Next-generation Sequencing Methods Research Articles

A review of multiple diagnostic approaches in the undiagnosed diseases network to identify inherited metabolic diseases.

The number of known inherited metabolic diseases (IMDs) has been expanding, and the rate of diagnosis is improving with the development of innovative approaches including next generation sequencing (NGS). However, a substantial proportion of IMDs remain undetected by traditional diagnostic approaches. We aim to highlight the spectrum of IMDs diagnosed by the Undiagnosed Diseases Network (UDN) and to learn from the UDN diagnostic processes that were able to detect IMDs. We conducted a retrospective analysis of 757 UDN participants diagnosed from 2015 until 2023 using the cohort database, which were divided into a cohort with IMDs (n = 194; 27%) and a cohort whose phenotypes were not explained by an IMD (n = 563; 73%), based on the International Classification of Inherited Metabolic Disorders (ICIMD). Then, we divided the causes of the metabolic 194 diagnoses into seven groups that included all the ICIMD categories. We inspected which clinical and laboratory approaches contributed to a final UDN diagnosis. We also present a UDN case example from each group to highlight the diagnostic yields that resulted from combining newer diagnostic approaches in the UDN and illustrate potential pitfalls of current NGS methods. These 194 cases of IMDs included examples from 21/25 (84%) of the ICIMD categories. Of the UDN subjects 164/194 (85%) were diagnosed with IMDs through NGS. The spectrum of IMDs detected in the UDN cohort is large and growing and appropriate use of newer multiple diagnostic approaches should further increase diagnosis of IMDs that are presently missed by the traditional laboratory screening methods.

A comprehensive study of common and rare genetic variants in spermatogenesis-related loci identifies new risk factors for idiopathic severe spermatogenic failure

Abstract STUDY QUESTION Can genome-wide genotyping data be analysed using a hypothesis-driven approach to enhance the understanding of the genetic basis of severe spermatogenic failure (SPGF) in male infertility? SUMMARY ANSWER Our findings revealed a significant association between SPGF and the SHOC1 gene, and identified three novel genes (PCSK4, AP3B1, and DLK1) along with 32 potentially pathogenic rare variants in 30 genes that contribute to this condition. WHAT IS KNOWN ALREADY SPGF is a major cause of male infertility, often with an unknown aetiology. SPGF can be due to either multifactorial causes, including both common genetic variants in multiple genes and environmental factors, or highly damaging rare variants. Next-generation sequencing (NGS) methods are useful for identifying rare mutations that explain monogenic forms of SPGF. Genome-wide association studies (GWAS) have become essential approaches for deciphering the intricate genetic landscape of complex diseases, offering a cost-effective and rapid means to genotype millions of genetic variants. Novel methods have demonstrated that GWAS datasets can be used to infer rare coding variants that are causal for male infertility phenotypes. However, this approach has not been previously applied to characterize the genetic component of a whole case-control cohort. STUDY DESIGN, SIZE, DURATION We employed a hypothesis-driven approach focusing on all genetic variation identified, using a GWAS platform and subsequent genotype imputation, encompassing over 6 million polymorphisms and a total of 1571 SPGF patients and 2431 controls. Both common (minor allele frequency, MAF > 0.01) and rare (MAF < 0.01) variants were investigated within a total of 1797 loci with a reported role in spermatogenesis. This gene panel was meticulously assembled through comprehensive searches in the literature and various databases focused on male infertility genetics. PARTICIPANTS/MATERIALS, SETTING, METHODS This study involved a European cohort using previously and newly generated data. Our analysis consisted of three independent methods: (1) variant-wise association analyses using logistic regression models, (2) gene-wise association analyses using combined multivariate and collapsing (CMC) burden tests, and (3) identification and characterisation of highly damaging rare coding variants showing homozygosity only in SPGF patients. MAIN RESULTS AND THE ROLE OF CHANCE The variant-wise analyses revealed an association between SPGF and SHOC1-rs12347237 (p = 4.15E-06, OR = 2.66), which was likely explained by an altered binding affinity of key transcription factors in regulatory regions and the disruptive effect of coding variants within the gene. Three additional genes (PCSK4, AP3B1, and DLK1) were identified as novel relevant players in human male infertility using the gene-wise burden test approach (p < 5.56E-04). Furthermore, we linked a total of 32 potentially pathogenic and recessive coding variants of the selected genes to 35 different cases. LARGE SCALE DATA Publicly available via GWAS catalog (Accession number: GCST90239721). LIMITATIONS, REASONS FOR CAUTION The analysis of low-frequency variants presents challenges in achieving sufficient statistical power to detect genetic associations. Consequently, independent studies with larger sample sizes are essential to replicate our results. Additionally, the specific roles of the identified variants in the pathogenic mechanisms of SPGF should be assessed through functional experiments. WIDER IMPLICATIONS OF THE FINDINGS Our findings highlight the benefit of using GWAS genotyping to screen for both common and rare variants potentially implicated in idiopathic cases of SPGF, whether due to complex or monogenic causes. The discovery of novel genetic risk factors for SPGF and the elucidation of the underlying genetic causes provide new perspectives for personalized medicine and reproductive counselling. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Spanish Ministry of Science and Innovation through the Spanish National Plan for Scientific and Technical Research and Innovation (PID2020-120157RB-I00) and the Andalusian Government through the research projects of “Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)” (ref. PY20_00212) and “Proyectos de Investigación aplicada FEDER-UGR 2023” (ref. C-CTS-273-UGR23). SGM was funded by the previously mentioned projects (ref. PY20_00212 and PID2020-120157RB-I00). AGJ was funded by MCIN/AEI/10.13039/501100011033 and FSE “El FSE invierte en tu futuro” (grant ref. FPU20/02926). IPATIMUP integrates the i3S Research Unit, which is partially supported by the Portuguese Foundation for Science and Technology (FCT), financed by the European Social Funds (COMPETE-FEDER) and National Funds (projects PEstC/SAU/LA0003/2013 and POCI-01-0145-FEDER-007274). SS is supported by FCT funds (10.54499/DL57/2016/CP1363/CT0019), ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, and is also partially supported by the Portuguese Foundation for Science and Technology (UIDP/00009/2020 and UIDB/00009/2020). SLarriba received support from Instituto de Salud Carlos III (grant: DTS18/00101], co-funded by FEDER funds/European Regional Development Fund (ERDF)-a way to build Europe-), and from “Generalitat de Catalunya” (grant 2021SGR052). SLarriba is also sponsored by the “Researchers Consolidation Program” from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES09/020). All authors declare no conflict of interest related to this study.

