MaterniCode: New Bioinformatic Pipeline to Detect Fetal Aneuploidies and Rearrangements Using Next-Generation Sequencing.

Objective: The present study is aimed at introducing and evaluating MaterniCode, a state-of-the-art bioinformatic pipeline for noninvasive prenatal testing (NIPT) that leverages the Ion Torrent semiconductor sequencing platform. The initiative strives to revolutionize prenatal diagnostics by offering a rapid and cost-effective method without sacrificing accuracy. Methods: Two distinct bioinformatic strategies were employed for fetal sex determination, one of which achieved 100% accuracy. We analyzed 1225 maternal blood samples for fetal aneuploidies, benchmarking against the industry standard Illumina VeriSeq™ NIPT Solution v2. The capability of MaterniCode to detect and characterize complex chromosomal anomalies was also assessed. Results: MaterniCode achieved near-perfect accuracy in fetal sex determination through chromosome Y (chrY )-specific gene analysis, whereas the alternative method, employing the ratio of high-quality mapped reads on chrY relative to all reads, delivered 100% accuracy. For fetal aneuploidy detection, both the integrated WisecondorX and NIPTeR algorithms demonstrated a 100% sensitivity and specificity rate, consistent with Illumina VeriSeq™ NIPT Solution v2. The pipeline also successfully identified and precisely mapped significant chromosomal abnormalities, exemplified by a 2.4 Mb deletion on chromosome 13 and a 3 Mb duplication on chromosome 2. Conclusion: MaterniCode has proven to be an innovative and highly efficient tool in the domain of NIPT, demonstrating excellent sensitivity and specificity. Its robust capability to effectively detect a wide range of complex chromosomal aberrations, including rare and subtle variations, positions it as a promising and valuable addition to prenatal diagnostic technologies. This enhancement to diagnostic precision significantly aids clinicians in making informed decisions during pregnancy management.

Mycobiome of the external ear canal of healthy cows.

Malassezia yeasts belong to the normal skin microbiota of a wide range of warm-blooded animals. However, their significance in cattle is still poorly understood. In the present study, the mycobiota of the external ear canal of 20 healthy dairy Holstein cows was assessed by cytology, culture, PCR, and next-generation sequencing. The presence of Malassezia was detected in 15 cows by cytology and PCR. The metagenomic analysis revealed that Ascomycota was the predominant phylum but M. pachydermatis the main species. The Malassezia phylotype 131 was detected in low abundance. Nor M. nana nor M. equina were detected in the samples.

The mycobiota of the external ear canal of healthy cows was assessed by cytology, culture, PCR, and NGS. The presence of Malassezia was detected by cytology and PCR. Ascomycota was the main phylum and M. pachydermatis the main species. The Malassezia phylotype 131 was also detected in the samples.

Study of the variation of the Malassezia load in the interdigital fold of dogs with pododermatitis.

The yeast Malassezia pachydermatis is a common inhabitant of the skin and mucosae of dogs. However, under certain circumstances this yeast can overgrow and act as an opportunistic pathogen causing otitis and dermatitis in dogs. Canine pododermatitis is a common disorder in dogs in which M. pachydermatis acts as an opportunistic pathogen. In the present study, the presence of Malassezia yeasts was assessed and quantified in samples collected from the interdigital space of dogs with pododermatitis before and after treatment, and from healthy dogs. The samples were subjected to two different cytological examinations, culture on Sabouraud glucose agar and modified Dixon's agar and a quantitative PCR targeting the internal transcribed spacer (ITS) genomic region. A selection of samples was analyzed by next generation sequencing (NGS) using the D1D2 domain of the large subunit of the ribosomal DNA as target. The pododermatitis samples before treatment showed higher cell counts, colony-forming units and ITS copies than the rest of samples. The NGS analysis revealed that Ascomycota was the main phylum in the healthy and post-treatment samples. However, Basidiomycota and M. pachydermatis was more abundant in the pododermatitis samples before treatment. These results support M. pachydermatis as an opportunistic agent in canine pododermatitis by a variety of methods, and demonstrate the correlation between cytologic and molecular methods for quantification.

