Next-generation Sequencing Methods Research Articles

The transcriptome response of Enterobacter sp. S-33 is modulated by low pH-stress.

Acidic environments naturally occur worldwide and uncontrolled use of agricultural practices may also cause acidification of soils. The development of acidic conditions disturbs the establishment of efficient microbial populations in their natural niches. The survival of Enterobacter species under acidic stress remains poorly understood. This study aimed to investigate the survival of an environmental isolate Enterobacter sp. S-33 under acidic stress and to identify the various genes involved in stress protection at the global gene transcription level. The obtained results provide new targets that will allow understanding the in-depth mechanisms involved in the adaptation of bacteria to environmental pH changes. We used the next-generation sequencing (NGS) method to analyze the expression (up-regulation & down-regulation) of genes under varying pH conditions. A total of 4214 genes were differentially expressed under acidic conditions (pH 5.0), with 294 up-regulated and 167 down-regulated. At pH 6.0, 50 genes were significantly expressed, of which 34 and 16 were identified as up-regulated and down-regulated, respectively. Many of the up-regulated genes were involved in carbohydrate metabolism, amino acid transport & metabolism, and the most down-regulated genes were related to post-translational modification, lipid transport & metabolism, etc. The observed transcriptomic regulation of genes and pathways identified that Enterobacter reduced its post-translational modification, lipid transport & metabolism, and increased carbohydrate metabolism, amino acid metabolism & transport, energy production & conversion to adapt and grow in acidic stress. The present work provides in-depth information on the characterization of genes associated with tolerance or adaptation to acidic stress of Enterobacter bacterium.

A comparative analysis between next-generation sequencing and conventional culture method to detect empyema-associated microorganisms: A systematic review

Empyema poses a significant global health concern, yet identifying responsible bacteria remains elusive. Recent studies question the efficacy of conventional pleural fluid culture in accurately identifying empyema-causing bacteria. The aim of this study was to compare diagnostic capabilities of next-generation sequencing (NGS) with conventional pleural fluid culture in identifying empyema-causing bacteria. Five databases (Google Scholar, Science Direct, Cochrane, Research Gate, and PubMed) were used to search studies comparing conventional pleural fluid culture with NGS for identifying empyema-causing bacteria using keywords. Positive results identified through conventional pleural fluid culture and NGS were extracted. In addition, bacterial profiles identified by NGS were also documented. Joanna-Briggs Institute (JBI) critical appraisal tool was employed to assess quality of included studies. Descriptive analysis was employed to present outcome of interests. From five databases, three studies, with 354 patients, were included. Findings from three studies showed that NGS outperformed conventional pleural fluid culture in detecting empyema-causing bacteria even in culture-negative samples. Moreover, dominant bacterial profiles identified through NGS included Streptococcus pneumoniae, Staphylococcus aureus, and anaerobic bacteria. In conclusion, NGS outperforms conventional pleural fluid culture in detection empyema-causing bacteria, yet further studies with larger samples and broader bacterial profiles are needed to increase confidence and urgency in its adoption over conventional pleural fluid culture.

Mutational Profiling by Next-Generation Sequencing in Patients with Metastatic Non-Small Cell Lung Carcinoma: Our Experience

