cleavage to detect the delE746_A750 subtype of EGFR exon 19 deletions. Sensitivity and specificity of the CBFR assay were analyzed with different concentrations of fluorescence reporter and different amounts of PCR product. The results demonstrated that increasing the fluorescent reporter to 4 μM and the PCR product to 5 μl enhanced sensitivity. The CBFR assay could detect EGFR exon 19 deletion even with a frequency of 1% in samples. In clinical NSCLC samples, optimized CBFR assay enabled visual detection of the delE746_A750 subtype in less than 1 h. The CBFR assay provides a sensitive, specific, and simple strategy designed based on a straightforward and inexpensive process. We suggest that the CBFR assay could serve as a diagnostic approach to detect mutations, deletions, and pathogens in underequipped laboratories and promote personalized therapeutic approaches. [ABSTRACT FROM AUTHOR]

Abstract The objective of this work was to develop a method to create and validate CRISPR-Cas systems and different gRNAs in soybean (Glycine max) embryos. Two model genes were used for simple mutation with one gRNA or partial gene deletion with two guides. The gRNAs were inserted into the CRISPR transformation vectors by a type IIS restriction enzyme or by subcloning and inserting the promoter + gRNA2 in the final transformation vector using the classic restriction enzyme cloning method. The vectors were successfully constructed for one and two gRNAs. Agrobacterium-mediated transient transformation in soybean was carried out to test the quality of gRNAs and of the system itself (expression cassette). Simple mutation and gene deletion were detected in the embryos transformed after DNA enrichment by enzyme digestion followed by polymerase chain reaction and sequencing, which indicates that the CRISPR-Cas system and guides were working. This protocol can be used to accelerate CRISPR-based genome editing strategies for genetic transformation in soybean.

Low-level tumor somatic DNA mutations in tissue and liquid biopsies obtained from cancer patients can have profound implications for development of metastasis, prognosis, choice of treatment, follow-up, or early cancer detection. Unless detected, such low-frequency DNA alterations can misinform patient management decisions or become missed opportunities for personalized medicine. Next-generation sequencing technologies and digital-PCR can resolve low-level mutations but require access to specialized instrumentation, time, and resources. Enzymatic-based approaches to detection of low-level mutations provide a simple, straightforward, and affordable alternative to enrich and detect such alterations and is broadly available to low-resource laboratory settings. This review summarizes the traditional uses of enzymatic mutation detection and describes the latest exciting developments, potential, and applications with specific reference to the field of liquid biopsy in cancer. [ABSTRACT FROM AUTHOR]

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer's 3′-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template's background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences. [ABSTRACT FROM AUTHOR]

• NSCLC small biopsy exfoliates tumour cells of good cellularity in fixative media. • Cell pellet collection is encouraged while TBLB samples are being processed. • Residual cell pellet retention post a thoracic small biopsy aids ancillary testing. • Combining cell pellets with traditional sampling is beneficial in advanced NSCLC. Lung cancer tissue obtained using small biopsies are relatively fragile, leaving behind some tiny tissue fragments or cell clusters in the fixative medium that are difficult to collect for processing as a paraffin-embedded tissue block. Usually, the cellular component of the residual fixative medium is discarded as medical waste as per routine laboratory protocol. No protocol exists for utilizing the cellular component of the residual fixative medium after processing the tissue blocks to improve lung cancer ancillary testing. This study aimed to undercover the potential value of these samples for lung cancer diagnosis and targeted therapy development. A protocol was developed for cell pellet sample collection from the residual fixative medium of a transbronchial forceps lung biopsy sample. Tumour cell number and fraction in a paired cell pellet and matching formalin-fixed paraffin-embedded tissue section were evaluated from 324 non-smallcell lung carcinoma (NSCLC) cases. We defined the adequacy of the cell pellet for molecular analysis as ≥ 200 tumour cells and ≥ 10 % tumour cells. Real-time polymerase chain reaction and next-generation sequencing were performed on adequate cell pellet samples. We discovered that the fixative medium of most transbronchial forceps lung biopsy samples was enriched in tumour cells. Among 324 biopsy samples, 70 (21.6%) exhibited inadequate formalin-fixed paraffin-embedded tissue sections, whereas 53 (75.7%) yielded adequate cell pellet samples. Somatic mutations detected in the formalin-fixed paraffin-embedded tissue section samples were also detected in the matching cell pellets. Cell pellets collected from the fixative medium of thoracic small biopsies are a beneficial supplemental material for ancillary testing. Combined use of cell pellets with traditional tissue-based samples can enhance the detection rate of informative mutations in patients with advanced NSCLC. [ABSTRACT FROM AUTHOR]