The lower airways microbiota and antimicrobial peptides indicate dysbiosis in sarcoidosis.

BACKGROUND: The role of the pulmonary microbiome in sarcoidosis is unknown. The objectives of this study were the following: (1) examine whether the pulmonary fungal and bacterial microbiota differed in patients with sarcoidosis compared with controls; (2) examine whether there was an association between the microbiota and levels of the antimicrobial peptides (AMPs) in protected bronchoalveolar lavage (PBAL). METHODS: Thirty-five sarcoidosis patients and 35 healthy controls underwent bronchoscopy and were sampled with oral wash (OW), protected BAL (PBAL), and left protected sterile brushes (LPSB). The fungal ITS1 region and the V3V4 region of the bacterial 16S rRNA gene were sequenced. Bioinformatic analyses were performed with QIIME 2. The AMPs secretory leucocyte protease inhibitor (SLPI) and human beta defensins 1 and 2 (hBD-1 and hBD-2), were measured in PBAL by enzyme-linked immunosorbent assay (ELISA). RESULTS: Aspergillus dominated the PBAL samples in sarcoidosis. Differences in bacterial taxonomy were minor. There was no significant difference in fungal alpha diversity between sarcoidosis and controls, but the bacterial alpha diversity in sarcoidosis was significantly lower in OW (p = 0.047) and PBAL (p = 0.03) compared with controls. The beta diversity for sarcoidosis compared with controls differed for both fungi and bacteria. AMP levels were significantly lower in sarcoidosis compared to controls (SLPI and hBD-1: p < 0.01). No significant correlations were found between alpha diversity and AMPs. CONCLUSIONS: The pulmonary fungal and bacterial microbiota in sarcoidosis differed from in controls. Lower antimicrobial peptides levels were seen in sarcoidosis, indicating an interaction between the microbiota and the innate immune system. Whether this dysbiosis represents a pathogenic mechanism in sarcoidosis needs to be confirmed in experimental studies. Video Abstract.

Exploring the Healthy Eye Microbiota Niche in a Multicenter Study.

PURPOSE: This study aims to explore and characterize healthy eye microbiota. METHODS: Healthy subjects older than 18 years were selected for this descriptive cross-sectional study. Samples were collected with an eSwab with 1 mL of Liquid Amies Medium (Copan Brescia, Italy). Following DNA extraction, libraries preparation, and amplification, PCR products were purified and end-repaired for barcode ligation. Libraries were pooled to a final concentration of 26 pM. Template preparation was performed with Ion Chef according to Ion 510, Ion 520, and Ion 530 Kit-Chef protocol. Sequencing of the amplicon libraries was carried out on a 520 or 530 chip using the Ion Torrent S5 system (Thermo Fisher; Waltham, MA, USA). Raw reads were analyzed with GAIA (v 2.02). RESULTS: Healthy eye microbiota is a low-diversity microbiome. The vast majority of the 137 analyzed samples were highly enriched with Staphylococcus, whereas only in a few of them, other genera such as Bacillus, Pseudomonas, and Corynebacterium predominate. We found an average of 88 genera with an average Shannon index of 0.65. CONCLUSION: We identified nine different ECSTs. A better understanding of healthy eye microbiota has the potential to improve disease diagnosis and personalized regimens to promote health.

3D chromatin remodelling in the germ line modulates genome evolutionary plasticity.

Chromosome folding has profound impacts on gene regulation, whose evolutionary consequences are far from being understood. Here we explore the relationship between 3D chromatin remodelling in mouse germ cells and evolutionary changes in genome structure. Using a comprehensive integrative computational analysis, we (i) reconstruct seven ancestral rodent genomes analysing whole-genome sequences of 14 species representatives of the major phylogroups, (ii) detect lineage-specific chromosome rearrangements and (iii) identify the dynamics of the structural and epigenetic properties of evolutionary breakpoint regions (EBRs) throughout mouse spermatogenesis. Our results show that EBRs are devoid of programmed meiotic DNA double-strand breaks (DSBs) and meiotic cohesins in primary spermatocytes, but are associated in post-meiotic cells with sites of DNA damage and functional long-range interaction regions that recapitulate ancestral chromosomal configurations. Overall, we propose a model that integrates evolutionary genome reshuffling with DNA damage response mechanisms and the dynamic spatial genome organisation of germ cells.