Introduction According to GLOBOCAN 2020, overall lung cancer is the second most common cancer in both sexes (11.4%) accounting for highest cancer-related mortality (18%).1 Adenocarcinoma subtype of non-small cell lung cancer (NSCLC) is the most common subtype and is divided into further molecular subtypes based on oncogenic driver mutations. Overall survival in these patients is poor with the use of conventional platinum-based double chemotherapy and various recent studies on targeted therapy studies have showed improved survival. Therefore, broad panel-based testing like next-generation sequencing (NGS) is strongly recommended to identify these targetable driver mutations. Aims and Objectives The aim of this study was to evaluate the mutational profile in patients with metastatic NSCLC (mNSCLC) by NGS method. Materials and Methods A hospital-based prospective observational study done on 88 patients under the Department of Medical Oncology, State Cancer Institute during a period of 1 year. All patients above 18 years of age diagnosed as mNSCLC having Eastern Cooperative Oncology Group performance status 0 to 2 and evaluated for mutational profiling by NGS method were included. Five gene panel tests including endothelial growth factor receptor (EGFR), echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK), BRAF, mesenchymal epithelial transition (MET), and ROS proto-oncogene 1 (ROS1) were used. Results and Observations Majority of mNSCLC cases were in the age group of 41 to 50 years (n = 30, 34.1%) with average age at presentation being 53.74 years. Male: female ratio was 1.14:1 and most patients were nonsmokers. Adenocarcinoma subtype of mNSCLC cases had the highest mutational burden (n = 55, 62.5%). EGFR (n = 32, 56.14%) was the most common mutation followed by EML4-ALK (n = 19, 33.33%). Most common EGFR mutation was in Exon 19. Other rare mutations were ROS1 (n = 4), BRAF V600E (n = 1), and MET (n = 1). Skeleton was the most common site of metastasis across all driver mutations. Conclusion EGFR and EML4-ALK were the commonest targetable mutations detected in the study. As there is very limited data from North Eastern region of India regarding mutational status in mNSCLC, this study opens up possibilities for further studies targeting multiple mutations to give us more comprehensive understanding of the mutational landscape of mNSCLC in this era of precision medicine.

Bacterial Metabarcoding of Raw Palm Sap Samples from Bangladesh with Nanopore Sequencing.

The traditional practice of harvesting and processing raw date palm sap is not only culturally significant but also provides an essential nutritional source in South Asia. However, the potential for bacterial or viral contamination from animals and environmental sources during its collection remains a serious and insufficiently studied risk. Implementing improved food safety measures and collection techniques could mitigate the risk of these infections. Additionally, the adoption of advanced food analytical methods offers the potential to identify pathogens and uncover the natural bacterial diversity of these products. The advancement of next-generation sequencing (NGS) technologies, particularly nanopore sequencing, offers a rapid and highly mobile solution. In this study, we employed nanopore sequencing for the bacterial metabarcoding of a set of raw date palm sap samples collected without protective coverage against animals in Bangladesh in 2021. We identified several bacterial species with importance in the natural fermentation of the product and demonstrated the feasibility of this NGS method in the surveillance of raw palm sap products. We revealed two fermentation directions dominated by either Leuconostoc species or Lactococcus species in these products at the first 6 h from harvest, along with opportunistic human pathogens in the background, represented with lower abundance. Plant pathogens, bacteria with the potential for opportunistic human infection and the sequences of the Exiguobacterium genus are also described, and their potential role is discussed. In this study, we demonstrate the potential of mobile laboratory solutions for food safety purposes in low-resource areas.

[Standardization of next-generation sequencing for detecting mutations associated with targeted therapy and immunotherapy based on dynamic pattern of expandable detection range].

Next-generation sequencing (NGS) has laid the foundation for precision oncology care. NGS technologynot only represents an innovation in the methodology but also brings about a revolution in the concept of detecting gene alterations for targeted therapy and immunotherapy of cancers. As basic biomedical research and drug development progress, the landscape of biomarkers associated with gene alterations continues to evolve. Thus, the standardization of NGS-based gene alterations detection should take into account the characteristics of NGS methods and the gene alteration biomarkers. To be specific, whether employed as in vitro diagnostic products or laboratory-developed tests, the detection range can be expanded in response to changes in the clinical evidence level of biomarkers during the process of assay development and clinical application. Such adjustment needs the analytical validation results for supplemented genes or mutant sites within a predefined detection system, which will maximally fulfill the evolving clinical demands in cancer diagnosis and treatment, simultaneously mitigate potential risks effectively. This article primarily discusses the standardization pathway for NGS testing of gene alterations in cancer by focusing on the characteristics of NGS methods, gene alteration biomarkers, and the current status of the standardization of NGS application.