Next-generation sequencing in diagnosis of monogenic cholestatic liver disorders: A single-center experience

Introduction Cholestasis in childhood is a rare clinical condition, yet a definitive diagnosis is crucial for initiating treatment of these curable diseases and preventing related morbidity and mortality. The most common cause of infant cholestasis is biliary atresia (25-40%), followed by monogenic cholestatic diseases (25%), metabolic diseases (20%), and cryptogenic cholestasis. This study focuses on assessing the clinical utility of next-generation sequencing (NGS) panels, including clinical exome sequencing and whole exome sequencing, in diagnosing cholestatic diseases when the etiology cannot be elucidated through conventional methods. Materials and Methods We conducted a retrospective examination of pediatric patients who sought care at a single-center pediatric gastroenterology department between August 2020 and March 2022, and were diagnosed with cholestasis. A total of 36 patients underwent a thorough investigation to rule out infectious, toxic, metabolic, structural, chromosomal, and endocrine causes. Patients whose diagnoses were established through traditional investigations were excluded from the study. The remaining 14 patients underwent either whole exome sequencing or targeted next-generation sequencing methods. Results A definitive diagnosis was achieved for 12 patients, while two patients remained undiagnosed despite comprehensive genetic examinations. The most commonly encountered diseases in this cohort were progressive familial intrahepatic cholestasis, linked to mutations in the ABCB11, ATP8B1, and TJP2 genes, as well as Dubin-Johnson syndrome associated with ABCC2 mutations. Next-generation sequencing demonstrated a diagnostic accuracy of 85.7% in patients for whom a diagnosis could not be established through extensive traditional workup. Conclusion Next-generation sequencing emerges as a valuable diagnostic tool in cases of cholestasis where traditional methods fall short in providing a definitive diagnosis. Moreover, our study unveiled three previously undocumented variants in the ABCB11 [c.1165G>C; p.(Ala389Pro) and c.783+1G>A] and ABCC2 [c.4246_4247del; p.(Lys1416ValfsTer46)] genes.

Abstract A019: Analytical and clinical validation of HPV-SEQ, an NGS-based liquid biopsy platform for detection and quantification of human papilloma virus circulating tumor DNA

