High-throughput Next-generation Sequencing Research Articles

Unveiling Inter- and Intra-Patient Sequence Variability with a Multi-Sample Coronavirus Target Enrichment Approach

Amid the global challenges posed by the COVID-19 pandemic, unraveling the genomic intricacies of SARS-CoV-2 became crucial. This study explores viral evolution using an innovative high-throughput next-generation sequencing (NGS) approach. By taking advantage of nasal swab and mouthwash samples from patients who tested positive for COVID-19 across different geographical regions during sequential infection waves, our study applied a targeted enrichment protocol and pooling strategy to increase detection sensitivity. The approach was extremely efficient, yielding a large number of reads and mutations distributed across 10 distinct viral gene regions. Notably, the genes Envelope, Nucleocapsid, and Open Reading Frame 8 had the highest number of unique mutations per 1000 nucleotides, with both spike and Nucleocapsid genes showing evidence for positive selection. Focusing on the spike protein gene, crucial in virus replication and immunogenicity, our findings show a dynamic SARS-CoV-2 evolution, emphasizing the virus–host interplay. Moreover, the pooling strategy facilitated subtle sequence variability detection. Our findings painted a dynamic portrait of SARS-CoV-2 evolution, emphasizing the intricate interplay between the virus and its host populations and accentuating the importance of continuous genomic surveillance to understand viral dynamics. As SARS-CoV-2 continues to evolve, this approach proves to be a powerful, versatile, fast, and cost-efficient screening tool for unraveling emerging variants, fostering understanding of the virus’s genetic landscape.

Distinct actions of testicular endocrine and lumicrine signaling on the proximal epididymal transcriptome.

The epididymal function and gene expression in mammals are under the control of the testis. Sex steroids are secreted from the testis and act on the epididymis in an endocrine manner. There is another, non-sex steroidal secreted signaling, named lumicrine signaling, in which testis-derived secreted proteins go through the male reproductive tract and act on the epididymis. The effects of such multiple regulations on the epididymis by the testis have been investigated for many genes. The recent development of high-throughput next-generation sequencing now enables us a further comparative survey of endocrine and lumicrine action-dependent gene expression. In the present study, testis-derived endocrine and lumicrine actions on epididymal gene expression were comparatively investigated by RNA-seq transcriptomic analyses. This investigation utilized experimental animal models in which testis-derived endocrine and/or lumicrine actions were interfered with, such as unilateral or bilateral orchidectomy. By bilateral orchidectomy, which interferes with both endocrine and lumicrine actions, 431 genes were downregulated. By unilateral orchidectomy, which also interferes with endocrine and lumicrine actions by the unilateral testis, but the endocrine action was compensated by the contralateral testis, 283 genes were downregulated. The content of such genes downregulated by unilateral orchidectomy was like those of lumicrine action-interfered efferent duct-ligation, W/Wv, and Nell2-/- mice. When genes affected by unilateral and bilateral orchidectomy were compared, 154 genes were commonly downregulated, whereas 217 genes were specifically downregulated only by bilateral orchidectomy, indicating the distinction between endocrine and lumicrine actions on the proximal epididymal transcriptome. Comparative transcriptome analyses also showed that the expressions of genes emerging since Amniota were notably impacted by bilateral orchidectomy, unilateral orchidectomy, and lumicrine action-interfering treatments; the degree of influence from these treatments varied based on the evolutionary stage beyond Amniota. These findings unveil an evolutional transition of regulated gene expression in the proximal epididymis by two different testis-derived signaling mechanisms.

Metagenomics for Pathogen Detection During a Mass Mortality Event in Songbirds.

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria forming the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes, including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events.