A longitudinal study of the pulmonary mycobiome in subjects with and without chronic obstructive pulmonary disease.

BACKGROUND: Few studies have examined the stability of the pulmonary mycobiome. We report longitudinal changes in the oral and pulmonary mycobiome of participants with and without COPD in a large-scale bronchoscopy study (MicroCOPD). METHODS: Repeated sampling was performed in 30 participants with and 21 without COPD. We collected an oral wash (OW) and a bronchoalveolar lavage (BAL) sample from each participant at two time points. The internal transcribed spacer 1 region of the ribosomal RNA gene cluster was PCR amplified and sequenced on an Illumina HiSeq sequencer. Differences in taxonomy, alpha diversity, and beta diversity between the two time points were compared, and we examined the effect of intercurrent antibiotic use. RESULTS: Sample pairs were dominated by Candida. We observed less stability in the pulmonary taxonomy compared to the oral taxonomy, additionally emphasised by a higher Yue-Clayton measure in BAL compared to OW (0.69 vs 0.22). No apparent effect was visually seen on taxonomy from intercurrent antibiotic use or participant category. We found no systematic variation in alpha diversity by time either in BAL (p-value 0.16) or in OW (p-value 0.97), and no obvious clusters on bronchoscopy number in PCoA plots. Pairwise distance analyses showed that OW samples from repeated sampling appeared more stable compared to BAL samples using the Bray-Curtis distance metric (p-value 0.0012), but not for Jaccard. CONCLUSION: Results from the current study propose that the pulmonary mycobiome is less stable than the oral mycobiome, and neither COPD diagnosis nor intercurrent antibiotic use seemed to influence the stability.

Nasopharyngeal Microbiota as an early severity biomarker in COVID-19 hospitalised patients.

This study aimed to analyse the diversity and taxonomic composition of the nasopharyngeal microbiota, to determine its association with COVID-19 clinical outcome. To study the microbiota, we utilized 16S rRNA sequencing of 177 samples that came from a retrospective cohort of COVID-19 hospitalized patients. Raw sequences were processed by QIIME2. The associations between microbiota, invasive mechanical ventilation (IMV), and all-cause mortality were analysed by multiple logistic regression, adjusted for age, gender, and comorbidity. The microbiota α diversity indexes were lower in patients with a fatal outcome, whereas the ß diversity analysis showed a significant clustering in these patients. After multivariate adjustment, the presence of Selenomonas spp., Filifactor spp., Actinobacillus spp., or Chroococcidiopsis spp., was associated with a reduction of more than 90% of IMV. Higher diversity and the presence of certain genera in the nasopharyngeal microbiota seem to be early biomarkers of a favourable clinical evolution in hospitalized COVID-19 patients.

PRGdb 4.0: an updated database dedicated to genes involved in plant disease resistance process.

The Plant Resistance Genes database (PRGdb; http://prgdb.org/prgdb4/) has been greatly expanded, keeping pace with the increasing amount of available knowledge and data (sequenced proteomes, cloned genes, public analysis data, etc.). The easy-to-use style of the database website has been maintained, while an updated prediction tool, more data and a new section have been added. This new section will contain plant resistance transcriptomic experiments, providing additional easy-to-access experimental information. DRAGO3, the tool for automatic annotation and prediction of plant resistance genes behind PRGdb, has been improved in both accuracy and sensitivity, leading to more reliable predictions. PRGdb offers 199 reference resistance genes and 586.652 putative resistance genes from 182 sequenced proteomes. Compared to the previous release, PRGdb 4.0 has increased the number of reference resistance genes from 153 to 199, the number of putative resistance genes from 177K from 76 proteomes to 586K from 182 sequenced proteomes. A new section has been created that collects plant-pathogen transcriptomic data for five species of agricultural interest. Thereby, with these improvements and data expansions, PRGdb 4.0 aims to serve as a reference to the plant scientific community and breeders worldwide, helping to further study plant resistance mechanisms that contribute to fighting pathogens.