Background: Blood plasma, one of the most studied liquid biopsies, contains various molecules that have biomarker potential for cancer detection, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA). As the vast majority of cell-free nucleic acids in circulation are non-cancerous, a laboratory workflow with a high detection sensitivity of tumor-derived nucleic acids is a prerequisite for precision oncology. One way to meet this requirement is by the combined analysis of cfDNA and cfRNA from the same liquid biopsy sample. So far, no study has systematically compared the performance of cfDNA and cfRNA co-purification to increase sensitivity. Results: First, we set up a framework using digital PCR (dPCR) technology to quantify cfDNA and cfRNA from human blood plasma in order to compare cfDNA/cfRNA co-purification kit performance. To that end, we optimized two dPCR duplex assays, designed to quantify both cfDNA and cfRNA with the same assays, by ensuring that primers and probes are located within a highly abundant exon. Next, we applied our optimized workflow to evaluate the co-purification performance of two manual and two semi-automated methods over a range of plasma input volumes (0.06–4 mL). Some kits result in higher nucleic acid concentrations in the eluate, while consuming only half of the plasma volume. The combined nucleic acid quantification systematically results in higher nucleic acid concentrations as compared to a parallel quantification of cfDNA and cfRNA in the eluate. Conclusions: We provide a framework to evaluate the performance of cfDNA/cfRNA co-purification kits and have tested two manual and two semi-automated co-purification kits in function of the available plasma input amount and the intended use of the nucleic acid eluate. We demonstrate that the combined quantification of cfDNA and cfRNA has a benefit compared to separate quantification. We foresee that the results of this study are instrumental for clinical applications to help increase mutation detection sensitivity, allowing improved disease detection and monitoring. [ABSTRACT FROM AUTHOR]

Typical grassland is the core of the Mongolian Plateau grassland belt, and is also an important ecological barrier in the north of China. It is of great significance to explore the real-time changes in grassland climate for the prevention and control of climate disasters, and for ecological protection. In this study, the spatial and temporal variation of temperature, precipitation and maximum wind speed in typical Mongolian Plateau grassland were studied using observation data from 16 meteorological stations from 1978 to 2020, using the linear trend method, cumulative anomaly method, Mann-Kendall test, sliding t-test and Morlet wavelet analysis. The results show that: (1) The typical grassland temperature has been increasing at a rate of 0.4 °C/10a (p < 0.001) over the past 40 years, with the most significant warming in spring and summer; a sudden change from low to high temperature occurred in 1992; the annual average temperature gradually increased from northeast to southwest, with significant warming in the southwest. (2) Annual precipitation decreased slightly at a rate of −2.39 mm/10a, with the most significant decrease in summer precipitation; a sudden change from more to less precipitation occurred in 1998; spatially, precipitation decreased gradually from east to west, with significant moisture reduction in its northern part. (3) The maximum wind speed decreased significantly at a rate of −0.33m/s/10a (p < 0.001), with the most pronounced decrease in spring; the maximum wind speed changed abruptly from strong to weak around 1991; spatially, the annual average maximum wind speed decreased gradually from northwest to southeast and northeast, with the most pronounced decrease in the south and northeast. (4) The wavelet analysis shows that the typical grassland area will still be in a warm, low-rainfall and weak-wind stage in the coming years. Using the above analysis, the typical grassland climate of the Mongolian Plateau has shown a clear trend of warm and dry, weak wind in the past 40 years. [ABSTRACT FROM AUTHOR]