Abstract Human papillomavirus (HPV) infection is the primary causative factor driving the rising incidence of oropharyngeal squamous cell carcinoma (OPSCC). High-risk HPV16 and HPV18 strains are responsible for the majority of HPV related OPSCC cases, with ∼90% and 3-5% respectively. As HPV positivity is associated with improved treatment response and prognosis, correct determination of HPV status is critical for risk stratification and treatment management. While diagnostic tumor biopsy remains a useful tool for HPV profiling, the drawbacks of this approach include pain and discomfort caused by its invasive nature, as well as inability to repeat multiple serial biopsies over time. Noninvasive detection of circulating tumor (ct)HPV-DNA in plasma has emerged as an attractive investigative strategy for early disease diagnosis, real-time response monitoring, and cancer surveillance. However, to date, there is no standard or routine liquid biopsy-based HPV testing for patients with OPSCC. The accuracy of HPV detection in plasma depends on the metrics of the specific method used. Comparison of the most common approaches for detection of ctHPV-DNA shows that next-generation sequencing (NGS)-based assays are superior to that of qPCR and ddPCR, especially in patients with low disease burden. Furthermore, as the majority of current liquid biopsy tests are designed to detect viral E6 and E7 genes, it may comprise a potential challenge for detection of patient-derived ctHPV-DNA among individuals treated with therapeutic vaccines expressing non-oncogenic E6/E7 constructs, actively evaluated in clinical trial settings. To this end, we have developed HPV-SEQ, a novel NGS method designed to achieve maximum sensitivity for HPV16/18 L1 gene in DNA libraries prepared from plasma of OPSCC patients. Here we describe the thorough analytical characterization and clinical validation studies of this lab-developed test conducted and certified under clinical laboratory improvement amendments regulations. HPV-SEQ assay were assessed using both contrived samples and biological specimens by measuring a set of performance attributes including limits of blank, limits of detection, limits of quantitation, accuracy and precision. The assay has demonstrated a robust quantitative detection across a wide ctHPV-DNA range, with low level of background signal (<0.05 copies/sample) in healthy donors and qualitative cut-off of >1.39 copies for both HPV strains (ensuring sufficient differentiation between negative and low positive samples), as well as outstanding sensitivity and specificity, approaching 100%. Given the rapid decrease in sequencing cost and effective integration of sequencing data in clinical care, NGS is likely to displace other HPV detection tests over the next few years. As such, our well-validated, ultra-sensitive, plasma-based HPV profiling method with optimal analytical performance may represent a significant advancement in risk stratification, treatment management, and surveillance for patients with OCSCC and other HPV- driven malignancies. Citation Format: Samaneh Eickelschulte, Anna Starus, David H Murray, Anja K Keyser, Oliver Schauer, Johannes Fredebohm, Frederick Jones, Alexander T Pearson, Everett E Vokes, Ari J Rosenberg, Nishant Agrawal, Evgeny Izumchenko. Analytical and clinical validation of HPV-SEQ, an NGS-based liquid biopsy platform for detection and quantification of human papilloma virus circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A019.

Illumina SBS Sequencing and DNBSEQ Perform Similarly for Single-Cell Transcriptomics

Background/Objectives: High-throughput single-cell RNA sequencing (scRNA-seq) workflows produce libraries that demand extensive sequencing. However, standard next-generation sequencing (NGS) methods remain expensive, contributing to the high running costs of single-cell experiments and often negatively affecting the sample numbers and statistical strength of such projects. In recent years, a plethora of new sequencing technologies have become available to researchers through several manufacturers, often providing lower-cost alternatives to standard NGS. Methods: In this study, we compared data generated from mouse scRNA-seq libraries sequenced with both standard Illumina sequencing by synthesis (Illumina SBS) and MGI’s DNA nanoball sequencing (DNBSEQ). Results: Our findings reveal similar overall performance using both technologies. DNBSEQ exhibited mildly superior sequence quality compared to Illumina SBS, as evidenced by higher Phred scores, lower read duplication rates and a greater number of genes mapping to the reference genome. Yet these improvements did not translate into meaningful differences in single-cell analysis parameters in our experiments, including detection of additional genes within cells, gene expression saturation levels and numbers of identified cells, with both technologies demonstrating equally robust performance in these aspects. The data produced by both sequencing platforms also produced comparable analytical outcomes for single-cell analysis. No significant difference in the annotation of cells into different cell types was observed and the same top genes were differentially expressed between populations and experimental conditions. Conclusions: Overall, our data demonstrate that alternative technologies can be applied to sequence scRNA-seq libraries, generating virtually indistinguishable results compared to standard methods, and providing cost-effective alternatives.

An Investigation into Diagnostic Strategies for Central Nervous System Infections Through the Integration of Metagenomic Next-Generation Sequencing and Conventional Diagnostic Methods.