Mitogenomes do not substantially improve phylogenetic resolution in a young non-model adaptive radiation of freshwater gastropods

BackgroundSpecies flocks in ancient lakes, and particularly those arising from adaptive radiation, make up the bulk of overall taxonomic and morphological diversity in these insular ecosystems. For these mostly young species assemblages, classical mitochondrial barcoding markers have so far been key to disentangle interspecific relationships. However, with the rise and further development of next-generation sequencing (NGS) methods and mapping tools, genome-wide data have become an increasingly important source of information even for non-model groups.ResultsHere, we provide, for the first time, a comprehensive mitogenome dataset of freshwater gastropods endemic to Sulawesi and thus of an ancient lake invertebrate species flock in general. We applied low-coverage whole-genome sequencing for a total of 78 individuals including 27 out of the 28 Tylomelania morphospecies from the Malili lake system as well as selected representatives from Lake Poso and adjacent catchments. Our aim was to assess whether mitogenomes considerably contribute to the phylogenetic resolution within this young species flock. Interestingly, we identified a high number of variable and parsimony-informative sites across the other ‘non-traditional’ mitochondrial loci. However, although the overall support was very high, the topology obtained was largely congruent with previously published single-locus phylogenies. Several clades remained unresolved and a large number of species was recovered polyphyletic, indicative of both rapid diversification and mitochondrial introgression.ConclusionsThis once again illustrates that, despite the higher number of characters available, mitogenomes behave like a single locus and thus can only make a limited contribution to resolving species boundaries, particularly when introgression events are involved.

Uncommon and Rare EGFR Mutations in Non-Small Cell Lung Cancer Patients with a Focus on Exon 20 Insertions and the Phase 3 PAPILLON Trial: The State of the Art.

Uncommon (ucEGFRmuts) and rare epidermal growth factor receptor (EGFR) mutations account for 10-15% of diagnosed cases and consist of a heterogeneous group represented by several clusters within exons 18-21 (e.g., exon 18 point mutations, exon 21 L861X, exon 20 S768I), as well as exon 20 insertions (Ex20ins). Their incidence is under molecular and clinical investigation following recent findings that reported an increase of sensitivity and specificity of next-generation sequencing (NGS) methods. Consequently, their detection allows for the selection of emerging treatment options to significantly improve patients' outcomes in these particular subgroups of EGFR-mutated advanced non-small cell lung cancer (NSCLC). Specifically, this commentary is focused on the notable progress of the Phase 3 PAPILLON study that showed primary efficacy results from amivantamab, a bispecific antibody with specific binding and affinity to extracellular domains of EGFR and MET, plus chemotherapy in the first-line setting for EGFR exon 20 insertion-mutated advanced or metastatic NSCLC patients, as compared with chemotherapy alone, thus becoming the new standard of care in this group of patients.

A rapid PCR-free next-generation sequencing method for comprehensive diagnosis of chromosome disease syndromes in prenatal samples.

The aim of this study is to investigate the application performance of rapid copy number variation sequencing (rCNV-seq) technology for the detection of chromosomal abnormalities during prenatal diagnosis. Samples were collected from 424 pregnant women who were at high-risk for noninvasive prenatal screening in Kunming Maternal and Child Care Hospital from January 2018 to May 2022. rCNV-seq technique was used to detect fetal chromosome abnormalities and compare the results with that of chromosomal karyotype analysis. The Result showed that 330 (77.83%, 330/424) cases indicated chromosomal abnormalities among 424 high-risk pregnant women who underwent rCNV-seq. Moreover, 94 (22.17%, 94/424) cases were discovered to have copy number variations. Among the 330 fetuses with chromosomal abnormalities, common autosomal aneuploidy was observed in 203 cases (47.87%, 203/424) and sex chromosome aneuploidy was observed in 91 cases (21.46%, 91/424). Moreover, the abnormalities in multiple chromosomes were discovered in 33 cases (7.78%, 33/424), and the rare autosomal aneuploidy was observed in 3 cases (0.71%, 3/424). There were 63 fetuses (14.86%, 63/424) with pathogenic CNVs among the 94 fetuses with variable copy numbers. Of the 245 pregnant women who voluntarily selected G-band karyotyping, 1 fetus with copy number variation had normal karyotype results, and the remaining women were consistent with rCNV-seq. Our study revealed that rCNV-seq has higher accuracy in detecting common trisomy and can also detect chromosomal microdeletions or microduplications that cannot be detected by G-banding karyotype analysis. There is no effective treatment for chromosomal diseases, so it is particularly important to prevent chromosomal diseases through genetic counseling and prenatal diagnosis of chromosomal diseases.