The sequential labeling model is commonly used for time series or sequence data where each instance label is classified using previous instance label. In this work, a sequential labeling model is proposed as a new approach to detect the type and index mutations simultaneously, using DNA sequences from lung cancer study cases. The methods used are One Dimensional Convolutional Neural Network (1D-CNN), Bidirectional Long Short-Term Memory (BiLSTM), and Bidirectional Gated Recurrent Unit (Bi-GRU). Each nucleotide in the patient’s DNA sequence is classified as either normal or with a certain type of mutation in which case, its index mutation is predicted. The mutation types detected are either substitution, insertion, deletion, or delins (deletion insertion) mutations. Based on the experiments that were conducted using EGFR gene, BiLSTM and Bi-GRU displayed better performance and were more stable than 1D-CNN. Further tests were carried out on the TP53, KRAS, CTNNB1, SMARCA4, CDKN2A, PTPRD, BRAF, ERBB2, and PTPRT gene. The proposed model reports F1-scores of 0.9596, and 0.9612 using Bi-GRU and BiLSTM, respectively. Based on the results the model can successfully detect the type and index mutations in the DNA sequence more accurately and faster without the need for other supporting data and tools, and does not require re-alignment to reference sequences. This will greatly facilitate the user in detecting type and index mutations faster by entering only the DNA sequence.

Mutation detecting is a routine work for sequencing data analysis and the trading of existing tools often involves the combinations of signals on a set of overlapped sequencing reads. However, the subclonal mutations, which are reported to contribute to tumor recurrence and metastasis, are sometimes eliminated by existing signals. When the clonal proportion decreases, signals often present ambiguous, while complicated interactions among signals break the IID assumption for most of the machine learning models. Although the mutation callers could lower the thresholds, false positives are significantly introduced. The main aim here was to detect the subclonal mutations with high specificity from the scenario of ambiguous sample purities or clonal proportions. We proposed a novel machine learning approach for filtering false positive calls to accurately detect mutations with wide spectrum subclonal proportion. We have carried out a series of experiments on both simulated and real datasets, and compared to several state-of-art approaches, including freebayes, MuTect2, Sentieon and SiNVICT. The results demonstrated that the proposed method adapts well to different diluted sequencing signals and can significantly reduce the false positive when detecting subclonal mutations. The codes have been uploaded at https://github.com/TrinaZ/TL-fpFilter for academic usage only. [ABSTRACT FROM AUTHOR]

Genes are composed of DNA and each gene has a specific sequence. Recombination or replication within the gene base ends in a permanent change in the nucleotide collection in a DNA called mutation and some mutations can lead to cancer. Breast adenocarcinoma starts in secretary cells. Breast adenocarcinoma is the most common of all cancers that occur in women. According to a survey within the United States of America, there are more than 282,000 breast adenocarcinoma patients registered each 12 months, and most of them are women. Recognition of cancer in its early stages saves many lives. A proposed framework is developed for the early detection of breast adenocarcinoma using an ensemble learning technique with multiple deep learning algorithms, specifically: Long Short-Term Memory (LSTM), Gated Recurrent Units (GRU), and Bi-directional LSTM. There are 99 types of driver genes involved in breast adenocarcinoma. This study uses a dataset of 4127 samples including men and women taken from more than 12 cohorts of cancer detection institutes. The dataset encompasses a total of 6170 mutations that occur in 99 genes. On these gene sequences, different algorithms are applied for feature extraction. Three types of testing techniques including independent set testing, self-consistency testing, and a 10-fold cross-validation test is applied to validate and test the learning approaches. Subsequently, multiple deep learning approaches such as LSTM, GRU, and bi-directional LSTM algorithms are applied. Several evaluation metrics are enumerated for the validation of results including accuracy, sensitivity, specificity, Mathew's correlation coefficient, area under the curve, training loss, precision, recall, F1 score, and Cohen's kappa while the values obtained are 99.57, 99.50, 99.63, 0.99, 1.0, 0.2027, 99.57, 99.57, 99.57, and 99.14 respectively. [ABSTRACT FROM AUTHOR]