The optimal strategy for detecting central nervous system infections (CNSI) in cerebrospinal fluid (CSF) samples remains unclear. In a one-year, multicenter retrospective study, we examined the efficacy of metagenomic next-generation sequencing (mNGS) in comparison to conventional pathogen diagnostic techniques for CSF in diagnosing CNSI. We calculated the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Youden index for each diagnostic approach. Additionally, receiver operating characteristic (ROC) curves were constructed, and the area under the curve (AUC) was determined to assess the diagnostic performance of each method. The study included 68 patients, comprising both adults and children, who were suspected of having CNSI. Through the application of comprehensive clinical interpretation (CCI), the sensitivity and specificity of mNGS were found to be 67.6% (95% confidence interval [CI]: 50.85-80.87%) and 45.8% (95% CI: 27.89-64.92%), respectively. In comparison, traditional pathogenic diagnostic methods indicated that the culture method demonstrated a sensitivity of 10.6% (95% CI: 4.63-22.6%) and a specificity of 100% (95% CI: 84.54-100%). Furthermore, the sensitivity and specificity of the peripheral blood nucleated cell count were determined to be 34.0% (95% confidence interval: 22.17-48.33%) and 57.1% (95% confidence interval: 36.54-75.53%), respectively. CSF nucleated cell count demonstrated a sensitivity of 66.0% (95% confidence interval [CI]: 51.67-77.83%) and a specificity of 61.9% (95% CI: 40.87-79.25%). In comparison, the CSF protein content exhibited a sensitivity of 63.8% (95% CI: 49.54-76.03%) and a specificity of 57.1% (95% CI: 36.54-75.53%). When combining mNGS with traditional methodologies, the overall sensitivity increased to 91.3% (95% CI: 79.67-96.56%), although the specificity was reduced to 18.2% (95% CI: 7.31-38.51%). The area under the ROC curve for culture, peripheral blood nucleated cell count, mNGS, CSF nucleated cell count, and CSF protein content were 0.8088, 0.6038, 0.6103, 0.5588, and 0.5588, respectively. The variation in CSF nucleated cell count did not significantly affect the diagnostic efficacy of mNGS. Currently, both mNGS and traditional diagnostic methods encounter substantial challenges in diagnosing CNSI.

The cumulative effect of compound heterozygous variants in TRPV3 caused Olmsted syndrome

BackgroundOlmsted syndrome (OS) is a rare genodermatosis predominantly inherited in an autosomal dominant manner, typically arising from gain-of-function (GOF) variants in the transient receptor potential channel vanilloid 3 (TRPV3). ObjectiveThis study aims to investigate potential mechanisms underlying OS in two cases presenting with an autosomal recessive inheritance pattern. MethodsNext-generation sequencing panel was employed to identify TRPV3 variants. TRPV3 plasmids carrying specific point variations were generated and transiently transfected into HEK293T cells. Electrophysiological patch-clamp techniques were utilized to record voltage-activated and ligand-activated currents. Celltiter-Glo luminescent assay was employed to analyze the cell viabilities. ResultsCompound heterozygous variants, c.1563G>C (p.W521C) and c.1376C>T (p.S459L), as well as c.1773G>C (p.L591F) and c.2186G>A (p.R729Q), were identified in the two OS patients respectively. Electrophysiological analysis of ligand-induced activation of TRPV3 variants demonstrated the closest correlation with clinical manifestations. All four variants displayed GOF channel activity characterized by increased sensitivity. Notably, W521C and L591F exhibited both heightened sensitivity and lower EC50 values for the TRPV3 agonist. Co-transfection with wild-type TRPV3 plasmids significantly rescued these effects. Cells co-transfected with the corresponding compound heterozygous variants exhibited intermediate electrophysiological characteristics. ConclusionsIn this study, we present two cases of OS by autosomal-recessive inheritance of TPRV3 variants. This study presents a notable observation of compound heterozygous GOF variants in TRPV3, highlighting their cumulative impact on clinical manifestations. Additionally, we advocate for the use of ligand-dependent ion channel activity assays to assess the pathogenicity of TRPV3 variants in OS.

A novel approach to detecting microduplication in split hand/foot malformation type 3 at the single-cell level: SHFM as a case study

BackgroundSplit hand/foot malformation (SHFM) is a congenital limb deficiency characterized by missing or shortened central digits. Several gene loci have been associated with SHFM. Identifying microduplications at the single-cell level is challenging in clinical practice, and traditional detection methods may lead to misdiagnoses in embryos and pregnant women.ResultsIn this research, we utilized a low cell count and whole-genome amplification products to employ single nucleotide polymorphism arrays, next-generation sequencing, and third-generation sequencing methods to detect copy number variants of microduplications in a SHFM3 case with limited DNA. Additionally, Karyomapping and combined linkage analysis were conducted to validate the results.ConclusionsThis study establishes a new strategy for identifying microduplications or microdeletions at the single-cell level in clinical preimplantation genetic testing, enhancing the efficiency and accuracy of diagnosing microduplication or microdeletion diseases during IVF-PGT and prenatal diagnosis.