Comprehensive analysis of the microbiome in Apis cerana honey highlights honey as a potential source for the isolation of beneficial bacterial strains.

Honey is a nutritious food made by bees from nectar and sweet deposits of flowering plants and has been used for centuries as a natural remedy for wound healing and other bacterial infections due to its antibacterial properties. Honey contains a diverse community of bacteria, especially probiotic bacteria, that greatly affect the health of bees and their consumers. Therefore, understanding the microorganisms in honey can help to ensure the quality of honey and lead to the identification of potential probiotic bacteria. Herein, the bacteria community in honey produced by Apis cerana was investigated by applying the next-generation sequencing (NGS) method for the V3-V4 hypervariable regions of the bacterial 16S rRNA gene. In addition, lactic acid bacteria (LAB) in the honey sample were also isolated and screened for in vitro antimicrobial activity. The results showed that the microbiota of A. cerana honey consisted of two major bacterial phyla, Firmicutes (50%; Clostridia, 48.2%) and Proteobacteria (49%; Gammaproteobacteria, 47.7%). Among the 67 identified bacterial genera, the three most predominant genera were beneficial obligate anaerobic bacteria, Lachnospiraceae (48.14%), followed by Gilliamella (26.80%), and Enterobacter (10.16%). Remarkably, among the identified LAB, Lactobacillus kunkeei was found to be the most abundant species. Interestingly, the isolated L. kunkeei strains exhibited antimicrobial activity against some pathogenic bacteria in honeybees, including Klebsiella spp., Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus. This underscores the potential candidacy of L. kunkeei for developing probiotics for medical use. Taken together, our results provided new insights into the microbiota community in the A. cerana honey in Hanoi, Vietnam, highlighting evidence that honey can be an unexplored source for isolating bacterial strains with potential probiotic applications in honeybees and humans.

Plasma miR-379 can predict treatment response to FOLFIRINOX and gemcitabine-nab-paclitaxel in advanced pancreatic cancer

BackgroundNovel biomarkers, such as plasma microRNAs (miRs), are needed to help guide clinical decision-making for the type of chemotherapy to use in patients with advanced pancreatic ductal adenocarcinoma (PDAC). This study assessed the ability of plasma miRs to predict optimal treatment response from FOLFIRINOX or gemcitabine-nab-paclitaxel in these patients. MethodsNext-generation sequencing (NGS) was performed for biomarker discovery in pre-treatment plasma samples from advanced PDAC patients subsequently treated with FOLFIRINOX (n = 12) or gemcitabine-nab-paclitaxel (n = 12). Selected candidate biomarkers were validated in 40 patients with advanced PDAC using RT-qPCR. Cox regression was then used to assess the predictive value of plasma miRs for either FOLFIRINOX or gemcitabine-nab-paclitaxel. ResultsIn the validation cohort, high plasma miR-379 expression was strongly predictive of treatment response (Pinteraction = 0.0004). Overall survival (OS) was significantly better with FOLFIRINOX vs. gemcitabine-nab-paclitaxel in those patients with lower plasma miR-379 expression (hazard ratio, 0.32 [95% confidence interval, 0.08 to 0.98]; P = 0.046). However, gemcitabine-nab-paclitaxel was associated with superior OS in patients with higher plasma miR-379 (hazard ratio, 0.28 [0.10 to 0.86]; P = 0.027). In contrast, miR-127, miR-155, and miR-200 showed no predictive value for treatment response for either chemotherapy regimen (Pinteraction = 0.12, Pinteraction = 0.83 and Pinteraction = 0.12, respectively). ConclusionsPlasma miR-379 appears clinically useful as a predictive biomarker to identify which patients with advanced PDAC benefit most from treatment with FOLFIRINOX or gemcitabine-nab-paclitaxel. Further validation in larger studies and clinical trials is now warranted.