Abstract 5054: Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications

Abstract Formalin-fixed paraffin-embedded (FFPE) biopsies are highly valuable and widely used tissue specimens for clinical diagnostics. However, obtaining sufficient and high-quality nucleic acid material from limited FFPE samples presents a challenge for downstream molecular analysis, such as next-generation sequencing (NGS). We present an optimized sequential extraction method that generates high-quality DNA and RNA from a single set of input tissues that is automatable and operation-friendly. This workflow performs well with reduced FFPE tissue input and efficiently supports various high-throughput clinical NGS applications. The DNA/RNA yield, quality, purity, and impacts on NGS assay performances were on par or better than an existing validated comparator extraction method. With comparable FFPE input, the new method demonstrated a superior extraction performance with significantly higher yield, quality, and purity. For DNA, NGS libraries were made with two different library preparation methods: a TA-ligation-based method and a single-strand-based method, followed by hybrid capturing and sequencing. The DNA from the new extraction method demonstrated a superior library conversion rate and improved target enrichment uniformity with both chemistries. This provides the potential of reduced input requirements, allowing very limited tissue, such as fine needle aspirate or core needle biopsy, for clinical NGS testing. The sequencing results were highly concordant between the existing and new extraction methods when the extracted DNA was subjected to a validated comprehensive genomic profiling (CGP) clinical test, demonstrating the quality and robustness of the extraction. For RNA, NGS libraries were made following the validated CGP clinical test with hybrid capturing, and the results were comparable between the two extraction methods. In conclusion, an FFPE extraction method was optimized to allow more clinical biopsy samples to be tested with different NGS workflows, providing a better diagnostic value for patient care. Citation Format: Rebecca Hong, Jieying Chu, Steven Hsiao, Daniel Whang, Steven P. Rivera, Heng Xie, Jiannan Guo. Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications [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 5054.

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 Low-cost High-throughput Targeted Sequencing for the Accurate Detection of Respiratory Tract Pathogen

Introduction: The current gold standard for SARS-CoV-2 diagnosis by real-time RT-PCR has limitations of gene numbers that can be detected. In this study, we developed a low-cost and high-throughput next-generation sequencing technology that can overcome the limitations of RT-PCR. Methodology: A targeted sequencing panel (TSP) consisting of approximately 500 amplicons was designed that can simultaneously detect a broad range of gene loci of SARS-CoV-2 and genes for the most common viruses of respiratory infectious viruses in a single run of up to 96 samples. 448 samples and 31 control samples were examined independently with both TSP and RT-PCR, results were compared for accuracy and other indicators. Results: TSP identified 50 SARS-CoV-2 positive samples with a 99.33% match to RT-PCR results. It is not surprising that TSP also identified multiple viral infections from 96 samples, whereas RT-PCR could not. TSP demonstrated its ability to conclude diagnosis for those undecided from RT-PCR tests. Conclusion: Our data demonstrated that TSP is a fast and accurate test for detecting multiple pathogen infections of the respiratory tract.

Long read sequencing on its way to the routine diagnostics of genetic diseases.

The clinical application of technological progress in the identification of DNA alterations has always led to improvements of diagnostic yields in genetic medicine. At chromosome side, from cytogenetic techniques evaluating number and gross structural defects to genomic microarrays detecting cryptic copy number variants, and at molecular level, from Sanger method studying the nucleotide sequence of single genes to the high-throughput next-generation sequencing (NGS) technologies, resolution and sensitivity progressively increased expanding considerably the range of detectable DNA anomalies and alongside of Mendelian disorders with known genetic causes. However, particular genomic regions (i.e., repetitive and GC-rich sequences) are inefficiently analyzed by standard genetic tests, still relying on laborious, time-consuming and low-sensitive approaches (i.e., southern-blot for repeat expansion or long-PCR for genes with highly homologous pseudogenes), accounting for at least part of the patients with undiagnosed genetic disorders. Third generation sequencing, generating long reads with improved mappability, is more suitable for the detection of structural alterations and defects in hardly accessible genomic regions. Although recently implemented and not yet clinically available, long read sequencing (LRS) technologies have already shown their potential in genetic medicine research that might greatly impact on diagnostic yield and reporting times, through their translation to clinical settings. The main investigated LRS application concerns the identification of structural variants and repeat expansions, probably because techniques for their detection have not evolved as rapidly as those dedicated to single nucleotide variants (SNV) identification: gold standard analyses are karyotyping and microarrays for balanced and unbalanced chromosome rearrangements, respectively, and southern blot and repeat-primed PCR for the amplification and sizing of expanded alleles, impaired by limited resolution and sensitivity that have not been significantly improved by the advent of NGS. Nevertheless, more recently, with the increased accuracy provided by the latest product releases, LRS has been tested also for SNV detection, especially in genes with highly homologous pseudogenes and for haplotype reconstruction to assess the parental origin of alleles with de novo pathogenic variants. We provide a review of relevant recent scientific papers exploring LRS potential in the diagnosis of genetic diseases and its potential future applications in routine genetic testing.