PlaBacter: Determination of Microbial Diversity on Plastic Polymers in Topçu Pond

Climate change is one of the biggest problems of our world, and one of the biggest measures taken to solve the climate crisis is to reduce the consumption of oil and oil derivatives, which are considered among fossil resources. However, with the globalising world and increasing population, oil companies have turned to the plastic and packaging sector in order to meet the increasing demand for plastic consumption. This situation inevitably increases the environmental damage of plastic waste. In particular, the inability to use water resources correctly and the pollution of water by wastes left in the environment adversely affect the lives of living things on earth. Within the scope of the study, it was aimed to determine the microbial diversity living on polyethylene terephthalate (PET), which is known as the raw material of tools such as plastic bottles, food containers, and fishing line, which are left as waste in the environment, especially in rivers and lake shores. In this context, Topçu Pond, which is an important water source in the field of agricultural irrigation in Yozgat province, was selected as the study area. Genomic DNA (gDNA) was isolated from biofilm layers formed on water, sediment, and PET samples collected from the study area. The bacterial communities living in the samples were identified by analysing the 16S rDNA of the gDNAs with the next-generation sequencing method. Thus, we conducted the first microbial diversity study in Topçu Pond and defined the bacterial composition on PET samples in the pond.

First De Novo genome assembly and characterization of Gaultheria prostrata.

Gaultheria Kalm ex L. (Ericaceae), a type of evergreen shrub, known as a natural source of methyl salicylate, possesses rich germplasm resources, strong habitat adaptability, significant ornamental value, and noteworthy pharmacological activities. However, due to the paucity of whole genomic information, genetically deep research in these areas remains limited. Consequently, we intend to obtain genome data through high-throughput sequencing, gene annotation, flow cytometry, transcription factors prediction and genetic marker analysis for a representative species of this genus, with Gaultheria prostrata selected for our study. In this study, we preliminarily obtained the genome of G. prostrata through next-generation sequencing methods. Utilizing 47.94 Gb of high-quality sequence data (108.95× coverage), assembled into 114,436 scaffolds, with an N50 length of 33,667 bp. The genome size assembled by SOAPdenovo, approximately 417 Mb, corresponded closely to predictions by flow cytometry (440 Mb) and k-mer analysis (447 Mb). The genome integrity was evaluated using BUSCO with 91%. The heterozygosity ratio was 0.159%, the GC content was 38.85%, and the repetitive regions encompassed over 34.6% of the genome. A total of 26,497 protein-coding genes have been predicted and annotated across Nr, Swissprot, GO, KEGG, and Pfam databases. Among these, 14,377 and 2,387 genes received functional annotation in Nr and Swissprot, respectively; 21,895, 24,424, and 22,330 genes were similarly annotated in GO, KEGG, and Pfam. Moreover, A total of 279,785 SSRs were identified and 345,270 primers for these SSRs were designed. Within the various nucleotide types of SSRs, AG/CT and AAG/CTT constituted the predominant dinucleotide and trinucleotide repeat types in G. prostrata. In addition, 1,395 transcription factors (TFs) from 75 TF families, 462 transcription regulators (TRs) from 33 TR families and 840 protein kinase (PKs) from 118 PK families were identified in this genome. We also performed phylogenetic analyses of G. prostrata and related species, including estimation of divergence times and expansion and contraction analyses, followed by positive selection analyses of orthologous gene pairs of G. prostrata and its close relative Vaccinium corymbosum. These results provide a reference for in-depth study of genus Gaultheria, contributing to future functional and comparative genomics analyses and providing supporting data for the development of molecular markers.

High prevalence of cardiotropic viruses in endomyocardial biopsies from patients with inflammatory cardiomyopathy demonstrated by a novel targeted NGS approach