Comprehensive Analysis of Clinically Relevant Copy Number Alterations (CNAs) Using a 523-Gene Next-Generation Sequencing Panel and NxClinical Software in Solid Tumors.

Copy number alterations (CNAs) are significant in tumor initiation and progression. Identifying these aberrations is crucial for targeted therapies and personalized cancer diagnostics. Next-generation sequencing (NGS) methods present advantages in scalability and cost-effectiveness, surpassing limitations associated with reference assemblies and probe capacities in traditional laboratory approaches. This retrospective study evaluated CNAs in 50 FFPE tumor samples (breast cancer, ovarian carcinoma, pancreatic cancer, melanoma, and prostate carcinoma) using Illumina's TruSight Oncology 500 (TSO500) and the Affymetrix Oncoscan Molecular Inversion Probe (OS-MIP) (ThermoFisher Scientific, Waltham, MA, USA). NGS analysis with the NxClinical 6.2 software demonstrated a high sensitivity and specificity (100%) for CNA detection, with a complete concordance rate as compared to the OS-MIP. All 54 known CNAs were identified by NGS, with gains being the most prevalent (63%). Notable CNAs were observed in MYC (18%), TP53 (12%), BRAF (8%), PIK3CA, EGFR, and FGFR1 (6%) genes. The diagnostic parameters exhibited high accuracy, including a positive predictive value, negative predictive value, and overall diagnostic accuracy. This study underscores NxClinical as a reliable software for identifying clinically relevant gene alterations using NGS TSO500, offering valuable insights for personalized cancer treatment strategies based on CNA analysis.

Abstract 322: Ultraplex genome and exome sequencing of rare single cells

Abstract Single-cell genome analysis enables researchers to gain novel insights into tumor heterogeneity. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging since the amount of genomic DNA present in a single cell is limited. PCR-based amplification methods normally have high error rates, low coverage uniformity, and extensive allelic drop-outs making interpretation of SNVs and CNVs challenging.Here we introduce a new whole-genome amplification method which utilizes an optimized isothermal DNA amplification reaction with a Hi-fidelity, error correcting polymerase and downstream library preparation protocol to allow pooling of amplified DNA. The key DNA amplification steps use multiple displacement amplification (MDA) technology to amplify gDNA directly from single cells, nuclei or previously isolated genomic DNA. During the MDA reaction, sample-barcodes are incorporated into the newly synthetized DNA, allowing the amplified DNA to be pooled and processed in to one ultraplex library. This method saves library preparation time, reduces DNA library costs and limits plastic consumable usage.Our current workflow allows 24 samples to be multiplexed in one library and 24 libraries to be produced for a total of 576 samples per sequencing flow cell. This method can be used to investigate chromosomal instability with low pass sequencing or SNV and CNV analysis following whole genome, exome-capture workflows. Citation Format: Ioanna Andreou, Samuel Rulli, Katja Heitz, Silke Huebner, Christin Meerschiff, Eric Lader. Ultraplex genome and exome sequencing of rare single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 322.

Abstract 2440: Cancer driver mutations as quantitative biomarkers of cancer risk interspecies analyses using CarcSeq