The complete chloroplast genome sequence of an invasive plant, Tragopogon dubius Scopoli (asteraceae)

Tragopogon dubius Scopoli is native to Europe and western Asia and is considered an invasive plant in China. In this study, the complete chloroplast genome of T. dubius was obtained using high-throughput next-generation sequencing technology. The whole chloroplast genome was 153,017 bp long with a GC content of 38% and comprised 130 genes (86 protein-coding genes, 36 tRNA genes, and 8 rRNA genes). Phylogenetic analysis based on the concatenated chloroplast protein-coding sequences showed that T. dubius is most closely related to Tragopogon pratensis. This study provides valuable genetic data for further phylogenetic analysis and molecular identification of species in the genus Tragopogon.

The effect of missing data on evolutionary analysis of sequence capture bycatch, with application to an agricultural pest

Sequence capture is a genomic technique that selectively enriches target sequences before high throughput next-generation sequencing, to generate specific sequences of interest. Off-target or ‘bycatch’ data are often discarded from capture experiments, but can be leveraged to address evolutionary questions under some circumstances. Here, we investigated the effects of missing data on a variety of evolutionary analyses using bycatch from an exon capture experiment on the global pest moth, Helicoverpa armigera. We added > 200 new samples from across Australia in the form of mitogenomes obtained as bycatch from targeted sequence capture, and combined these into an additional larger dataset to total > 1000 mitochondrial cytochrome c oxidase subunit I (COI) sequences across the species’ global distribution. Using discriminant analysis of principal components and Bayesian coalescent analyses, we showed that mitogenomes assembled from bycatch with up to 75% missing data were able to return evolutionary inferences consistent with higher coverage datasets and the broader literature surrounding H. armigera. For example, low-coverage sequences broadly supported the delineation of two H. armigera subspecies and also provided new insights into the potential for geographic turnover among these subspecies. However, we also identified key effects of dataset coverage and composition on our results. Thus, low-coverage bycatch data can offer valuable information for population genetic and phylodynamic analyses, but caution is required to ensure the reduced information does not introduce confounding factors, such as sampling biases, that drive inference. We encourage more researchers to consider maximizing the potential of the targeted sequence approach by examining evolutionary questions with their off-target bycatch where possible—especially in cases where no previous mitochondrial data exists—but recommend stratifying data at different genome coverage thresholds to separate sampling effects from genuine genomic signals, and to understand their implications for evolutionary research.

Myeloid Neoplasms: Better Understanding of their Molecular Pathogenesis with Improvised Genomic Testing: A Ray of Hope for Better Clinical Outcomes

With the increase in incidence and prevalence of myeloid neoplasms in India, it has become a necessity to understand its molecular mechanisms, acquisition of genomic alterations, and understand its primary and secondary resistance pathways which ultimately impact the decision of therapeutics. The objective of this review is to investigate the molecular aspects of this disease type and identify the biomarkers that help with diagnosis, risk assessment, prognosis, and selecting the best line of treatment for a specific myeloid neoplasm. Advancements and innovations in molecular technologies from simplest Real-Time PCR to high throughput next-generation sequencing have played a vital role in screening the most common mutations and fusions to the novel and rare. Molecular technologies have helped to enumerate the genomic landscape of myeloid malignancies. The understanding of both- the mechanisms and the technology is a strong combination as it has helped revolutionize precision oncology and helped in giving better therapeutic choices with better clinical outcomes. The importance of cellular morphology, clinical symptoms, and molecular pathology in assessing the risk of myeloid malignancies is emphasized and summarized in the review. The review concludes that understanding molecular pathogenesis can be improved by using clinical-pathological-molecular strategies for diagnosis and therapy decision-making.