Abstract Background Viral infection of the heart muscle is a common cause of myocarditis and viral persistence can lead to chronic inflammatory cardiomyopathies (DCMi). A number of viruses including parvovirus B19 (B19V), adenovirus (AdV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes virus 6 (HHV-6) and enteroviruses (EV) are known to infect the myocardium, but their prevalence and role in persisting DCMi are not well understood. Virological etiologies can only be determined via endomyocardial biopsy (EMB), but the low tissue amount limits the number of conventional viral nucleic acid tests (virus-specific PCR) that can be performed in parallel. This study aimed to detect known and novel cardiotropic viruses in EMB of DCMi patients using a targeted next generation sequencing (NGS) approach. Methods A cohort of 33 patients with inflammatory cardiomyopathy (DCMi) (LVEF=29±12%) were retrospectively analyzed and compared to previous results of virus-specific PCR. DNA and RNA extracted from EMB were used for sequencing library preparation, and viral nucleic acids were enriched via a custom myBaits hybridization capture approach. Enriched libraries were sequenced on an Illumina MiSeq-platform in paired-end mode (500.000 reads/sample), and reads were taxonomically classified via Kraken2 and mapped against viral reference genomes using BWA-MEM2 or HiSat2, and subsequently deduplicated. Viral reads detected via both Kraken2 and mapping were classified as valid. Results Targeted NGS confirmed 19 out of 22 (86%) of the previous PCR-based viral detections. However, NGS was able to detect B19V DNA and RNA in an additional 11 and 15 patient samples respectively; CMV DNA/RNA in additional 5 and 4 samples respectively; EBV DNA/RNA in additional 5 and 1 samples respectively; and HHV-6 DNA/RNA in additional 3 and 1 samples respectively. Furthermore, viral reads of adenovirus type 2, adenovirus-associated virus type 2 (AAV-2), herpes viruses 7 and 8, and human parvovirus 4 were also found among the cohort. Thus, targeted NGS was able to identify 82 viral infections in a cohort of 33 DCMi patients, while only 22 were found using routine PCR tests. For routinely tested B19V, the use of the new NGS diagnostic approach improved sensitivity by 42% and specificity by 72%. Conclusions The presented NGS method represents a powerful new tool for the diagnosis of viral and inflammatory heart disease. The sensitivity is significantly higher than with conventional PCR approaches and the taxonomic classification is more specific. This enables the detection of previously false-negative routine viruses as well as potentially new cardiotropic pathogens from limited bioptic patient material – thus significantly increasing the diagnostic yield of EMB. In addition, our study demonstrates a high prevalence of non-enteroviral cardiotropic viruses in DCMi patients, shedding new light on the etiology of this heterogenous diseases and paving the way for more specific future treatments.

Single Nucleotide Recognition and Mutation Site Sequencing Based on a Barcode Assay and Rolling Circle Amplification

Single nucleotide polymorphisms (SNPs) present significant challenges in microbial detection and treatment, further raising the demands on sequencing technologies. In response to these challenges, we have developed a novel barcode-based approach for highly sensitive single nucleotide recognition. This method leverages a dual-head folded complementary template probe in conjunction with DNA ligase to specifically identify the target base. Upon recognition, the system triggers rolling circle amplification (RCA) followed by the self-assembly of CdSe quantum dots onto polystyrene microspheres, enabling a single-particle fluorescence readout. This approach allows for precise base identification at individual loci, which are then analyzed using a bio-barcode array to screen for base changes across multiple sites. This method was applied to sequence a drug-resistant mutation site in Helicobacter pylori (H. pylori), demonstrating excellent accuracy and stability. Offering high precision, high sensitivity, and single nucleotide resolution, this approach shows great promise as a next-generation sequencing method.

Genetic Diversity of the Species of the Genus Deschampsia P.Beauv. (Poaceae) Based on the Analysis of the ITS Region: Polymorphism Proves Distant Hybridization.

The species of the genus Deschampsia are difficult for identification, and the genus is difficult for taxonomic treatment. The regions of 35S rRNA genes were studied for the species of the genus Deschampsia of different geographical origin with a method of sequencing by Sanger (ITS1-5.8S rRNA gene-ITS2, 14 species) and with a method of a locus-specific next-generation sequencing (NGS) on the Illumina platform (ITS1-5.8S rRNA, 7 species). All species of Deschampsia formed one clade; the species, referred by some authors on the basis of morphological characters to the species D. cespitosa s.l., entered one subclade. Subantarctic species formed a separate subclade and their ribotypes formed their own subnetwork. Avenella flexuosa, earlier referred to Deschampsia, entered the other clade, though this species contains some ribotypes common with some Deschampsia species. Deschampsia pamirica and related mountain species have their own specific ribotype groups. On the network of the ribotypes, one can see that D. cespitosa from Great Britain forms a network with some species, but D. cespitosa from the USA forms its own network. Ribotype analysis of each sample revealed traces of introgression with Deyeuxia/Calamagrostis in D. cespitosa and with A. flexuosa and probable introgression of Northern and subantarctic species.

The potential role of next-generation sequencing in identifying MET amplification and disclosing resistance mechanisms in NSCLC patients with osimertinib resistance.