Abstract Unmet needs in cancer risk assessment include the ability to predict rodent life-time tumor responses from short-term exposures and a scientific basis for rodent to human extrapolation. As an approach to address these needs, the use of hotspot cancer driver mutations (CDMs) as biomarkers of cancer risk was integrated with an error-corrected, next generation sequencing (NGS) approach. We have developed and investigated the sensitivity of a high-throughput NGS method capable of detecting a panel of the most prevalent point mutations observed in human oncogenes and tumor suppressor genes. This method, CarcSeq, involves performing multiple, high-fidelity PCR reactions to amplify the most prevalent mutations reported to occur in human tumors, tagging the amplicons with 9 bp unique identifier sequences at each end, constructing libraries using Illumina ChIP Seq kits, sequencing the libraries on an Illumina NextSeq500 platform and generating single-strand consensus sequences (SSCS), bioinformatically, as a means of error-correction. We validated CarcSeq mutant fraction measurements through observations related to known cancer driver tissue-specificities and mutation spectra for normal human breast and lung, ductal carcinoma, and lung adenocarcinoma samples. Performance of a reconstruction experiment established CarcSeq has a sensitivity of 104. The approach has now been adapted to analyze analogous conserved codons in rat and mouse. For each of the three species, the developed panel includes 13-15 amplicons encompassing known cancer driver genes (e.g., KRAS, PIK3CA, and TP53) encompasses the equivalent of 30 known human hotspot codons and has a combined target sequence length of ~1 kb. Amplicons were generated from DNA isolated from 16-week-old, untreated rodent tissue samples with known differences in spontaneous tumor incidence: female Wistar, Sprague Dawley, and F344 rat mammary samples and male and female B6C3F1 and CD-1 mouse lung samples. The rodent panel was optimized to generate ~100,000 SSCS/amplicon. CDMs were observed at conserved codons in young, untreated rodents, and results are consistent with what was expected based on strain differences in spontaneous tumorigenesis. Citation Format: Kelly L. Harris, Binsheng Gong, Barbara Parsons, Jennifer Faske, Joshua Xu. Cancer driver mutations as quantitative biomarkers of cancer risk interspecies analyses using CarcSeq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2440.

A universal molecular control for DNA, mRNA and protein expression

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

Evaluation of next-generation sequencing versus next-generation flow cytometry for minimal-residual-disease detection in Chinese patients with multiple myeloma

PurposeTo evaluate the efficacy of next-generation sequencing (NGS) in minimal-residual-disease (MRD) monitoring in Chinese patients with multiple myeloma (MM).MethodsThis study analyzed 60 Chinese MM patients. During MRD monitoring in these patients’ post-therapy, clonal immunoglobulin heavy chain (IGH) rearrangements were detected via NGS using LymphoTrack assays. MRD monitoring was performed using NGS or next-generation flow cytometry (NGF), and the results were compared. Additionally, the sensitivity and reproducibility of the NGS method were assessed.ResultsThe MRD detection range of the NGS method was 10–6–10–1, which suggested good linearity, with a Pearson correlation coefficient of 0.985 and a limit of detection of 10–6. Intra- and inter-assay reproducibility analyses showed that NGS exhibited 100% reproducibility with low variability in clonal cells. At diagnosis, unique clones were found in 42 patients (70.0%) with clonal IGH rearrangements, which were used as clonality markers for MRD monitoring post-therapy. Comparison of NGS and NGF for MRD monitoring showed 79.1% concordance. No samples that tested MRD-positive via NGF were found negative via NGS, indicating the higher sensitivity of NGS. MRD could be detected using NGS in 6 of 7 samples before autologous hematopoietic stem-cell transplantation, and 5 of them tested negative post-transplantation. In contrast, the NGF method could detect MRD in only 1 sample pre-transplantation.ConclusionCompared with NGF, NGS exhibits higher sensitivity and reproducibility in MRD detection and can be an effective strategy for MRD monitoring in Chinese MM patients.

Co-infecting viruses of species Bovine rhinitis B virus (Picornaviridae) and Bovine nidovirus 1 (Tobaniviridae) identified for the first time from a post-mortem respiratory sample of a sheep (Ovis aries) in Hungary