Comprehensive landscape of non-CODIS STRs in global populations provides new insights into challenging DNA profiles

The worldwide implementation of short tandem repeats (STR) profiles in forensic genetics necessitated establishing and expanding the CODIS core loci set to facilitated efficient data management and exchange. Currently, the mainstay CODIS STRs are adopted in most general-purpose forensic kits. However, relying solely on these loci failed to yield satisfactory results for challenging tasks, such as bio-geographical ancestry inference, complex DNA mixture profile interpretation, and distant kinship analysis. In this context, non-CODIS STRs are potent supplements to enhance the systematic discriminating power, particularly when combined with the high-throughput next-generation sequencing (NGS) technique. Nevertheless, comprehensive evaluation on non-CODIS STRs in diverse populations was scarce, hindering their further application in routine caseworks. To address this gap, we investigated genetic variations of 178 historically available non-CODIS STRs from ethnolinguistically different worldwide populations and studied their characteristics and forensic potentials via high-coverage whole genome sequencing (WGS) data. Initially, we delineated the genomic properties of these non-CODIS markers through sequence searching, repeat structure scanning, and manual inspection. Subsequent population genetics analysis suggested that these non-CODIS STRs had comparable polymorphism levels and forensic utility to CODIS STRs. Furthermore, we constructed a theoretical next-generation sequencing (NGS) panel comprising 108 STRs (20 CODIS STRs and 88 non-CODIS STRs), and evaluated its performance in inferring bio-geographical ancestry origins, deconvoluting complex DNA mixtures, and differentiating distant kinships using real and simulated datasets. Our findings demonstrated that incorporating supplementary non-CODIS STRs enabled the extrapolation of multidimensional information from a single STR profile, thereby facilitating the analysis of challenging forensic tasks. In conclusion, this study presents an extensive genomic landscape of forensic non-CODIS STRs among global populations, and emphasized the imperative inclusion of additional polymorphic non-CODIS STRs in future NGS-based forensic systems.

Novel compound heterozygous MYO15A splicing variants in autosomal recessive non-syndromic hearing loss.

Hereditary hearing loss is a highly heterogeneous disorder. This study aimed to identify the genetic cause of a Chinese family with autosomal recessive non-syndromic sensorineural hearing loss (ARNSHL). Clinical information and peripheral blood samples were collected from the proband and its parents. Two-step high-throughput next-generation sequencing on the Ion Torrent platform was applied to detect variants as follows. First, long-range PCR was performed to amplify all the regions of the GJB2, GJB3, SLC26A4, and MT-RNR1 genes, followed by next-generation sequencing. If no candidate pathogenetic variants were found, the targeted exon sequencing with AmpliSeq technology was employed to examine another 64 deafness-associated genes. Sanger sequencing was used to identify variants and the lineage co-segregation. The splicing of the MYO15A gene was assessed by in silico bioinformatics prediction and minigene assays. Two candidate MYO15A gene (OMIM, #602,666) heterozygous splicing variants, NG_011634.2 (NM_016239.3): c.6177 + 1G > T and c.9690 + 1G > A, were identified in the proband, and these two variants were both annotated as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Further bioinformatic analysis predicted that the c.6177 + 1G > T variant might cause exon skipping and that the c.9690 + 1G > A variant might activate a cryptic splicing donor site in the downstream intronic region. An in vitro minigene assay confirmed the above predictions. We identified a compound heterozygous splicing variant in the MYO15A gene in a Han Chinese family with ARNSHL. Our results broaden the spectrum of MYO15A variants, potentially benefiting the early diagnosis, prevention, and treatment of the disease.