GreeNC 2.0: a comprehensive database of plant long non-coding RNAs.

The Green Non-Coding Database (GreeNC) is one of the reference databases for the study of plant long non-coding RNAs (lncRNAs). Here we present our most recent update where 16 species have been updated, while 78 species have been added, resulting in the annotation of more than 495 000 lncRNAs. Moreover, sequence clustering was applied providing information about sequence conservation and gene families. The current version of the database is available at: http://greenc.sequentiabiotech.com/wiki2/Main_Page.

Ago1 controls myogenic differentiation by regulating eRNA-mediated CBP-guided epigenome reprogramming.

The role of chromatin-associated RNAi components in the nucleus of mammalian cells and in particular in the context of developmental programs remains to be elucidated. Here, we investigate the function of nuclear Argonaute 1 (Ago1) in gene expression regulation during skeletal muscle differentiation. We show that Ago1 is required for activation of the myogenic program by supporting chromatin modification mediated by developmental enhancer activation. Mechanistically, we demonstrate that Ago1 directly controls global H3K27 acetylation (H3K27ac) by regulating enhancer RNA (eRNA)-CREB-binding protein (CBP) acetyltransferase interaction, a key step in enhancer-driven gene activation. In particular, we show that Ago1 is specifically required for myogenic differentiation 1 (MyoD) and downstream myogenic gene activation, whereas its depletion leads to failure of CBP acetyltransferase activation and blocking of the myogenic program. Our work establishes a role of the mammalian enhancer-associated RNAi component Ago1 in epigenome regulation and activation of developmental programs.

Malat-1-PRC2-EZH1 interaction supports adaptive oxidative stress dependent epigenome remodeling in skeletal myotubes.

PRC2-mediated epigenetic function involves the interaction with long non-coding RNAs (lncRNAs). Although the identity of some of these RNAs has been elucidated in the context of developmental programs, their counterparts in postmitotic adult tissue homeostasis remain uncharacterized. To this aim, we used terminally differentiated postmitotic skeletal muscle cells in which oxidative stress induces the dynamic activation of PRC2-Ezh1 through Embryonic Ectoderm Develpment (EED) shuttling to the nucleus. We identify lncRNA Malat-1 as a necessary partner for PRC2-Ezh1-dependent response to oxidative stress. We show that in this pathway, PRC2-EZH1 dynamic assembly, and in turn stress induced skeletal muscle targeted genes repression, depends specifically on Malat-1. Our study reports about PRC2-RNA interactions in the physiological context of adaptive oxidative stress response and identifies the first lncRNA involved in PRC2-Ezh1 function.

The impact of chromosomal fusions on 3D genome folding and recombination in the germ line.

The spatial folding of chromosomes inside the nucleus has regulatory effects on gene expression, yet the impact of genome reshuffling on this organization remains unclear. Here, we take advantage of chromosome conformation capture in combination with single-nucleotide polymorphism (SNP) genotyping and analysis of crossover events to study how the higher-order chromatin organization and recombination landscapes are affected by chromosomal fusions in the mammalian germ line. We demonstrate that chromosomal fusions alter the nuclear architecture during meiosis, including an increased rate of heterologous interactions in primary spermatocytes, and alterations in both chromosome synapsis and axis length. These disturbances in topology were associated with changes in genomic landscapes of recombination, resulting in detectable genomic footprints. Overall, we show that chromosomal fusions impact the dynamic genome topology of germ cells in two ways: (i) altering chromosomal nuclear occupancy and synapsis, and (ii) reshaping landscapes of recombination.

The pulmonary mycobiome-A study of subjects with and without chronic obstructive pulmonary disease.