Purpose: The gold standard for identification of post-zygotic variants (PZVs) is droplet digital polymerase chain reaction or high-depth sequencing across multiple tissues types. These approaches are yet to be systematically implemented for monogenic disorders. We developed PZV detection pipelines for correct classification of de novo variants., Method: Our pipelines detect PZV in parents (gonosomal mosaicism [pGoM]) and children (somatic mosaicism, "M3"). We applied them to research exome sequencing (ES) data from the Australian Cerebral Palsy Biobank (n = 145 trios) and Simons Simplex Collection (n = 405 families). Candidate mosaic variants were validated using deep amplicon sequencing or droplet digital polymerase chain reaction., Results: 69.2% (M3 trio ), 63.9% (M3 single ), and 92.7% (pGoM) of detected variants were validated, with 48.6%, 56.7%, and 26.2% of variants, respectively, meeting strict criteria for mosaicism. In the Australian Cerebral Palsy Biobank, 16.6% of probands and 20.7% of parents had at least 1 true-positive somatic or pGoM variant, respectively. A large proportion of PZVs detected in Simons Simplex Collection parents (79.8%) and child (94.5%) were not previously reported. We reclassified 3.7% to 8.0% of germline de novo variants as mosaic., Conclusion: Many PZVs were incorrectly classified as germline variants or missed by previous approaches. Systematic application of our pipelines could increase genetic diagnostic rate, improve estimates of recurrence risk in families, and benefit novel disease gene identification., Competing Interests: Conflict of Interest The authors declare that they have no competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract We develop the Oncogene Concatenated Enriched Amplicon Nanopore Sequencing (OCEANS) method, in which variants with low variant allele frequency (VAFs) are amplified and subsequently concatenated for Nanopore Sequencing. OCEANS allows accurate detection of somatic mutations with VAF limits of detection between 0.05 and 1%. We construct 4 distinct multi-gene OCEANS panels targeting recurrent mutations in acute myeloid leukemia, melanoma, non-small- cell lung cancer, and hepatocellular carcinoma and validate them on clinical samples. By demonstrating detection of low VAF single nucleotide variant mutations using Nanopore Sequencing, OCEANS is poised to enable same-day clinical sequencing panels.

Aiming at the mutation characteristics of runoff sequence under changing environment, this paper adopted the multi-scale linear fitting method to detect the mutation point of annual runoff sequence of four hydrological stations on the upper reaches of Fenhe River and detected the mutation precursor signals based on the evolution law of recovery rate, recovery force, variance and autocorrelation coefficient of the system. The results show that the mutation point in the annual runoff sequence of Shangjingyou Station, Fenhe Reservoir Station, Zhais-hang Station and Lancun Station are in 1975, 1966, 1966 and 1966 respectively. The runoff has been reduced by 22.81%, 19.75%, 24.68% and 31.14% respectively. The mutation precursor signals are detected in 1973, 1958, 1958 and 1958 respectively based on the recovery rate and recovery force of the system. The mutation precursor signals are detected in 1968, 1960, 1960 and 1960 respectively based on the variance and autocorrelation coefficient. The mutation precursor signals are all premised within ten years of the mutation year, which has a reliable early warning effect on the mutation. [ABSTRACT FROM AUTHOR]

Liquid biopsy technology is non-invasive and convenient, and is currently an emerging technology for cancer screening. Among them, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 12a (Cas12a) based nucleic acid detection technology has the advantages of high sensitivity, rapidity, and easy operation. However, CRISPR-Cas12a does not discriminate single-base mismatches of targets well enough to meet the needs of clinical detection. Herein, we developed the Triple-Mismatch Differentiating (TMD) assay. This assay amplified the small thermodynamic difference in mismatches at one site at the level of CRISPR-Cas12a activation to a significant thermodynamic difference at three sites at both the level of CRISPR-Cas12a activation and trans-cleavage, which greatly improves the ability of CRISPR-Cas12a to discriminate between base mismatches. Our manipulation greatly improved the specificity of the CRISPR-Cas12a system while maintaining its inherent sensitivity and simplicity, increasing the detection limit to 0.0001%. When testing samples from pancreatic cancer patients, our results were highly consistent with NGS sequencing results. We believe that the TMD assay will provide a new technology for early cancer detection and will be widely used in the clinical practice. [Display omitted] [ABSTRACT FROM AUTHOR]

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10-8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs.