Сравнительный анализ микробиоты ткани яичка и уретры у пациентов с азооспермией методом секвенирования нового поколения (NGS)

Aim. To determine the role of taxonomic bacterial diversity of the urethral and testicular microbiota in the development of male infertility using high-throughput next-generation sequencing (NGS). Material and methods. In the period between 2018 and 2022 we examined 53 patients. Three study groups were formed: Infertile patients with nonobstructive azoospermia and concomitant varicocele (n = 13) – group 1; Infertile patients with obstructive azoospermia (n = 29) – group 2; Fertile patients with confirmed paternity and testicular pathology requiring histological verification to exclude testicular oncopathology (n = 11) – group 3. Each patient underwent TESE with testicular both and urethral tissue sampling in order to compare the bacterial landscape and control the purity of the method. The obtained material was sequenced using a high-throughput method (NGS). Results. According to our findings, the testicular tissue of obstructive azoospermia patients had significantly depleted bacterial diversity in comparison to nonobstructive azoospermia and concomitant varicocele ones. Also, the fertile group turned out to be the most diverse in its taxonomic community. These results may suggest the bacterial microbiome’s influence on men’s reproductive health. Discussion. Our urogenital bacterial diversity analysis showed that human testicular tissue is not a microbiologically sterile environment and also presented new data associated with testicular tissue and its possible relations with male infertility. Conclusion. A focus on the qualitative and quantitative composition of the testicular microbial community may form the new basis in the diagnosis and treatment of male infertility associated with various types of azoospermia

Cross-population applications of genomics to understand the risk of multifactorial traits involving inflammation and immunity.

The interplay between genetic and environmental factors plays a significant role in interindividual variation in immune and inflammatory responses. The availability of high-throughput low-cost genotyping and next-generation sequencing has revolutionized our ability to identify human genetic variation and understand how this varies within and between populations, and the relationship with disease. In this review, we explore the potential of genomics for patient benefit, specifically in the diagnosis, prognosis and treatment of inflammatory and immune-related diseases. We summarize the knowledge arising from genetic and functional genomic approaches, and the opportunity for personalized medicine. The review covers applications in infectious diseases, rare immunodeficiencies and autoimmune diseases, illustrating advances in diagnosis and understanding risk including use of polygenic risk scores. We further explore the application for patient stratification and drug target prioritization. The review highlights a key challenge to the field arising from the lack of sufficient representation of genetically diverse populations in genomic studies. This currently limits the clinical utility of genetic-based diagnostic and risk-based applications in non-Caucasian populations. We highlight current genome projects, initiatives and biobanks from diverse populations and how this is being used to improve healthcare globally by improving our understanding of genetic susceptibility to diseases and regional pathogens such as malaria and tuberculosis. Future directions and opportunities for personalized medicine and wider application of genomics in health care are described, for the benefit of individual patients and populations worldwide.

Genome-Wide Association Study of Preharvest Sprouting in Wheat.

Genome-wide association study (GWAS) is widely used to characterize genes or quantitative trait loci (QTLs) associated with preharvest sprouting and seed dormancy. GWAS can identify both previously discovered and novel QTLs across diverse genetic panels. The high-throughput SNP arrays or next-generation sequencing technologies have facilitated the identification of numerous genetic markers, thereby significantly enhancing the resolution of GWAS. Although various methods have been developed, the fundamental principles underlying these techniques remain constant. Here, we provide a basic technological flow to perform seed dormancy assay, followed by GWAS using population structure control, and compared itwith previous identified QTLs and genes.

Comprehensive Molecular Genetic Analysis in Glioma Patients by Next Generation Sequencing.