In this study, a picornavirus and a nidovirus were identified from a single available nasopharyngeal swab (NPS) sample of a freshly deceased sheep, as the only vertebrate viruses found with viral metagenomics and next-generation sequencing methods. The sample was originated from a mixed feedlot farm in Hungary where sheep and cattle were held together but in separate stalls. Most of the sheep had respiratory signs (coughing and increased respiratory effort) at the time of sampling. Other NPS were not, but additional enteric samples were collected from sheep (n = 27) and cattle (n = 11) of the same farm at that time. The complete/nearly complete genomes of the identified viruses were determined using RT-PCR and Nanopore (MinION-Flonge) / Dye-terminator sequencing techniques. The results of detailed genomic and phylogenetic analyses indicate that the identified picornavirus most likely belongs to a type 4 genotype of species Bovine rhinitis B virus (BRBV-4, OR885914) of genus Aphthovirus, family Picornaviridae while the ovine nidovirus (OvNV, OR885915) – as a novel variant – could belong to the recently created Bovine nidovirus 1 (BoNV) species of genus Bostovirus, family Tobaniviridae. None of the identified viruses were detectable in the enteric samples using RT-PCR and generic screening primer pairs.Both viruses are well-known respiratory pathogens of cattle, but their presence was not demonstrated before in other animals, like sheep. Furthermore, neither BRBV-4 nor BoNVs were investigated in European cattle and/or sheep flocks, therefore it cannot be determined whether the presence of these viruses in sheep was a result of a single host species switch/spillover event or these viruses are circulating in not just cattle but sheep populations as well.Further studies required to investigate the spread of these viruses in Hungarian and European sheep and cattle populations and to identify their pathogenic potential in sheep.

A capture-based method of prenatal cell-free DNA screening for autosomal recessive non-syndromic hearing loss.

This study aimed to develop and validate a prenatal cell-free DNA (cfDNA) screening method that uses capture-based enrichment to genotype fetal autosomal recessive disorders. This method was applied in pregnancies at high risk of autosomal recessive non-syndromic hearing loss (ARNSHL) to assess its accuracy and effectiveness. This assay measured the allele counts in both white blood cell DNA and cfDNA from the blood samples of pregnant women using a capture-based next-generation sequencing method. It then applied a binomial model to infer the fetal genotypes with the maximum likelihood. Ninety-four pregnant couples that were carriers of variants of ARNSHL in GJB2 or SLC26A4 were enrolled. The fetal genotypes deduced using this screening method were compared with the results of genetic diagnosis using amniocentesis. Of the 94 couples, 65 carried more than one variant, resulting in 170 single-nucleotide polymorphism (SNP) loci to be inferred in the fetuses. Of the 170 fetal SNP genotypes, 150 (88.2%) had high confidence calls and 139 (92.7%) of these matched the genotypes obtained by amniocentesis result. Out of the remaining 20 (11.8%) cases with low-confidence calls, only 14 (70.0%) were concordant with genetic diagnosis using amniocentesis. The concordance rate was 100% for sites where the maternal genotype was wild-type homozygous. The discordance was site-biased, with each locus showing a consistent direction of discordance. Genetic diagnosis identified a total of 19 wild-type homozygotes, 46 heterozygotes, 19 compound heterozygotes, and 10 pathogenic homozygotes. This screening method correctly genotyped 81.9% (77/94) of fetuses and demonstrated a sensitivity of 89.7% and a specificity of 89.2% for correctly identifying ARNSHL. This capture-based method of prenatal screening by cfDNA demonstrated strong potential for fetal genotyping of autosomal recessive disorders.

Analysis of the CYP21A2 gene pathogenic variants in CAH patients from Surgut using next-generation sequencing (NGS)