Osimertinib, one of the third-generation EGFR-tyrosine kinase inhibitors (TKIs) designed to target EGFR T790M mutation, significantly improves the prognosis of lung cancer. However, drug resistance still happens and MET amplification is responsible for one of the main causes. Fluorescence in situ hybridization (FISH) is the gold standard for MET amplification detection, but fundamentally limited by observer subjectivity. Herein, we assessed the value of next-generation sequencing (NGS) method in MET amplification detection in non-small cell lung cancer (NSCLC), as well as revealed the mutation profiling of NSCLC patients with osimertinib resistance to provide some valuable clues to the mechanisms of resistance. A total of 317 cancer tissue samples from 317 NSCLC patients at time of progression following osimertinib were submitted to NGS and only 96 tissues were tested by FISH simultaneously. With FISH results as gold standard, enumeration algorithm was applied to establish the optimal model for identifying MET amplification using gene copy number (GCN) data. The optimal model for identifying MET amplification was constructed based on the GCN of MET, BRAF, CDK6 and CYP3A4, which achieved a 74.0% overall agreement with FISH and performed well in identifying MET amplification except polysomy with a sensitivity of 85.7% and a specificity of 93.9%. The inconsistency between NGS and FISH occurred mainly in polysomy subtype, while MET GCN ≥ 5 could be reliably recognized by NGS. Moreover, the most frequently mutated genes in NSCLC patients with osimertinib resistance were EGFR (59.94%), followed by TP53 (43.85%), NRG1 (9.46%), PIK3CA (6.31%), and ATM (5.36%). The known resistance mechanisms, including MET amplification, EGFR (C797S, L718Q/R), TP53, CDK4, CDK6, CDKN2A, BRAF, KRAS, NRAS and PIK3CA mutations were also disclosed in our cohort. NGS assay can achieve a high concordance with FISH in MET amplification detection and has advantages in portraying various genetic alterations, which is of worthy in clinical promotion.

The chronic wound characterisation study and biobank: a study protocol for a prospective observational cohort investigation of bacterial community composition, inflammatory responses and wound-healing trajectories in non-healing wounds

IntroductionChronic wounds affect 1%–2% of the global population, with rising incidence due to ageing and lifestyle-related diseases. Bacterial biofilms, found in 80% of chronic wounds, and scattered single-cell bacteria may...

Structural alterations of the EGFR gene in glioblastoma samples as a prognostic factor and molecular target for therapy

Introduction. Epidermal growth factor receptor (EGFR) is a transmembrane protein of the receptor tyrosine kinase family that is activated in various cancers (non-small cell lung cancer, colorectal cancer, head and neck tumors). In glial brain tumors, increased EGFR expression levels are characteristic of the most aggressive subtype, glioblastoma. Frequent structural changes of EGFR in glioblastoma are amplification of the chromosome region where the EGFR gene is located, point mutations, as well as deletion of exons 2–7 of the EGFR gene leading to the formation of EGFRvIII transcript.Aim. To determine structural changes of the EGFR gene (point mutations and amplification of the EGFR gene, EGFRvIII transcript) in tumor samples using different methods and to evaluate their potential clinical significance.Materials and methods. The study included 75 patients with brain gliomas (70 of them glioblastoma) aged 34 to 78 years (mean age 56 years). DNA and RNA isolation was performed from fresh frozen tumor tissue, as well as from peripheral blood leukocytes. EGFR gene mutations were determined by next-generation sequencing (NGS), and β allele frequency (BAF) comparative analysis (normal-tumor) was performed to determine the copy number of chromosome 7 regions. Quantitative polymerase chain reaction was used to confirm the EGFR gene amplification in tumor samples, and reverse transcription-PCR was used to detect EGFRvIII variant.Results. The NGS method revealed 11/70 (16 %) mutations in coding regions of EGFR gene in glioblastoma samples, the EGFR gene amplification was detected in 26/70 (37 %) cases; no structural changes of the EGFR gene were detected in 5 glioma samples (astrocytoma, oligodendroglioma). All cases of EGFR gene amplification detected by NGS were confirmed by quantitative polymerase chain reaction. To search for EGFRvIII transcript, 31 tumor RNA samples were examined, of which EGFR amplification was present in 12 samples. EGFRvIII transcript was detected only in samples with EGFR gene amplification – 4/12 (33 %). To assess the clinical significance of structural gene alterations, the frequency of occurrence in primary and recurrent glioblastoma samples was compared.Conclusion. The NGS method allows to detect both point mutations and amplification of the EGFR gene. The EGFR gene amplification was associated with EGFRvIII mutation in 33 % of cases. No statistically significant differences in the frequency of structural changes in the EGFR gene between primary and relapsed glioblastomas were found.

Detection of KIT Mutations in Systemic Mastocytosis: How, When, and Why.

More than 90% of patients affected by mastocytosis are characterized by a somatic point mutation of KIT, which induces ligand-independent activation of the receptor and downstream signal triggering, ultimately leading to mast cell accumulation and survival. The most frequent mutation is KIT p.D816V, but other rarer mutations can also be found. These mutations often have a very low variant allele frequency (VAF), well below the sensitivity of common next-generation sequencing (NGS) methods used in routine diagnostic panels. Highly sensitive methods are developing for detecting mutations. This review summarizes the current indications on the recommended methods and on how to manage and interpret molecular data for the diagnosis and follow-up of patients with mastocytosis.