Glioma is caused by multiple genomic alterations. The evolving classification of gliomas emphasizes the significance of molecular testing. Next generation sequencing (NGS) offers the assessment of parallel combinations of multiple genetic alterations and identifying actionable mutations that guide treatment. This study comprehensively analyzed glioma patients using multi-gene NGS panels, providing powerful insights to inform diagnostic classification and targeted therapies. We conducted a targeted panel-based NGS analysis on formalin-fixed and paraffin-embedded nucleic acids extracted from a total of 147 glioma patients. These samples underwent amplicon capture-based library preparation and sequenced using the Oncomine Comprehensive Assay platform. The resulting sequencing data were then analyzed using the bioinformatics tools. A total of 301 mutations, were found in 132 out of 147 tumors (89.8%). These mutations were in 68 different genes. In 62 tumor samples (42.2%), copy number variations (CNVs) with gene amplifications occurred in 25 genes. Moreover, 25 tumor samples (17.0%) showed gene fusions in 6 genes and intragenic deletion in a gene. Our analysis identified actionable targets in several genes, including 11 with mutations, 8 with CNVs, and 3 with gene fusions and intragenic deletion. These findings could impact FDA-approved therapies, NCCN guideline-based treatments, and clinical trials. We analyzed precisely diagnosing the classification of gliomas, detailing the frequency and co-occurrence of genetic alterations and identifying genetic alterations with potential therapeutic targets by NGS-based molecular analysis. The high-throughput NGS analysis is an efficient and powerful tool to comprehensively support molecular testing in neurooncology.

A Protocol for the Detection of Fusion Transcripts Using RNA-Sequencing Data.

Fusion transcripts are formed when two genes or their mRNAs fuse to produce a novel gene or chimeric transcript. Fusion genes are well-known cancer biomarkers used for cancer diagnosis and as therapeutic targets. Gene fusions are also found in normal physiology and lead to the evolution of novel genes that contribute to better survival and adaptation for an organism. Various in vitro approaches, such as FISH, PCR, RT-PCR, and chromosome banding techniques, have been used to detect gene fusion. However, all these approaches have low resolution and throughput. Due to the development of high-throughput next-generation sequencing technologies, the detection of fusion transcript becomes feasible using whole genome sequencing, RNA-Seq data, and bioinformatics tools. This chapter will overview the general computational protocol for fusion transcript detection from RNA-sequencing datasets.

Comparative 16s metagenomic analysis of prokaryotic diversity in freshwater and permanent snow-line glacial lakes in Türkiye

Freshwater lakes are critical to healthy ecosystems, providing vital services like drinking water and recreation for surrounding communities. Microorganisms within these ecosystems play essential roles, driving biogeochemical cycles for elements like carbon, nitrogen, and sulfur. This study utilized a metagenomic approach to examine the prokaryotic communities of three freshwater lakes in T?rkiye: the Eber and Bey?ehir lakes, located at close altitudes (967 m and 1,115 m, respectively), which serve as primary water sources for nearby communities, and Lake Uludag Buzlu (2,390 m) that lies at the permanent snow border within the Uludag glacial lake system. Metagenomics allowed us to identify species, genetic structures, and the functional roles of microorganisms. Employing high-throughput next-generation sequencing (NGS) technology, we analyzed 16S ribosomal DNA (rDNA) sequences (V3-V4 regions) from the lake samples. EzBioCloud software facilitated the analysis of prokaryotic diversity obtained using Illumina NovaSeq technology. While Eber and Bey?ehir lakes had similar diversity, Bacillota dominated in the higher-altitude Lake Uludag Buzlu. Genus-level analysis revealed Parabacteroides as the most prevalent in Lake Uludag Buzlu, contrasting with Limnohabitans dominance in Lake Eber; Lake Bey?ehir exhibited co-dominance of Limnohabitans and Planktophila.

Сравнительная характеристика тестикулярной и уретральной микробиоты у пациентов с различными видами азооспермии и сопутствующим варикоцеле.