BACKGROUND: The fungal part of the pulmonary microbiome (mycobiome) is understudied. We report the composition of the oral and pulmonary mycobiome in participants with COPD compared to controls in a large-scale single-centre bronchoscopy study (MicroCOPD). METHODS: Oral wash and bronchoalveolar lavage (BAL) was collected from 93 participants with COPD and 100 controls. Fungal DNA was extracted before sequencing of the internal transcribed spacer 1 (ITS1) region of the fungal ribosomal RNA gene cluster. Taxonomic barplots were generated, and we compared taxonomic composition, Shannon index, and beta diversity between study groups, and by use of inhaled steroids. RESULTS: The oral and pulmonary mycobiomes from controls and participants with COPD were dominated by Candida, and there were more Candida in oral samples compared to BAL for both study groups. Malassezia and Sarocladium were also frequently found in pulmonary samples. No consistent differences were found between study groups in terms of differential abundance/distribution. Alpha and beta diversity did not differ between study groups in pulmonary samples, but beta diversity varied with sample type. The mycobiomes did not seem to be affected by use of inhaled steroids. CONCLUSION: Oral and pulmonary samples differed in taxonomic composition and diversity, possibly indicating the existence of a pulmonary mycobiome.

External ear canal mycobiome of some rabbit breeds.

The genus Malassezia is part of the normal skin mycobiota of a wide range of warm-blooded animals. In this genus, M. cuniculi is the only species described from rabbits. However, Malassezia species are rarely studied in lagomorphs. In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples. Although no growth was observed in the cultured plates, cytological examination revealed the presence of round cells similar to those of Malassezia yeasts. For metagenomics analysis, the D1/D2 domain of the large subunit of the ribosomal DNA (LSU rDNA) was PCR amplified and the resulting reads were mapped against a custom-made cured database of 26S fungal sequences. NGS analysis revealed that Basidiomycota was the most abundant phylum in all the samples followed by Ascomycota. Malassezia was the most common genus presenting the highest abundance in the external ear canal. Malassezia phylotype 131 and M. cuniculi were the main sequences detected in the external auditory canal of rabbits. The study included both lop-eared and erect-eared rabbits and no differences were observed in the results when comparing both groups. This is the first attempt to study the external ear canal mycobiome of rabbits of different breeds using NGS. LAY SUMMARY: In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples.

Transcriptome-wide expression profiling of Sporothrix schenckii yeast and mycelial forms and the establishment of the Sporothrix Genome DataBase.

Sporothrix schenckii is a dimorphic fungus existing as mould in the environment and as yeast in the host. The morphological shift between mycelial/yeast phases is crucial for its virulence, but the transcriptional networks implicated in dimorphic transition are still not fully understood. Here, we report the global transcriptomic differences occurring between mould and yeast phases of S. schenckii, including changes in gene expression profiles associated with these distinct cellular phenotypes. Moreover, we also propose a new genome annotation, which reveals a more complex transcriptional architecture than previously assumed. Using RNA-seq, we identified a total of 17 307 genes, of which 11 217 were classified as protein-encoding genes, whereas 6090 were designated as non-coding RNAs (ncRNAs). Approximately ~71 % of all annotated genes were found to overlap and the different-strand overlapping type was the most common. Gene expression analysis revealed that 8795 genes were differentially regulated among yeast and mould forms. Differential gene expression was also observed for antisense ncRNAs overlapping neighbouring protein-encoding genes. The release of transcriptome-wide data and the establishment of the Sporothrix Genome DataBase (http://sporothrixgenomedatabase.unime.it) represent an important milestone for Sporothrix research, because they provide a strong basis for future studies on the molecular pathways involved in numerous biological processes.

Three-Dimensional Genomic Structure and Cohesin Occupancy Correlate with Transcriptional Activity during Spermatogenesis.

Mammalian gametogenesis involves dramatic and tightly regulated chromatin remodeling, whose regulatory pathways remain largely unexplored. Here, we generate a comprehensive high-resolution structural and functional atlas of mouse spermatogenesis by combining in situ chromosome conformation capture sequencing (Hi-C), RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) of CCCTC-binding factor (CTCF) and meiotic cohesins, coupled with confocal and super-resolution microscopy. Spermatogonia presents well-defined compartment patterns and topological domains. However, chromosome occupancy and compartmentalization are highly re-arranged during prophase I, with cohesins bound to active promoters in DNA loops out of the chromosomal axes. Compartment patterns re-emerge in round spermatids, where cohesin occupancy correlates with transcriptional activity of key developmental genes. The compact sperm genome contains compartments with actively transcribed genes but no fine-scale topological domains, concomitant with the presence of protamines. Overall, we demonstrate how genome-wide cohesin occupancy and transcriptional activity is associated with three-dimensional (3D) remodeling during spermatogenesis, ultimately reprogramming the genome for the next generation.