Copyright of Polish Otorhinolaryngological Review / Polski Przegląd Otorynolaryngologiczny (Index Copernicus) is the property of Index Copernicus International and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Chronic renal disease associated with X-linked Alport syndrome (XLAS) is relatively rare. However, due to the lack of specificity in the pathologic and clinical manifestations of the disease, it is easy to be misdiagnosed. In this study, we included three Chinese families with XLAS and used targeted NGS to find gene variants. In family X1, the 36-year-old male proband had hematuria, massive proteinuria, sensorineural deafness and ESRD at 33. In silico prediction showed the novel c.1424-4C > G variant reduced the score of the normal 3' splice site from 0.47 to 0.00 (according to BDGP). Transcriptional analysis from his peripheral blood cells indicated that it caused the insertion of an amino acid [p.(Lys474_Gly475insVal)]. In family X2, the proband was a 32-year-old male, who had hematuria, proteinuria, hypertension, hearing loss and progressed into ESRD at 30 years. He carried a novel missense variant c.2777G > T p.(Gly926Val). In family X3, the proband, a 16-year-old male, had hematuria, massive proteinuria, sensorineural deafness and ESRD; the results of renal pathological findings were consistent with AS. He carried a novel variant c.4529-2A > T, so did his mother with ESRD and probable XLAS. Bioinformatic analysis with BDGP showed that it abolished the acceptor site from 0.83 to 0.00. RT-PCR analysis from his kidney tissue indicated that it caused exon 50 skipping and exon 50 skipping along with inserting a cryptic exon derived from intron 49 p.[Gly1510Aspfs*11, Gly1510Alafs*35]. Another novel missense variant c.1552G > A p.(Gly518Arg) was identified in his mother and his aunt. No skewed X-chromosome inactivation was involved in these two female patients. In conclusion, four novel variants in COL4A5 were identified and transcriptional analysis is essential to investigate the pathogenicity of intronic variants. Thus we found a rare event in a female patient with XLAS caused by two COL4A5 variants in trans. [ABSTRACT FROM AUTHOR]

Chronic renal disease associated with X-linked Alport syndrome (XLAS) is relatively rare. However, due to the lack of specificity in the pathologic and clinical manifestations of the disease, it is easy to be misdiagnosed. In this study, we included three Chinese families with XLAS and used targeted NGS to find gene variants. In family X1, the 36-year-old male proband had hematuria, massive proteinuria, sensorineural deafness and ESRD at 33. In silico prediction showed the novel c.1424-4C > G variant reduced the score of the normal 3’ splice site from 0.47 to 0.00 (according to BDGP). Transcriptional analysis from his peripheral blood cells indicated that it caused the insertion of an amino acid [p.(Lys474_Gly475insVal)]. In family X2, the proband was a 32-year-old male, who had hematuria, proteinuria, hypertension, hearing loss and progressed into ESRD at 30 years. He carried a novel missense variant c.2777G > T p.(Gly926Val). In family X3, the proband, a 16-year-old male, had hematuria, massive proteinuria, sensorineural deafness and ESRD; the results of renal pathological findings were consistent with AS. He carried a novel variant c.4529-2A > T, so did his mother with ESRD and probable XLAS. Bioinformatic analysis with BDGP showed that it abolished the acceptor site from 0.83 to 0.00. RT-PCR analysis from his kidney tissue indicated that it caused exon 50 skipping and exon 50 skipping along with inserting a cryptic exon derived from intron 49 p.[Gly1510Aspfs*11, Gly1510Alafs*35]. Another novel missense variant c.1552G > A p.(Gly518Arg) was identified in his mother and his aunt. No skewed X-chromosome inactivation was involved in these two female patients. In conclusion, four novel variants in COL4A5 were identified and transcriptional analysis is essential to investigate the pathogenicity of intronic variants. Thus we found a rare event in a female patient with XLAS caused by two COL4A5 variants in trans.