Introduction. Everywhere, about 15% of couples of reproductive age have the problem of infertility, with the male factor being the main cause in 50% of cases. The lack of effective treatment for male infertility is largely due to the multifactorial etiology of this disease. One of these factors is infections of the urogenital tract, the diagnostic search for which requires the development of new methods. Today, next generation sequencing (NGS), using the nucleotide sequence in the 16S rRNA region of the bacterial genome, is the most modern and highly sensitive method for determining the microbiome. Aim: to study and compare the structure of the testicular and urethral microbiota in patients with various types of azoospermia and concomitant varicocele. Materials and methods. The study included 107 infertile patients with various types of azoospermia, in some cases, with concomitant varicocele, who applied to the Center for Reproductive and Cellular Medicine of the State Budgetary Healthcare Institution of the Children's City Clinical Hospital in Krasnodar, Ministry of Health of the Republic of Krasnodar region. As a result of sample preparation, 31 samples did not meet the quality criteria and were excluded from the study. In all cases, patients underwent micro-TESE (multifocal testicular biopsy), as well as collection of biological material from the urethra. When studying the structure of the microbiome of testicular tissue, amplicons of the bacterial 16S rRNA gene were analyzed using high-throughput next-generation sequencing (NGS). Data were processed using the QIIME program (version 1.9.1). Results. Comparative characteristics of the structure of the microbiota of the urethra and testicles were carried out using average values in groups, at the level of phyla and families. When comparing the urethral microbiota, a similarity in the species composition of the microbiota was revealed in patients with nonobstructive azoospermia (NOA) and obstructive azoospermia (ОА), while the species composition of the testicular microbiota in patients with ОА is similar to that in patients with varicocele. As a result of data analysis, 6 phyla and 12 families of bacteria were identified that are common to both testicular and urethral tissue. When comparing the frequency of occurrence of these bacteria, differences were identified in the comparison groups. Conclusions. The microbiota of the urethra in men has a wide taxonomic composition, which has characteristics in infertile patients, caused, in our opinion, by disruption of the GTB, in particular, in patients with varicocele. The structure of the microbiota of testicular tissue has a decrease in species diversity in patients with non-obstructive azoospermia with the dominant Enterobacteriaceae_A family, in contrast to patients with obstructive azoospermia and patients with NOA and concomitant varicocele. The current situation and the discrepancy in scientific publications and expert opinions require further expansion of the range of scientific interdisciplinary research.

Identification of differentially expressed circRNAs in prostate cancer of different clinical stages by RNA sequencing

Circular RNAs (circRNAs) are linked to cancer, but it's still not clear what role they play in prostatic cancer. Through high-throughput sequencing, the goal of this study was to compare how circRNAs are expressed at different stages of prostate cancer. 12 patients attending the Department of Urology at the Second Affiliated Hospital of Nanchang University between June 2020 and October 2021 were used for RNA sequencing, and 14 patients were used for real-time fluorescent quantitative PCR (qRT-PCR). The expression profiles of prostate cancer circRNAs were constructed by sequencing with the help of next-generation high-throughput sequencing technology, and the differentially expressed circRNAs were analyzed by targeting microRNA (miRNA) loci and Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the genes from which circRNAs originated. Finally, the expression of target circRNAs in two prostate tissues was verified by qRT-PCR. Following high-throughput sequencing, 13,047 circRNAs were identified, and 605 circRNAs with significant differential expression were identified, of which 361 circRNAs were up-regulated, and 244 circRNAs were down-regulated. Analysis of circRNA-originated genes using GO and the KEGG enrichment analysis showed that circRNA host genes can regulate and influence multiple signaling pathways in prostate cancer with important biological functions. And the circRNA–miRNA network was constructed. The highest number of differentially expressed circRNA-binding miRNAs were: hsa_circ_000 7582 (52), hsa_circ_000 6198 (37), hsa_circ_000 6759 (28), hsa_circ_000 5675 (25), and hsa_circ_000 2172 (22). Moreover, we further screened out the circRNA (hsa_circ_0005692) that was significantly differentially expressed and common to all groups and verified by qRT-PCR that the expression of the target circRNA (hsa_circ_0005692) was significantly downregulated in prostate cancer compared with benign prostatic hyperplasia (BPH) tissues.