A Walkthrough to the Use of GreeNC: The Plant lncRNA Database.

Experimentally validated plant lncRNAs have been shown to regulate important agronomic traits such as phosphate starvation response, flowering time, and interaction with symbiotic organisms, making them of great interest in plant biology and in breeding. We developed a pipeline to annotate lncRNAs and applied it to 37 plant species and 6 algae, resulting in the annotation of more than 120,000 lncRNAs. To facilitate the study of lncRNAs for the plant research community, the information gathered is organized in the Green Non-Coding Database (GreeNC, http://greenc.sciencedesigners.com/) . This chapter contains a detailed explanation of the content of GreeNC and how to access both programmatically and with a web browser.

PRGdb 3.0: a comprehensive platform for prediction and analysis of plant disease resistance genes.

The Plant Resistance Genes database (PRGdb; http://prgdb.org) has been redesigned with a new user interface, new sections, new tools and new data for genetic improvement, allowing easy access not only to the plant science research community but also to breeders who want to improve plant disease resistance. The home page offers an overview of easy-to-read search boxes that streamline data queries and directly show plant species for which data from candidate or cloned genes have been collected. Bulk data files and curated resistance gene annotations are made available for each plant species hosted. The new Gene Model view offers detailed information on each cloned resistance gene structure to highlight shared attributes with other genes. PRGdb 3.0 offers 153 reference resistance genes and 177 072 annotated candidate Pathogen Receptor Genes (PRGs). Compared to the previous release, the number of putative genes has been increased from 106 to 177 K from 76 sequenced Viridiplantae and algae genomes. The DRAGO 2 tool, which automatically annotates and predicts (PRGs) from DNA and amino acid with high accuracy and sensitivity, has been added. BLAST search has been implemented to offer users the opportunity to annotate and compare their own sequences. The improved section on plant diseases displays useful information linked to genes and genomes to connect complementary data and better address specific needs. Through, a revised and enlarged collection of data, the development of new tools and a renewed portal, PRGdb 3.0 engages the plant science community in developing a consensus plan to improve knowledge and strategies to fight diseases that afflict main crops and other plants.

Mammalian Comparative Genomics Reveals Genetic and Epigenetic Features Associated with Genome Reshuffling in Rodentia.

Understanding how mammalian genomes have been reshuffled through structural changes is fundamental to the dynamics of its composition, evolutionary relationships between species and, in the long run, speciation. In this work, we reveal the evolutionary genomic landscape in Rodentia, the most diverse and speciose mammalian order, by whole-genome comparisons of six rodent species and six representative outgroup mammalian species. The reconstruction of the evolutionary breakpoint regions across rodent phylogeny shows an increased rate of genome reshuffling that is approximately two orders of magnitude greater than in other mammalian species here considered. We identified novel lineage and clade-specific breakpoint regions within Rodentia and analyzed their gene content, recombination rates and their relationship with constitutive lamina genomic associated domains, DNase I hypersensitivity sites and chromatin modifications. We detected an accumulation of protein-coding genes in evolutionary breakpoint regions, especially genes implicated in reproduction and pheromone detection and mating. Moreover, we found an association of the evolutionary breakpoint regions with active chromatin state landscapes, most probably related to gene enrichment. Our results have two important implications for understanding the mechanisms that govern and constrain mammalian genome evolution. The first is that the presence of genes related to species-specific phenotypes in evolutionary breakpoint regions reinforces the adaptive value of genome reshuffling. Second, that chromatin conformation, an aspect that has been often overlooked in comparative genomic studies, might play a role in modeling the genomic distribution of evolutionary breakpoints.