The RAS family of small GTPases is mutated in roughly a fifth of human cancers. Hotspot point mutations at codons G12, G13, and Q61 account for 95% of all these mutations, which are well established to render the encoded proteins oncogenic. In humans, this family comprises three genes: HRAS, NRAS, and KRAS. Accumulating evidence argues that oncogenic RAS point mutations may be initiating, as they are often truncal in human tumours and capable of inducing tumorigenesis in mice. As such, there is great interest in detecting oncogenic mutation in the RAS genes to understand the origins of cancer, as well as for early detection purposes. To this end, we previously adapted the microbial ultra-sensitive Maximum Depth Sequencing (MDS) assay for the murine Kras gene, which was capable of detecting oncogenic mutations in the tissues of mice days after carcinogen exposure, essentially capturing the very first step in tumour initiation. Given this, we report here the adaption and details of this assay to detect mutations in a human KRAS sequence at an analytic sensitivity of one mutation in a million independently barcoded templates. This humanized version of MDS can thus be exploited to detect oncogenic mutations in KRAS at an incredible sensitivity and modified for the same purpose for the other RAS genes. [ABSTRACT FROM AUTHOR]

Background: Phenylketonuria (PKU) is the most common aminoacidopathy with an autosomal recessive inheritance pattern. A global PKU prevalence is estimated about 6.002 in 100,000 newborns. In Iran, the prevalence of PKU is estimated at about 1 in 4,698, and it shows an increasing trend from north (0.0015%) to south (0.02%) of the country. Untreated PKU causes mental retardation, microcephaly, and seizure. PAH gene mutations located at chromosome 12q23 are responsible for the classical type of this disease. The spectrum of PAH mutations is varied in different ethnicities and different parts of the world. The aim of this study was to investigate the frequency of PAH mutation in the Mazandaran province, which could be useful for genetic counseling and prenatal diagnosis. Methods: A total of 66 individuals from 33 families from two provinces (9 families from Golestan and 24 families from Mazandaran) from north of Iran participated in this study. After genomic DNA extraction, PAH gene analysis was carried out using DNA sequencing of both coding and non-coding regions by ABI 3130XL genetic analyzer. Results: Twenty-six different mutations were identified in the PAH gene in this study. Four mutations including IVS10-11 (c.1066-11G>A), c.727C>T (p.Arg243X), c.898G>T (p.Ala300Ser), and c.601C>T (p.His201Tyr) were the most common mutations with 37.48% frequency in Mazandaran province. Most frequent mutations in Golestan province were IVSI0-11 (c.1066-11G>A), c.722delG (p.Arg241fs), c.842C>T (p.Pro281Leu), and IVSII+5 (G>A) with frequency 58.57%. Conclusions: The results from the present study verify heterogeneity of the PAH gene and may help to diagnose tests for carrier detection and prenatal diagnosis of the PKU disease in Iranian population. [ABSTRACT FROM AUTHOR]

Abstract Next-generation sequencing technologies have enabled a dramatic expansion of clinical genetic testing both for inherited conditions and diseases such as cancer. Accurate variant calling in NGS data is a critical step upon which virtually all downstream analysis and interpretation processes rely. Just as NGS technologies have evolved considerably over the past 10 years, so too have the software tools and approaches for detecting sequence variants in clinical samples. In this review, I discuss the current best practices for variant calling in clinical sequencing studies, with a particular emphasis on trio sequencing for inherited disorders and somatic mutation detection in cancer patients. I describe the relative strengths and weaknesses of panel, exome, and whole-genome sequencing for variant detection. Recommended tools and strategies for calling variants of different classes are also provided, along with guidance on variant review, validation, and benchmarking to ensure optimal performance. Although NGS technologies are continually evolving, and new capabilities (such as long-read single-molecule sequencing) are emerging, the “best practice” principles in this review should be relevant to clinical variant calling in the long term.