The epidemiological and infectious characteristics of novel types of Coxiella burnetii co-infected with Coxiella-like microorganisms from Xuyi County, Jiangsu province, China

Coxiella burnetii (C. burnetii) is the causative agent of Q fever, a type of zoonoses withwidespread distribution. In 2019, a case of Q fever was diagnosed by metagenomic next-generation sequencing (mNGS) method in Xuyi County (Jiangsu province, China). The seroprevalence of previous fever patients and the molecular epidemiology of Coxiella in wild hedgehogs and harbouring ticks around the confirmed patient were detected to reveal the genetic characteristics and pathogenicity of the Coxiella strains. Four of the 90 serum samples (4.44%) were positive for specific C. burnetii IgM antibody, suggesting that local humans are at risk of Q fever. The positive rates of C. burnetii in hedgehogs and ticks were 21.9% (7/32) and 70.5% (122/173), respectively. At least 3 strains of Coxiella were found prevalent in the investigated area, including one new genotype of pathogenic C. burnetii (XYHT29) and two non-pathogenic Coxiella-like organisms (XYHT19 and XYHT3). XYHT29 carried by ticks and wild hedgehogs successfully infected mice, imposing a potential threat to local humans. XYHT19, a novel Coxiella-like microorganism, was first discovered in the world to co-infect with C. burnetii in Haemaphysalis flava. The study provided significant epidemic information that could be used for prevention and control strategies against Q fever for local public health departments and medical institutions.

A comparative genomic and phenotypic study of Vibrio cholerae model strains using hybrid sequencing

Next-generation sequencing methods have become essential for studying bacterial biology and pathogenesis, often depending on high-quality, closed genomes. In this study, we utilized a hybrid sequencing approach to assemble the genome of C6706, a widely used Vibrio cholerae model strain. We present a manually curated annotation of the genome, enhancing user accessibility by linking each coding sequence to its counterpart in N16961, the first sequenced V. cholerae isolate and a commonly used reference genome. Comparative genomic analysis between V. cholerae C6706 and N16961 uncovered multiple genetic differences in genes associated with key biological functions. To determine whether these genetic variations result in phenotypic differences, we compared several phenotypes relevant to V. cholerae pathogenicity like genetic stability, acid sensitivity, biofilm formation and motility. Notably, V. cholerae N16961 exhibited greater motility and reduced biofilm formation compared to V. cholerae C6706. These phenotypic differences appear to be mediated by variations in quorum sensing and cyclic di-GMP signalling pathways between the strains. This study provides valuable insights into the regulation of biofilm formation and motility in V. cholerae.

DICER1 Variants in Pediatric and Young Adult Thyroid Nodules.

Background: Recent studies have suggested that pathogenic variants of the DICER1 gene could be a driver of alterations in some pediatric thyroid nodules, but data are still limited. The aim of this study was to detect variants in the DICER1 gene in a large cohort of pediatric thyroid nodules and then correlate them with clinicopathological data, with a focus on the disease prognosis in patients with thyroid carcinoma. Methods: This retrospective cohort study consisted of 350 pediatric and young adult patients (aged 2-21 years) with thyroid nodules, from whom 275 fresh-frozen thyroid nodule samples and 92 fine-needle aspiration biopsy (FNAB) samples were collected. After an analysis of variants in major genetic alterations of thyroid tumors, variants in the DICER1 gene were identified using next-generation sequencing and multiplex ligation-dependent probe amplification methods. Peripheral blood was analyzed from patients with DICER1-positive tumors. The results of genetic analysis were then correlated with clinicopathological data. Results: Variants in the DICER1 gene were detected in a total of 24/350 (6.9%; 95%CI [4.4;10.0]) pediatric and young adult patients, respectively, in 10/119 (8.4%; [4.1;14.9]) patients with benign fresh-frozen tissue, in 8/141 (5.7%; [1.9;9.5]) with papillary thyroid carcinoma (PTC) and in 6/86 (7.0%; [4.1;14.6]) patients with FNAB. No other gene alteration was found in DICER1-positive samples. Germline DICER1 variants were identified in 11/24 (45.8%; [25.6;67.2]) patients. Two somatic (biallelic) variants in the DICER1 gene were found in 9/24 (37.5%; [18.8;59.4]) thyroid nodules. Somatic deletions of at least 3 Mbp long were revealed in 2/24 (8.3%; [1.0;27.0]) cases. DICER1-positive PTCs were significantly associated with the follicular subtype of PTC (p = 0.001), encapsulation (p = 0.006) and were larger in size (p = 0.035), but with no extrathyroidal extension (p = 0.039), and less frequent lymph node metastases (p = 0.003) compared with DICER1-negative PTCs. Patients with DICER1-positive PTC had an excellent response to treatment in 75% of cases. Conclusions: Variants of the DICER1 gene are frequently found in the thyroid nodules of pediatric and young adult patients. In our patients, DICER1-positive PTCs showed low invasiveness. Our findings support considering more conservative management for DICER1-positive low-risk PTCs.