Background21-hydroxylase deficiency is present in 90–95% of cases of congenital adrenal hyperplasia (CAH). Eleven major pathogenic variants account for 93% of all identified variants in the CYP21A2 gene in various clinical forms of the disease. Each population has its own range of significant pathogenic variants. We aimed to study the frequency of pathogenic variants in the CYP21A2 gene using NGS technology and real-time PCR in Surgut patients with different clinical forms of CAH. NGS was performed on 70 patients with salt-wasting and non-classical clinical forms of 21-hydroxylase deficiency, verified by direct Sanger sequencing and PCR–RFLP analysis.ResultsEleven different pathogenic variants were found in 68.57% (48/70) of patients. Among 92.86% (13/14) of patients with salt-wasting CAH, variants were found to be homozygous, with CYP21A2 gene deletion as the most frequent mutation (46.4% or 13/28 alleles). In the group with non-classical CAH, pathogenic variants were identified only in 60.71% (34/56) of patients. V282L was discovered to be the most common variant in heterozygous carriers (45.45%, 15/33). NGS method identified 2 variants that were not determined by the standard method for major mutations detection: p.C170* and p.W22X, accounting for 3% of all known pathogenic variants.ConclusionOur data make it possible to clarify the specific spectrum of CYP21A2 gene pathogenic variants in CAH patients from Surgut. The NGS method allows for the identification of rare pathogenic variants (3%) in the CYP21A2 gene that are not included in the conventional PCR–RFLP analysis.

CyanoStrainChip: A Novel DNA Microarray Tool for High-Throughput Detection of Environmental Cyanobacteria at the Strain Level.

Detecting cyanobacteria in environments is an important concern due to their crucial roles in ecosystems, and they can form blooms with the potential to harm humans and nonhuman entities. However, the most widely used methods for high-throughput detection of environmental cyanobacteria, such as 16S rRNA sequencing, typically provide above-species-level resolution, thereby disregarding intraspecific variation. To address this, we developed a novel DNA microarray tool, termed the CyanoStrainChip, that enables strain-level comprehensive profiling of environmental cyanobacteria. The CyanoStrainChip was designed to target 1277 strains; nearly all major groups of cyanobacteria are included by implementing 43,666 genome-wide, strain-specific probes. It demonstrated strong specificity by in vitro mock community experiments. The high correlation (Pearson's R > 0.97) between probe fluorescence intensities and the corresponding DNA amounts (ranging from 1-100 ng) indicated excellent quantitative capability. Consistent cyanobacterial profiles of field samples were observed by both the CyanoStrainChip and next-generation sequencing methods. Furthermore, CyanoStrainChip analysis of surface water samples in Lake Chaohu uncovered a high intraspecific variation of abundance change within the genus Microcystis between different severity levels of cyanobacterial blooms, highlighting two toxic Microcystis strains that are of critical concern for Lake Chaohu harmful blooms suppression. Overall, these results suggest a potential for CyanoStrainChip as a valuable tool for cyanobacterial ecological research and harmful bloom monitoring to supplement existing techniques.

Characterization of the complete mitochondrial genome of Paecilomyces variotii and comparative evolutionary mitochondriomics of 36 fungi.

Paecilomyces variotii has important economic value in stimulating crop growth, biodegradation, and other aspects. Up to now, there are no research reports on its mitochondrial genome. The mitochondrial genome of Paecilomyces variotii was determined with the next-generation sequencing method (Illumina, NovaSeq), and its characteristics were analyzed using various bioinformatics approaches. The length of complete mitochondrial genome sequence of P. variotii is 40,965bp and consists of 14 protein-coding genes, 2 ribosomal RNA genes, 1 ribosomal protein S3 gene, 26 transport RNA genes. The results of phylogenetics analysis using Bayesian inference and Maximum likelihood methods showed that P. variotii belongs to the Eurotiales order in the Thermoascaceae family, and 9 genera within the Eurotiomycetes class were effectively distinguished with high support rates (bootstrap value > 92% and posterior probabilities > 99%). The analysis of synonymous substitution rates and nonsynonymous substitution rates indicated that the Ka/Ks values of the 14 PCGs in the mitochondrial genomes of the two orders in the Eurotiomycetes class ranged from 0 to 0.4333. This study revealed the structural and sequence information characteristics of the mitochondrial genome of P. variotii, and the phylogenetic results strongly support its classification within the family Thermoascaceae, consistent with traditional morphological taxonomy studies. The 14 PCGs in the mitochondrial genomes of the two orders in the Eurotiomycetes class are subject to strong purifying (negative) selection. The results of this research provides an important molecular basis for the development of genomics, evolutionary genetics and molecular markers of P. variotii in the future.