Abstract Background Stargardt disease 1 (STGD1; MIM 248200) is a monogenic form of autosomal recessive genetic disease caused by mutation in ABCA4. This gene has a major role in hydrolyzing N-retinylidene-phosphatidylethanolamine to all-trans-retinal and phosphatidylethanolamine. The purpose of this study is to identify the frequency of putative disease-causing mutations associated with Stargardt disease in a South Indian population. Methods A total of 28 clinically diagnosed Stargardt-like phenotype patients were recruited from south India. Ophthalmic examination of all patients was carefully carried out by a retina specialist based on the stages of fundus imaging and ERG grouping. Genetic analysis of ABCA4 was performed for all patients using Sanger sequencing and clinical exome sequencing. Results This study identified disease-causing mutations in ABCA4 in 75% (21/28) of patients, 7% (2/28) exhibited benign variants and 18% (5/28) were negative for the disease-causing mutation. Conclusion This is the first study describing the genetic association of ABCA4 disease-causing mutation in South Indian Stargardt 1 patients (STGD1). Our findings highlighted the presence of two novel missense mutations and an (in/del, single base pair deletion & splice variant) in ABCA4. However, genetic heterogeneity in ABCA4 mutants requires a larger sample size to establish a true correlation with clinical phenotype.

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer’s 3′-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template’s background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

Vegetation is the main body of the terrestrial ecosystem and is a significant indicator of environmental changes in the regional ecosystem. As an essential link connecting South Asia and Southeast Asia, the Lancang-Mekong River Basin(LMRB) can provide essential data support and a decision-making basis for the assessment of terrestrial ecosystem environmental changes and the research and management of hydrology and water resources in the basin by monitoring changes in its vegetation cover. This study takes the Lancang-Mekong River Basin as the study area, and employs the Sen slope estimation, Mann–Kendall test, and Hurst exponent based on the MODIS NDVI data from 2000 to 2021 to study the spatial and temporal evolution trend and future sustainability of its NDVI. Besides, the nonlinear characteristics such as mutation type and mutation year are detected and analyzed using the BFAST01 method. Results demonstrated that: (1) In the past 22 years, the NDVI of the Lancang-Mekong River Basin generally exhibited a fluctuating upward trend, and the NDVI value in 2021 was the largest, which was 0.825, showing an increase of 4.29% compared with 2000. However, the increase rate was different: China has the most considerable NDVI growth rate of 7.25%, followed by Thailand with an increase of 7.21%, Myanmar and Laos as the third, while Cambodia and Vietnam have relatively stable vegetation changes. The overall performance of NDVI is high in the south and low in the north, and is dominated by high and relatively high vegetation coverage, of which the area with vegetation coverage exceeding 0.8 accounts for 62%. (2) The Sen-MK trend showed that from 2000 to 2021, the area where the vegetation coverage in the basin showed a trend of increase and decrease accounted for 66.59% and 18.88%, respectively. The Hurst exponent indicated that the areas where NDVI will continue to increase, decrease, and remain unchanged in the future account for 60.14%, 25.29%, and 14.53%, respectively, and the future development trend of NDVI is uncertain, accounting for 0.04%. Thus, more attention should be paid to areas with a descending future development trend. (3) BFAST01 detected eight NDVI mutation types in the Lancang-Mekong River Basin over the past 22 years. The mutations mainly occurred in 2002–2018, while 2002–2004 and 2014–2018 were the most frequent periods of breakpoints. The mutation type of “interruption: increase with negative break” was changed the most during this period, which accounts for 36.54%, and the smallest was “monotonic decrease (with negative break)”, which only accounts for 0.65%. This research demonstrates that combining the conventional trend analysis method with the BFAST mutation test can more accurately analyze the spatiotemporal variation and nonlinear mutation of NDVI, thus providing a scientific reference to develop ecological environment-related work.

Reverse genetics approaches are critical for uncovering complex biological processes and genetic engineering. Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) system (CRISPR/Cas9) has been widely used for generating mutants in a large number of species. The Nile tilapia (Oreochromis niloticus), a gonochoristic teleost with XX/XY sex determination system, is a good model for studying fish sex determination and reproduction. Here, we describe a detailed procedure for gene mutation in tilapia by CRISPR/Cas9. This protocol covers selection of target sites, in vitro RNA transcription, artificial insemination and microinjection of one cell stage embryos. We also provide details for detection of somatic and germline transmitted mutations, fast establishment of mutant lines and discuss some practical advices. This protocol will facilitate broader applications of CRISPR/Cas9 in studies of tilapia as well as other aquaculture fishes. [ABSTRACT FROM AUTHOR]

Since the emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in December 2019, the scientific community has been sharing data on epidemiology, diagnostic methods, and whole-genomic sequences almost in real time. The latter have already facilitated phylogenetic analyses, transmission chain tracking, protein modeling, the identification of possible therapeutic targets, timely risk assessment, and identification of novel variants. We have established and evaluated an amplification-based approach for whole-genome sequencing of SARS-CoV-2. It can be used on the miniature-sized and field-deployable sequencing device Oxford Nanopore MinION, with sequencing library preparation time of 10 min. We show that the generation of 50,000 total reads per sample is sufficient for a near complete coverage (>90%) of the SARS-CoV-2 genome directly from patient samples even if virus concentration is low (Ct 35, corresponding to approximately 5 genome copies per reaction). For patient samples with high viral load (Ct 18–24), generation of 50,000 reads in 1–2 h was shown to be sufficient for a genome coverage of >90%. Comparison to Illumina data reveals an accuracy that suffices to identify virus mutants. AmpliCoV can be applied whenever sequence information on SARS-CoV-2 is required rapidly, for instance for the identification of circulating virus mutants.

Background: Thalassemia is one of the most prevalent worldwide autosomal recessive disorders characterized by a great molecular and clinical expression heterogeneity. Alpha and beta-thalassemia are the main two types observed in case of mutations affecting alpha and beta-globin genes respectively. Delta-thalassemia is noted when mutations occur on the delta-globin gene. In Tunisia, β-thalassemia prevalence is estimated at 2.21% of carriers. However, few reports investigated the delta-globin gene. Objectives: In this work, we aimed to perform a molecular study to help define the molecular spectrum of δ-thalassemia mutations in Tunisia. Patients and methods: The study involved 7558 patients among whom we selected 179 individuals with abnormal HbA2 values or fractions. Hemoglobin analysis was performed using Capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). DNA sequencing was performed on ABI prism 310 Genetic Analyzer Applied Biosystems. CUPSAT (Cologne University Protein Stability Analysis Tool) was used for the prediction of protein stability changes upon missense mutations and mutants were modeled via DeepView—SwissPdbViewer and POV-Ray softwares for molecular dynamics simulation studies. Results: We identified four mutations: HbA2-Yialousa described for the first time in Tunisia (in 72.72% of cases) and 3 mutations reported for the first time in the world: (i) c.442 T > C Stop147Arg ext 15aa-stop observed in 18.18% of cases, (ii) c.187 G > C (Ala62Pro) noted in 4.54% of cases and (iii) c.93-1G > C found in 4.54% of cases. Conclusion: Our data provide genetic basis that would be especially useful in screening for beta-thalassemia trait during delta-beta thalassemia associations. [ABSTRACT FROM AUTHOR]

Circulating tumor DNA (ctDNA), a fraction of cell-free DNA (cfDNA) in the circulatory system, is released from tumor cells and thus carries tumor-specific genetic signatures. Using blood-derived ctDNA to detect somatic mutations has shown great value in guiding cancer targeted therapy. Isolation and detection efficiencies are the key factors affecting the performance of ctDNA detection. To optimize and standardize our clinical practice, in this study, we analyzed the isolation efficiency of four commercial cfDNA purification kits: QIAamp circulating nucleic acid kit, AmoyDx® Circulating DNA kits, Microdiag® circulating DNA isolation kit, and MagMAX cell-free DNA isolation kit; and the detection efficiency of two mainstream domestic EGFR gene mutation detection kits: MicroDiag EGFR gene mutation detection kit and Fluorometric real-time PCR Detection Kit for the analysis of EGFR gene mutations. Reference materials and plasma samples collected from lung cancer patients and healthy volunteers were used for the analysis. Our results showed that QIAamp circulating nucleic acid kit and Microdiag® circulating DNA kit had the highest recovery rate (up to 21.25 ng/mL) for short DNA fragments of about 173 bp which is the peak length of ctDNA. For ctDNA detection, the MicroDiag®EGFR gene mutation detection kit showed the highest detection rate and sensitivity for detecting EGFR mutations at a mutant frequency of 0.5%. This work provides a reliable choice of commercial kits for the clinical application of ctDNA. [ABSTRACT FROM AUTHOR]